# Development of an adverse outcome pathway for radiation-induced microcephaly via expert consultation and machine learning

Thomas Jaylet, Roel Quintens, Mohamed Abderrafi Benotmane, Jukka Luukkonen, Ignacia Braga Tanaka, Chrystelle Ibanez, Christelle Durand, Magdalini Sachana, Omid Azimzadeh, Christelle Adam-Guillermin, et al.

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

## To cite this version:

Thomas Jaylet, Roel Quintens, Mohamed Abderrafi Benotmane, Jukka Luukkonen, Ignacia Braga Tanaka, et al., Development of an adverse outcome pathway for radiation-induced microcephaly via expert consultation and machine learning. International Journal of Radiation Biology, 2022, 98 (12), pp.1752-1762. 10.1080/09553002.2022.2110312. hal-03966042

HAL Id: hal-03966042

https://hal.science/hal-03966042

Submitted on 21 Jul 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished 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>
Development of an Adverse Outcome Pathway for radiation-induced
microcephaly via expert consultation and machine learning

Thomas Jaylet<sup>a</sup>, Roel Quintens<sup>b</sup>, Mohamed Abderrafi Benotmane<sup>b</sup>, Jukka Luukkonen<sup>c</sup>, Ignacia Braga Tanaka III<sup>d</sup>, Chrystelle Ibanez<sup>e</sup>, Christelle Durand<sup>e</sup>, Magdalini Sachana<sup>f</sup>, Omid Azimzadeh<sup>g</sup>, Christelle Adam-Guillermin<sup>h</sup>, Knut Erik Tollefsen<sup>i,j,k</sup>, Olivier Laurent<sup>l</sup>, Karine Audouze<sup>a</sup>, Olivier Armant<sup>m*</sup>

<sup>a</sup>Université Paris Cité, T3S, Inserm UMRS 1124, Paris, France ; <sup>b</sup>Radiobiology Unit, Belgian Nuclear Research Centre, SCK-CEN, Mol, Belgium ; <sup>c</sup>University of Eastern Finland, Kuopio Campus, Department of Environmental and Biological Sciences, P.O. Box 1627, FI-70211 Kuopio, Finland ; <sup>d</sup>Department of Radiobiology, Institute for Environmental Sciences, 1-7 lenomae, Obuchi, Rokkasho-mura, Kamikita-gun, Aomori 039-3212, Japan ; <sup>e</sup>PSE-SANTE/SESANE/LRTOX Institut de Radioprotection et de Sûreté Nucléaire (IRSN), F-92262, Fontenay-aux-Roses, France ; <sup>f</sup>Organisation for Economic Co-operation and Development (OECD), Environment Health and Safety Division, 75775 CEDEX 16 Paris, France ; <sup>g</sup>Federal Office for Radiation Protection (Bfs), Section Radiation Biology, 85764 Neuherberg, Germany ; <sup>h</sup>PSE-SANTE/SDOS/LMDN, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Cadarache, 13115 Saint-Paul-Lez-Durance, France ; <sup>i</sup>Norwegian Institute for Water Research (NIVA), Økernveien 94, N-0579, Oslo, Norway ; <sup>j</sup>Norwegian University of Life Sciences (NMBU), Post box 5003, N-1432 Ås, Norway ; <sup>k</sup>Centre for Environmental Radioactivity, Norwegian University of Life Sciences (NMBU), Post box 5003, N-1432 Ås, Norway ; <sup>l</sup>PSE-SANTE/SESANE/LEPID, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), F-92262, Fontenay-aux-Roses, France ; <sup>m</sup>PSE-ENV/SRTE/LECO, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Cadarache, 13115 Saint-Paul-Lez-Durance, France.

*corresponding author: olivier.armant@irsn.fr

28

Notes on contributors
30 **Olivier Armant (OA)**, Ph.D, is a senior scientist at the French Institute of Radioprotection and Nuclear Safety (IRSN). His studies focus on the molecular mechanisms deregulated upon exposure to low dose of radiations during embryonic development and post-accidental effects on wild-life in Chernobyl and Fukushima. He is a member of the High-Level Group on Low-Dose Research (HLG-LDR) Joint Topical Group Rad-Chem.
31
32
33
34

35 **Karine Audouze (KA)**, Ph.D, is associate professor at the Université Paris Cité in France, where she is leading the SysTox group. She is member of the OECD Extended Advisor Group on Molecular Screening and Toxicogenomics (EAGMST), of the High-Level Group on Low-Dose Research (HLG-LDR) Rad/Chem Joint Topical and of the OECD AOP knowledge base (AOP-KB) group.
36
37
38
39

40 **Omid Azimzadeh (OAz)**, Ph.D, is a senior researcher in the section of Radiation Biology at the German Federal Office for Radiation Protection (BfS) and an Adjunct Professor (Docent) of Radiation Biology at Technical University Munich. His research interests focus on the adverse effects of radiation exposure on normal tissues with a special emphasis on omics analysis. He is a member of a working group CricuDis (Circulatory Diseases from Radiation Exposure) at the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR).
41
42
43
44
45

46 **Mohamed Abderrafi Benotmane (MAB)**, Ph.D, is a researcher project leader at the Radiobiology Unit at the Belgian Nuclear Research Centre 'SCK-CEN'. He is mainly interested in medical applications making use of radiation for diagnostic and therapy. Over the years he has been mainly involved in central nervous system impairments following exposure to radiation at young age prenatally and at early postnatal periods. The outcome of his research is of high societal impact for the protection of the brain of the fetus and the young child. He coordinated the FP7 EU project CEREBRAD (GA 295552) (2011-2015) and participated to many other EU projects (DoReMi, OPERRA, CONCERT, and MEDIRAD). He is a member of the High Council for Health in Belgium. He is delegate at the High-level group on low-dose research (HLG-LDR), and contributes as Radiobiology expert in several commissions (MELODI, WHO, OECD, ENA).
47
48
49
50
51
52
53
54
55
56

57 **Christelle Durand (CD)**, Ph.D, is a scientist at the French Institute of Radioprotection and Nuclear Safety (IRSN). Her research focuses on studying the consequences of postnatal brain exposure to IR on behavioral functions and to identify the molecular and cellular mechanisms associated with these potential disorders in mice.
58
59
60

61 **Chrystelle Ibanez (CI)**, Ph.D, is a research scientist at the French Institute of Radioprotection and Nuclear Safety (IRSN). She leads neurotoxicology and radiotoxicology research projects after contamination via inhalation. She has studied the effects and mechanisms of uranium
62
63
64

transport and tungsten particles to the brain. Here current research projects focus on the effect of combined radiological and chemical exposures on brain toxicity parameters.

65

66

**Olivier Laurent (OL)**, Ph.D, is a senior scientist at the French Institute of Radioprotection and Nuclear Safety (IRSN). He conducts occupational and environmental epidemiological studies in people exposed to low doses of radiation and other environmental stressors. He is member of the High-Level Group on Low-Dose Research (HLG-LDR) Joint Topical Group Rad-Chem.

67

68

69

70

71

**Roel Quintens (RQ)**, Ph.D, is a senior scientist and project leader at the Radiobiology Unit of SCK-CEN where he leads two main research themes: (1) the mechanistic analysis of prenatal radiation-induced defects (e.g. microcephaly and epilepsy) in brain development modeled in mice and human brain organoids, and (2) the identification of novel transcriptional biomarkers (splice variants, circular RNAs) of exposure and cancer cell sensitivity to different radiation qualities. He is member of the Strategic Research Agenda working group of MELODI.

72

73

74

75

76

77

**Magdalini Sachana (MS)**, Ph.D, is a Policy Analyst in the Environmental Health and Safety Division of the OECD's Environmental Directorate. She manages the development and implementation of policies in the field of chemical safety and contributes to the OECD Test Guidelines, Pesticide and Hazard Assessment Programs. Dr. Sachana, among other projects, is contributing to the coordination of the OECD project on the *in vitro* battery for developmental neurotoxicity and the AOP Program.

78

79

80

81

82

83

**Ignacia Braga Tanaka III (IT)**, DVM, Ph.D, is an Associate Senior Scientist at the Institute for Environmental Sciences (IES) in Rokkasho, Aomori, Japan. She is a trained veterinarian and here research work focus on the late effects of chronic exposure to low dose-rate radiation on life span, cancer and non-neoplastic diseases in various exposure scenarios. She is member of the ICRP Task Groups 110 (Veterinary Medicine) and 121 (Offspring and next generations).

84

85

86

87

88

**Knut Erik Tollefsen (KET)**, Ph.D, is a Chief Scientist at the Norwegian Institute for Water Research (NIVA) and an Adjunct Professor at the Norwegian University of Life Sciences (NMBU). He is a Norwegian delegate at the High-level group on low-dose research (HLG-LDR), OECD Extended Advisor Group on Molecular Screening and Toxicogenomics (EAGMST), co-chair of the HLG-LDR Rad/Chem AOP Joint Topical Group and coordinates NIVAS's Computational Toxicology Program, NCTP (www.niva.no/tcp).

89

90

91

92

93

94

95

**Thomas Jaylet (TJ)**, is Ph.D student in bioinformatics at Université Paris-Cité. His thesis is part of the RadoNorm project (under grant agreement No 900009) and aims to improve the

96
understanding of ionizing radiation exposure through the development of AOPs and integrative systems toxicology methods. He is also involved in the development of the AOP-helpFinder tool to facilitate the documentation of AOPs.

**Jukka Luukkonen (JL)**, Ph.D, is a university lecturer in the Radiation Research Group, University of Eastern Finland, with responsibilities on research and teaching related to the radiation biology. He has over 15 years of experience conducting studies with combined exposure to radiation and other agents.

ORCID ID

Olivier Armant 0000-0001-7101-9209

Karine Audouze 0000-0001-7525-4089

Omid Azimzadeh 0000-0001-8984-0388

M Abderrafi Benotmane 0000-0002-8985-1578

Jukka Luukkonen 0000-0002-0957-003X

Roel Quintens 0000-0002-4359-3403

Ignacia Tanaka 0000-0002-9526-0184

Knut Erik Tollefsen 0000-0002-7534-0937
# Development of an Adverse Outcome Pathway for radiation-induced microcephaly via expert consultation and machine learning

**Background:** Brain development during embryogenesis and in early postnatal life is particularly complex and involves the interplay of many cellular processes and molecular mechanisms, making it extremely vulnerable to exogenous insults, including ionizing radiation (IR). Microcephaly is one of the most frequent neurodevelopmental abnormalities that is characterized by small brain size, and is often associated with intellectual deficiency. Decades of research span from epidemiological data on *in utero* exposure of the A-bomb survivors, to studies on animal and cellular models that allowed deciphering the most prominent molecular mechanisms leading to microcephaly. The Adverse Outcome Pathway (AOP) framework is used to organize, evaluate and portray the scientific knowledge of toxicological effects spanning different biological levels of organizations, from the initial interaction with molecular targets to the occurrence of a disease or adversity. In the present study, the framework was used in an attempt to organize the current scientific knowledge on microcephaly progression in the context of ionizing radiation (IR) exposure. This work was performed by a group of experts formed during a recent workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. Here we report on the development of a putative AOP for congenital microcephaly resulting from IR exposure based on discussions of the working group and we emphasize the use of a novel machine-learning approach to assist in the screening of the available literature to develop AOPs.

**Conclusion:** The expert consultation led to the identification of crucial biological events for the progression of microcephaly upon exposure to IR, and highlighted current knowledge gaps. The machine learning approach was successfully used to screen the existing knowledge and helped to rapidly screen the body of evidence and in particular the epidemiological data. This systematic review approach also ensured that the analysis was sufficiently comprehensive to identify the most relevant data and facilitate rapid and consistent AOP development. We anticipate that as machine learning approaches become more user-friendly through easy-to-use web interface, this would allow AOP development to become more efficient and less time consuming.
149

150

Keywords: Adverse Outcome Pathway; AOP; microcephaly; brain; development;

ionizing radiation

151

## Abbreviation list

152

AOP(s) Adverse Outcome Pathway(s)

153

AO Adverse outcome

154

BH Bradford Hill criteria

155

DDR DNA damage response

156

DSB(s) DNA double-strand break(s)

157

KE(s) Key event(s)

158

KER(s) Key event relationship(s)

159

Gy Gray

160

HR Homologous recombination

161

IR Ionizing radiation

162

LET Linear energy transfer

163

MIE Molecular initiating event

164

NHEJ Nonhomologous end-joining

165

NPP Nuclear power plant

166

qAOP(s) Quantitative Adverse Outcome Pathway(s)

167

RGCs Radial glia cell(s)

168

Sv Sievert

169

TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

170

WoE Weight of evidence

171

WG Working group

172

ZIKV Zika virus

173

174

## Epidemiological studies on IR-induced microcephaly

175

176

177

178

Microcephaly is a medical condition characterized by a small head size less than the expected average for age, gender and considered population. Based on the literature, its definition may vary qualitatively (e.g. exclusion of other syndromes) or quantitatively (2 or 3 standard deviations below the mean, which may also reflect
severity, with dependence on the chosen reference population) (Passemard et al. 2013; Becerra-Solano et al. 2021). Microcephaly is classified either as primary when brain development is altered during embryogenesis, or acquired when the brain is normal at birth but suffer damages postnatally resulting in impaired growth (Passemard et al. 2013). Both cases result in reduced brain size/volume and is frequently associated with intellectual disability (Woods 2004). Several risk factors have been identified for microcephaly, including infections during pregnancy (e.g. syphilis, herpes simplex, HIV, Zika), maternal exposure to ionizing radiation or chemicals (e.g. mercury, arsenic, alcohol), pre- and perinatal injuries to the developing brain (trauma, hypoxia-ischemia) and genetic abnormalities. This wide range of risk factors can partly explain the variations in incidence rates observed in normal populations (from 1 to 150 cases per 100,000 birth) (Passemard et al. 2013; Becerra-Solano et al. 2021). In addition, the consideration of additional criteria (e.g. mental disability) as indicators of microcephaly, can create even more disparities in the number of cases accounted in the epidemiological cohorts from different populations. Recent studies dealing with microcephaly are related to Zika virus (ZIKV) infection during gestation, where the estimated prevalence rate is of 2.3% (95% CI = 1.0-5.3%) among all pregnancies (Coelho and Crovella 2017).

Associations between microcephaly and ionizing radiation (IR) have first been suspected from a series of early clinical observations of newborns with small head size and ophthalmic defects with or without mental disability, when pregnant women were exposed to pelvic irradiation (more than 10 mSv of X-rays) (Goldstein and Murphy 1929; Goldstein 1930). Data from Atomic-bomb survivors of Hiroshima and Nagasaki exposed *in utero* between 8-25 weeks of gestation showed statistically significant associations between IR exposure and small head sizes (defined here as head
circumference inferior to two standard deviations below the sex and age specific mean of the studied population of 1473 children) (Plummer 1952; Miller and Blot 1972; Otake and Schull 1993; Otake and Schull 1998). Some craniostenosis cases were potentially included which raised some controversy (Otake and Schull 1998). The most radiosensitive period of embryonic development of the human brain was established as between 8-15 weeks of gestation (Miller and Blot 1972; Otake and Schull 1998). More recently, three papers reported increased prevalence rates of microcephaly (defined as occipito-frontal circumferences of at least three standard deviations below the standard for age and sex proposed by the World Health Organization, https://www.who.int/tools/child-growth-standards) in children of women residing in areas impacted by the Chernobyl fall-out of the Rivne Province of Ukraine at the time of pregnancy. However, as these papers considered geographically aggregated data rather than individual data (Wertelecki et al. 2014; Wertelecki et al. 2017; Wertelecki et al. 2018), they currently provide a lower weight of evidence compared to the atomic bomb survivor studies in Hiroshima and Nagasaki.

## 219 Aims of the AOP working group on IR induced microcephaly

The Adverse Outcome Pathway (AOP) framework allows the description of events from the molecular initiation of the toxic effects to the adverse effects observed in the organism at levels relevant for human and biota populations. It describes the fundamental biological events from a Molecular Initiating Event (MIE) and propagation through essential Key Events (KEs) across the different biological scales of organization to result in an Adverse Outcome (AO). The Key Event Relationships (KERs) represent links between the KEs and are supported by experimental data evaluated as empirical evidence using modified (tailored) Bradford Hill (BH) criteria (Becker et al. 2015). Thus, the AOP describes in a simple way the scientific knowledge
of the most critic toxic effects at different biological levels of organization from
molecular mechanisms to the occurrence of the disease or adverse effects of regulatory
or research focus (Villeneuve et al. 2014; OECD 2018). This concept can also be used
to model the combined effects caused by single- or mixtures of different stressors to
identify and quantify the links between the biological events and the health outcomes
(Beyer et al. 2014; Salbu et al. 2019).

The Organization for Economic Cooperation and Development (OECD)
launched in 2012 a program on the development and assessment of AOPs to support
chemical risk assessment. Through this program, guidance and a dedicated web-based
platform (www.aopwiki.org) emerged to facilitate AOP development and uptake from
the scientific and regulatory communities. As the development of AOP are relatively
new to the field of low dose radiation research (Chauhan et al. 2022), a workshop
dedicated to AOP of IR effects was organized jointly by the Multidisciplinary European
Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE)
on April 12-16, 2021. The aim was to foster interactions between radiobiologists,
radioecotoxicologists and other communities, including chemical toxicologists, in order
to define the particularities of the AOP when applied to the radiation field and prioritize
the development of AOP relevant for health and environmental effects (Chauhan et al.
2021). Here we report on the discussion of a working group (WG) dedicated to the
development and proposing of an AOP for fetal neurotoxicity resulting from IR
exposure. The WG is composed of scientists knowledgeable in the field of brain
development, low dose radiation research (including epidemiology), chemical toxicity
research and computational science. Developmental toxicity encompasses many
pathologies, and it was rapidly recognized that brain development is particularly
vulnerable to IR. Microcephaly provided a favorable case study as : i) the outcome
(decrease in brain size) is clearly defined and measurable, ii) epidemiological data is
available to demonstrate an increased risk of microcephaly following *in utero* exposure
to IR, iii) mechanistic understanding of the effects is well documented, and iv)
microcephaly was not documented in AOP-Wiki (https://aopwiki.org).

Discussions centered first on the exposure situation to IR. It was acknowledged
that most of the evidence, primarily epidemiological data, were obtained for acute
exposure at a relative low dose of IR (> 0.1 Gy) during embryonic development and that
most effects were studied using low LET IR, including both gamma and X-rays.
Virtually all data were obtained from human and rodent studies (mouse and rat),
although the existence of two studies on wild animals (monkey and birds in Fukushima
and Chernobyl, respectively (Møller et al. 2011; Hayama et al. 2017)) was
acknowledged, but not considered sufficiently supportive for expanding the taxonomic
application domains of the AOP. During the review of the available human data, the
group considered accidental *in utero* exposures to the dose > 0.1 Gy as the preferential
scenario, since occupational and environmental exposures are unlikely (Applegate et al.
2021). This dose is in the range of the threshold value for which therapeutic abortion
should be considered in human (0.1 Gy) (Nakagawa et al. 1997). The frequency and
severity of microcephaly is dose-dependent in epidemiological data and animal
experiments, but the dose-threshold of occurrence of microcephaly is different in human
and mouse (above 0.3 Gy in the mouse (Verreet, Verslegers, et al. 2016) and above 0.1
Gy for the human (Otake and Schull 1998)).

Data at a lower dose (< 0.1 Gy) are scarce, and microcephaly was proposed to be
an endpoint without dose threshold. To perform a comprehensive review, the group
decided to consider all available data irrespective of the dose, in order to detect potential
early molecular or cellular key events of microcephaly. It was also discussed that dose
and dose rates would be important for the development of quantitative AOPs (qAOP),
the quantitative characterization of stressor-response and response-response
relationships along an AOP (Conolly et al. 2017; Song et al. 2020; Moe et al. 2021),
although such effort was not undertaken in the present work. In this context, dose
dependencies were considered as potential strategy to assess the weight of evidence
(WoE) between adjacent KE events according to the modified Bradford-Hill criteria
(Becker et al. 2015).

The group considered different possible MIEs for the microcephaly AOP.
‘Deposition of energy’ (found as MIE 1686 in AOP-Wiki) was proposed as a possible
consensus between the different WGs during the workshop (Chauhan et al. 2021). In the
context of microcephaly, such MIE can be linked directly to the first KE ‘increased
DNA damages’ for which compelling evidence of essentiality exist. A coherent parallel
can be made with an already existing MIE, ‘deposition of particles’, as ‘Deposition of
energy’ conveys the notion of interactions between photons (or high LET particles) and
living matter at the origin of the effects observed at the molecular level. However, this
MIE does not distinguish non-ionizing from ionizing radiation exposures, while the
body of evidence collected for microcephaly comprises only IR. Alternative proposed
MIE were ‘deposition of ionizing energy’ (MIE 1550) or ‘increase, DNA damages’
itself (KE 1194). However, for the sake of coherence and transposability with the
MELODI/Alliance WGs it was decided to keep ‘deposition of energy’ as the MIE for
microcephaly and highlighted that further discussion and harmonization of potential
MIEs could be relevant within the radiation community.

The integration of the effects observed at different levels of biological
organization, central for the AOP framework, was identified as closely linked to the
concept of systems biology. The existence of a high-throughput dataset describing the
effects of IR on gene expression leading to microcephaly in the mouse embryonic brain (Mfossa et al. 2020) was discussed as an interesting approach to identify KEs and characterize the KERs, but such analysis was beyond the reach of the group. Instead, the screening of additional databases useful for AOP development, such as the Comparative Toxicogenomics Database (CTD), GeneCards, AOP-Wiki and PubMed databases were highlighted as useful approaches to capture and assess evidence systematically. The integration of these data to the results of the scoping review, also performed by the WG, was thus proposed as an innovative way to incorporate the existing knowledge to the microcephaly AOP and ensure its comprehensive assembly.

## 313 Identification of key events through weight of evidence considerations

AOP development is based on the available knowledge from studies that support each KE and KER. The analysis of the evidence collected in the scientific literature is a tedious and challenging process made by experts that must understand, organize and evaluate the WoE along the AOP. This process is at the basis of the mapping of the knowledge made during the construction of AOPs, and is made through the effective assessment of biological plausibility, causality and consistency of the empirical evidence according to the modified Bradford-Hill criteria, as described by Becker *et al.* (Becker et al. 2015). The WG performed the scoping review of the literature in several steps: i) the constitution of a preliminary AOP (or AOP ‘skeleton’) based on experts’ knowledge, ii) consolidation of the AOP by evaluation of the scientific evidence, and iii) proposition of a hypothetical AOP based on consensus among experts. The second step is the heart of the compilation of evidence to develop AOPs. The WG decided to prioritize the evaluation of scientific articles focusing on microcephaly induced by IR, but, as these articles are limited in number, functional studies from genetic mutations leading to microcephaly were also included. In addition, genetic studies on
microcephaly were considered important to provide evidence of essentiality and causality between KEs. The group decided to focus pragmatically on the effects of IR on neurogenesis, as a first step towards a more comprehensive and integrated evaluation of the effects on other type of cells in the brain like oligodendrocyte, astrocyte or endothelial cells implicated in the vascularization of the brain and transport across the blood-brain barrier.

### Increased DNA damage leads to TP53 activation and apoptosis of progenitors in the developing brain

Controlling the number and differentiation of progenitor cells during brain development is critical to generate the appropriate number of neurons (and other type of cells) and to achieve normal expansion of the cerebral cortex (Noctor et al. 2004; Kriegstein and Alvarez-Buylla 2009; Xing et al. 2021)(Pinson and Huttner 2021). The best documented cause of primary microcephaly is the loss of radial glial cells (RGCs) and intermediate progenitors by apoptosis (Baala et al. 2007; Sessa et al. 2008). Such losses occur by the induction of genomic instability (Marthiens et al. 2013) and the accumulation of unrepairable DNA damages (through cell division) via mutation in critical genes involved in the DNA damage response (DDR) (Buck et al. 2006; O'Driscoll and Jeggo 2008; Zhou et al. 2013; Bianchi et al. 2017), or defective chromosomal segregation during mitosis that leads to prolonged cell arrest and TP53-mediated apoptosis (Chen et al. 2014; Insolera et al. 2014; Phan et al. 2021). In addition, other mutations can lead to excessive and inappropriate TP53 activation in the developing brain, ultimately leading to microcephaly (or microcephaly-associated syndromes) because of increased apoptosis of neural progenitors (Bowen and Attardi 2019).

As a well-known mutagen, IR induces microcephaly. Compelling evidence from human
epidemiological studies and laboratory animals exposed *in utero* demonstrated the link between radiation-induced DNA damage and apoptosis of progenitors in the mouse cerebral cortex as the origin of microcephaly (Nowak et al. 2006; Saha et al. 2014; Barazzuol and Jeggo 2016; Kashiwagi et al. 2018; Mfossa et al. 2020). Microcephaly is observed after exposure throughout the neurogenesis period, from E11 to E17 in the mice and between weeks 8 and 25 of gestation in humans (Verreet, Verslegers, et al. 2016), and is apparent in the domain of the low dose radiation (near 0.2 Gy for humans and 0.3 Gy for the mouse). In both species, the decreased brain size was strongly correlated to altered cognitive performance (Verreet, Rangarajan, et al. 2016; Verreet, Verslegers, et al. 2016). Importantly, the central role of TP53-induced apoptosis was demonstrated by transcriptome-wide expression studies of TP53 early transcriptional response to radiation exposure in mouse (Quintens et al. 2015; Mfossa et al. 2020) and zebrafish (Jaafar et al. 2013). Moreover, the partial rescue of the microcephaly phenotype when conditional *Trp53* knock out mice are exposed to IR, provides a direct causal link between radiation-induced apoptosis and microcephaly (Mfossa et al. 2020).

The evidence describing the effects of IR on DNA is already present in two different AOP (https://aopwiki.org, AOP#272, AOP#293) and it is out of the scope of this work to present the detailed mechanisms of DNA damages and repair (see for instance (Lu et al. 2019; Tang et al. 2022)). However, it remain important to consider that during neurogenesis, the repair of DNA double-strand breaks (DSBs) via nonhomologous end-joining (NHEJ) or homologous recombination (HR) depend on the developmental stage, as DSB in early progenitors (E13 in the mouse) are mainly repaired by HR, while progenitors present at later stage requires NHEJ (Orii et al. 2006; Li et al. 2020). The causality between the KE increased DSB – TP53 activation – apoptosis, is well described and the biological plausibility is considered strong. Increased DSB and
379 apoptosis are two KEs already present in AOP-Wiki (respectively KE 1194 and KE
380 1262 or KE 1365), but TP53 activation was not yet proposed as a KE (except KE 1923
381 ‘Altered gene expression, TP53 dependent apoptosis pathway). Many other functional
382 studies evidenced a role of DDR and TP53-dependent responses similar to those elicited
383 by IR, suggesting the activation of identical underlaying mechanisms (Pilaz et al. 2016;
384 Mao et al. 2016; Baumgartner et al. 2018; Shi et al. 2019; Keil et al. 2020).

385 Beside apoptosis, mitotic spindle defects (Shimada et al. 2016) and endoplasmic
386 reticulum stress (Passemard et al. 2019) were also implicated in microcephaly in the
387 context of IR exposure. Finally, data showed that TP53 have also a role in cell-fate
388 determination during neurogenesis, independent of its classical function in the induction
389 of apoptosis. Indeed, *in vivo* study using *Trp53* mutant mice demonstrated that Trp53
390 hyperactivation led to a precocious differentiation of RGC into neurons during
391 embryonic development (Liu et al. 2013; Mfossa et al. 2020). In this case, the pool of
392 RGC is depleted too early (Gruber et al. 2011), limiting the production of neurons
393 throughout neurogenesis and thereby contributing to a decreased brain size. Given all
394 these data, the WG considered TP53 activation as a genuine KE essential for the
395 appearance of microcephaly via two independent KE, namely apoptosis and premature
396 neuronal differentiation.

397 *Oxidative stress*

398 Deposition of ionizing energy may induce oxidative stress, an imbalance between
399 oxidants and antioxidants in favor of the former. In addition, IR can directly damage
400 DNA but also indirectly through the production of reactive oxygen species (Kim et al.
401 2019). Oxidative stress produces mainly single-strand DNA breaks, whereas more
402 detrimental effects of radiation are thought to be contributed by the radicals formed
along the radiation track where they induce clustered DNA-damage (Sharma et al. 2016). Although oxidative stress has been associated with a variety of diseases, such as cancer and cardiac diseases, there is currently only limited evidence of its involvement in radiation-induced microcephaly. This limited evidence includes the occurrence of oxidative stress and cell cycle arrest in hippocampal neuronal progenitor cells (Oh et al. 2013) and increased neuroinflammation in the brain of adult Wistar albino rats (Kale et al. 2018). Although it is challenging to directly link a measure of oxidative stress to DNA damage, some studies have demonstrated quite clearly a key role played by oxidative stress in radiosensitivity and IR-induced DDR, which remain to be investigated in the case of microcephaly (Hong et al. 2013; Yazlovitskaya et al. 2015) (Nikitaki et al. 2015). In addition to acute DNA damage by radiation-induced oxidants, several studies have found radiation to cause persistent oxidative stress (Datta et al. 2012; Cosar et al. 2012), which in turn might result in a prolonged increase in DNA damage. Taken together, we acknowledge that oxidative stress might be involved in parallel or as a complementary pathway that can be described by an AOP network, but we did not include it as a KE in the AOP due to the low weight of evidence of this process during microcephaly progression.

## Neuroinflammation

Neuroinflammation is considered as a key component of the stress response in the brain after exposure to various stressors in adults and during embryogenesis. It has therefore been considered as a possible KE during the construction of the preliminary AOP for microcephaly. Recent studies associating neuroinflammation and microcephaly are related to ZIKV infection during gestation. Indeed, many studies highlighted that infections by ZIKV during pregnancy is characterized by polyfunctional immune activation associated with increased levels of inflammatory cytokines in the amniotic
fluid of ZIKV-positive pregnant women, including the presence of interferon-gamma-
inducible protein 10, interleukin-6 (IL-6) and IL-8, vascular endothelial growth factor
(VEGF), monocyte chemoattractive protein 1 (MCP-1) and granulocyte colony-
stimulating factor (G-SF) (Ghouzzi et al. 2017). This inflammatory response is
associated with neuronal damages during neurogenesis, leading to microcephaly (Tappe
et al. 2016; Ornelas et al. 2017; Lima et al. 2019). In the context of IR, several studies
reported a microglial activation after exposure of embryonic, young or adult rodents to
IR, which elicited neuroinflammation associated with neuronal loss and cognitive
deficit (Quintens et al. 2015; Verreet, Verslegers, et al. 2016; Osman et al. 2020;
Constanzo et al. 2020). Gene expression changes in irradiated microglia of juvenile
mice highly overlap with those seen in the developing mouse brain and converge on the
TP53 pathway and inflammation (Osman et al. 2020; Mfossa et al. 2020). Importantly,
pharmacological inhibition of several cytokines can confer neuroprotection depending
on the stage of development and type of injury, highlighting the contribution of
neuroinflammation in the appearance of neurological disorders (Hagberg et al. 2015). In
addition there is a strong relationship between chronic inflammation and oxidative
damage after exposure to IR (Najafi et al. 2017; Yahyapour et al. 2018). Indeed, after
exposure to very high dose of IR (> 1Gy), necrosis is more common than apoptosis,
which leads to the release of damage-associated molecular patterns by necrotic cells
which stimulate more T-cells and dendritic cells, amplifying the production of reactive
oxygen species (ROS) and inflammatory mediators (Multhoff et al. 2011; Yahyapour et
al. 2018). In contrast, at lower doses of IR, macrophages detect apoptotic cells and can
release anti-inflammatory cytokines such as transforming growth factor beta (TGF-β)
that keep inflammation under control. Disruption of non-cell autonomous signaling like
TGF-β can have a role in the progression of microcephaly as they are also essential for
453 cortical migration during development (Zhang et al. 2016). The role of these factors in
454 the sense of bystander effects of radiation has so far not been demonstrated. The
455 interaction between inflammation and oxidative stress is thus complex. It is noteworthy
456 that infection of human neural progenitor cells by Zika virus (Ghouzzi et al. 2017),
457 irradiation of the mouse embryonic brain (Quintens et al. 2015) and mutation in the
458 DDR gene citron kinase (Di Cunto et al. 2000), all results in TP53-mediated apoptosis
459 of the developing neuronal progenitors (Quintens 2017). As radiation-induced apoptosis
460 triggers inflammation (Boyd et al. 2021), it is possible that neuroinflammation
461 contributes to microcephaly, but this process remains poorly documented and could also
462 be considered as secondary compared to apoptosis. The WG decided thus to exclude
463 neuroinflammation from the list of possible KE until a causative role has been
464 established.

465 **Radiation-induced microcephaly, test-case application of a machine learning**
466 **method**

467 Given the enormous amount of existing knowledge in the scientific literature and other
468 data sources, the identification of relevant biological information to build AOP is a
469 complex and time-consuming task, especially in deciphering sparse data. Thus, to assist
470 in the AOP development process, the WG discussed that the use of artificial intelligence
471 (AI) and automated data mining methods can represent a complementary approach to
472 the expert manual review of the available data. From this perspective, an innovative AI
473 tool, called AOP-helpFinder, that combines graph theory and text mining, was
474 developed to automatically explore the scientific abstracts from the PubMed database
475 (Carvaillo et al. 2019; Jornod et al. 2021). This tool was previously successfully applied
476 in several studies to identify and extract associations between stressors (bisphenol S,
477 bisphenol F and pesticides) and KEs of interest (Carvaillo et al. 2019; Rugard et al.
2020; Jornod et al. 2021). Epidemiological studies linking microcephaly and IR exposures being sparse in the literature, and then difficult to identify, we used the AOP-helpFinder tool to quickly identify and extract, in a systematic manner, all published existing information that could help AOP development. To do so, the user-friendly AOP-helpFinder webserver was applied using the key words 'ionizing radiation' and biological events 'Microcephaly' and 'Small head size' (http://aop-helpfinder.u-paris-sciences.fr/, accessed on October, 19), with the options "Reduced search: 20%" and "Refinement filter: yes", in order to extract as much information as possible. Doing so the co-occurrence of 'ionizing radiation' with 'Microcephaly' or 'Small head size' in the abstract of PubMed database was scanned. Other phenotypical terminology such as brain atrophy, gliosis, or reduced neurogenesis where considered less precise. After automatic computational exploration of 125,226 abstracts related to IR from PubMed database, the AOP-helpFinder tool allowed to gather 47 articles associating IR and microcephaly (or small head size). Among these 47 links, a manual curation of the abstracts allowed to keep only the 9 epidemiological articles dealing with exposure to IR in humans. All these 9 publications were fully evaluated and discussed in the WG. Two types of epidemiological studies were discriminated, either related to A-bomb survivors in Hiroshima and Nagasaki (Plummer 1952; Miller and Blot 1972; Otake and Schull 1998) or exposures after the Chernobyl NPP accident (Wertelecki et al. 2014; Wertelecki et al. 2017; Wertelecki et al. 2018). After removing potential redundancy within these studies (including metanalysis and confirmational studies), the WG proposed to incorporate this knowledge via non-adjacent KER between the MIE 'energy deposition' and microcephaly, as these studies demonstrated a clear link between the two events, but without intermediate KEs.
502 **Preliminary AOP based on expert consultation**

503 Based on the discussion in the WG, it was possible to propose a preliminary AOP
504 related to radiation-induced microcephaly (Figure1)..

505 The MIE (MIE1686, Deposition of Energy) leads to the increased DNA damage at the
506 molecular scale (KE 1194, Increase, DNA damage), which is considered in this AOP as
507 the main route by which IR exerts their effects at higher levels of biological
508 organization. The large amount of data describing the link between IR and DNA
509 damages already present in AOP-Wiki, and the additional evidences gathered in the
510 context of neurogenesis (Table1), led to WG to propose a ‘High’ WoE for this KE.
511 ‘Activation of TP53’ (KE 1974) is proposed as a novel essential KE. The existing KE
512 1923 ‘altered gene expression, P53 dependent apoptosis pathway’ was not used as it is
513 restricted to apoptosis. This KE is first supported by a time course analysis by
514 immunoblot of phosphorylated TP53 on (E14) primary mouse striatal stem irradiated
515 acutely to 1 Gy and 2 Gy of X-rays (Kashiwagi et al. 2018). The authors also quantified
516 TP53BP1 by immunofluorescence and describe that the peak of DNA damages (10 min
517 after irradiation) precedes, as expected, the peak of TP53 activation (3h to 5h after
518 irradiation). Similar data were obtained by Limoli et al. (Limoli et al. 2004), but with
519 higher dose (5 Gy). In the context of microcephaly, the link between increased DNA-
520 damages and TP53 activation was demonstrated by immunofluorescence using γH2Ax
521 and TP53BP1 on brain cryosections and immunoblotting against phosphorylated TP53
522 in Citk-/- E14.5 mouse embryos (Bianchi et al. 2017; Mfossa et al. 2020). Together, this
523 WoE for ‘Activation of TP53’ (KE 1974) was considered high. TP53 activation is
524 necessary for the induction of two possible cellular outcomes in the developing brain,
525 increased apoptosis (KE1365, ‘Increase apoptosis’) or ‘Premature cell differentiation’
526 (KE 1979), which could both contribute to ‘microcephaly’ (AO 1978). The WG
identified the KE1365, 'Increase apoptosis' (already present in AOP-Wiki), with high
level of confidence in the context of brain development, as described by analysis using
cleaved Caspase-3 (CC3) (Shimada et al. 2016; Mfossa et al. 2020), TUNEL assay
(Saha et al. 2014; Barazzuol and Jeggo 2016; Bianchi et al. 2017; Mfossa et al. 2020) on
mouse brain cryosections, western blotting (Kashiwagi et al. 2018) and microscopic
analysis of pyknotic nuclei (Nowak et al. 2006; Etienne et al. 2012). The KE 'Premature
cell differentiation' (KE 1979) was supported by immunostaining of the neuronal
markers on mouse brain cryosections (Liu et al. 2013; Mfossa et al. 2020). We noted the
existence of the KE 1560 'altered differentiation', but considered that this KE was not
adequate as altered differentiation can also lead to defective cell function and this term
does not convey the notion of temporality during development. In addition, both IR-
induced apoptosis and premature differentiation were ablated in conditional *Trp53*
knock out mouse, which adds to the WoE (Mfossa et al. 2020). But, together, as few
studies were identified, this WoE was considered moderate. Finally, the abundant
epidemiological data in the field of radiation is incorporated as a non-adjacent KER
between energy deposition and the adverse outcome (AOP 441, https://aopwiki.org).

The proposed AOP represents the foundation on which more dedicated work will be
needed. Indeed, despite the willingness of the WG to follow the modified Bradford-Hill
criteria to assess the WoE for the KER, this process was only initiated, and it was
evident that additional effort will be needed to compile the evidence in a systematic and
transparent manner. Additional potential KE were considered in the initial phase of the
AOP construction and might be considered for future development of the AOP towards
an AOP network. For transparency the complete list of the unused events is provided in
Supplementary Table 1.
551 **Conclusion**

552 In this work, we proposed an AOP for radiation-induced microcephaly and highlighted
553 the important molecular and cellular processes leading to this adverse outcome. The
554 WG considered accidental exposure to IR (> 0.1 Gy) of low LET energy (gamma and
555 X-rays) during pregnancy (between 8 and 25 weeks post-fertilization in human), as a
556 well-known risk factor for microcephaly. The effects of IR on RGs and intermediate
557 progenitors in the developing brain were considered as the main KEs leading to
558 microcephaly. The AOP proposed here is a simplistic representation of the known
559 mechanisms leading to microcephaly after exposure to IR. It relies on epidemiological
560 data and is completed by mechanistic evidences that support the relationship between
561 IR and microcephaly. As such, this AOP can facilitate the predictive extrapolation to
562 other animal species, given that the processes of neurogenesis are to a certain extent
563 similar and the KERs conserved, and can thus help understand the effects on brain size
564 observed on wild animals after the Fukushima and Chernobyl nuclear accidents (Møller
565 et al. 2011; Hayama et al. 2017).

566 Besides a classical knowledge gap in the field of low-dose (< 0.1 Gy) and low-dose rate
567 (< 5 mGy/h), the development of the AOP also emphasized the need for more studies on
568 cell fate after irradiation. Indeed, if apoptosis is well documented, the effects of IR on
569 premature neuronal differentiation, cell migration, cytokinesis defects and their link
570 with cellular senescence, oxidative stress and neuroinflammation remain scarce, as well
571 as their contribution to microcephaly. The WG also identified the effect on non-
572 neuronal tissues as a major gap, especially for the vascular system and microglia. More
573 functional analysis (i.e. mutants, transgenesis and other mechanistic data) are thus
574 required to delineate causes and consequences in the context of DNA damage induced
575 by IR. It is also noteworthy that most AOP related to DNA damage in the AOP-Wiki
database relate to cancer effects. If cancer is indeed a major outcome following
exposure to a relative low to moderate dose of IR, non-cancer effects (including
developmental effects but also cardiovascular or cognitive effects in adults) remain less
studied and should not be neglected. The final proposed AOP is relatively simple and
more dedicated work is required to assess WoE in a systematic and transparent manner
as well as identifying additional MIEs and KEs relevant for the same AO. This effort
would entail developing additional AOPs that collectively can form an AOP network
where the relevance of each AOP can be assessed in terms of developing quantitative
stressor-response and response-response relationships. In this regard, automated
analysis by machine learning approaches will help to screen the huge literature rapidly,
and permits consolidation of KEs, but limits still exists for this automated process as
different AO and KE terminology can affect the search. More generally, system biology
approaches including omics data will be useful to consolidate the molecular events of
the AOP, and will be of use by bringing a quantitative aspect to the AOP.

590

591 Acknowledgements:

592 This project was supported by funding from the Euratom research and training program 2019-
593 2020 under grant agreement No. 900009 (RadoNorm) ; the Research Council of Norway (RCN)
594 through its Centre for Excellence funding scheme (Project No. 223268 and project No. 268294
595 ‘MixRisk’) and NIVAS Computational Toxicology Program, NCTP (www.niva.no/nctp, RCN
596 project No. 160016).

597

598 Disclosure statement

599 All authors declare that they have no competing interests. The opinions expressed and
600 arguments employed herein are those of the authors and do not necessarily reflect the
601 official views of the OECD or of the governments of the OECD Member Countries.

602

603 References:
604 Applegate KE, Findlay Ú, Fraser L, Kinsella Y, Ainsbury L, Bouffler S. 2021.
605 Radiation exposures in pregnancy, health effects and risks to the embryo/foetus-
606 information to inform the medical management of the pregnant patient. J Radiol Prot.
607 41(4). https://doi.org/10.1088/1361-6498/ac1c95

608 Baala L, Briault S, Etchevers HC, Laumonnier F, Natiq A, Amiel J, Boddaert N, Picard
609 C, Sbiti A, Asermouh A, et al. 2007. Homozygous silencing of T-box transcription
610 factor EOMES leads to microcephaly with polymicrogyria and corpus callosum
611 agenesis. Nat Genet. 39(4):454-456. https://doi.org/10.1038/ng1993

612 Barazzuol L, Jeggo PA. 2016. In vivo sensitivity of the embryonic and adult neural stem
613 cell compartments to low-dose radiation. J Radiat Res. 57(Suppl 1):i2-i10.
614 https://doi.org/10.1093/jrr/rrw013

615 Baumgartner M, Olthof AM, Aquino GS, Hyatt KC, Lemoine C, Drake K, Sturrock N,
616 Nguyen N, al Seesi S, Kanadia RN. 2018. Minor spliceosome inactivation causes
617 microcephaly, owing to cell cycle defects and death of self-amplifying radial glial cells.
618 Development. 145(17):dev166322. https://doi.org/10.1242/dev.166322

619 Becerra-Solano LE, Mateos-Sánchez L, López-Muñoz E. 2021. Microcephaly, an
620 etiopathogenic vision. Pediatr Neonatol. 62(4):354-360.
621 https://doi.org/10.1016/j.pedneo.2021.05.008

622 Becker RA, Ankley GT, Edwards SW, Kennedy SW, Linkov I, Meek B, Sachana M,
623 Segner H, Van Der Burg B, Villeneuve DL, et al. 2015. Increasing Scientific
624 Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill
625 Considerations for Evaluating Weight of Evidence. Regul Toxicol Pharmacol.
626 72(3):514-537. https://doi.org/10.1016/j.yrtph.2015.04.004

627 Beyer J, Petersen K, Song Y, Ruus A, Grung M, Bakke T, Tollefsen KE. 2014.
628 Environmental risk assessment of combined effects in aquatic ecotoxicology: A
629 discussion paper. Marine Environmental Research. 96:81-91.
630 https://doi.org/10.1016/j.marenvres.2013.10.008

631 Bianchi FT, Tocco C, Pallavicini G, Liu Y, Vernì F, Merigliano C, Bonaccorsi S, El-
632 Assawy N, Priano L, Gai M, et al. 2017. Citron Kinase Deficiency Leads to
633 Chromosomal Instability and TP53-Sensitive Microcephaly. Cell Rep. 18(7):1674-
634 1686. https://doi.org/10.1016/j.celrep.2017.01.054

635 Bowen ME, Attardi LD. 2019. The role of p53 in developmental syndromes. J Mol Cell
636 Biol. 11(3):200-211. https://doi.org/10.1093/jmcb/mjy087

637 Boyd A, Byrne S, Middleton RJ, Banati RB, Liu G-J. 2021. Control of
638 Neuroinflammation through Radiation-Induced Microglial Changes. Cells. 10(9):2381.
639 https://doi.org/10.3390/cells10092381

640 Buck D, Malivert L, de Chasseval R, Barraud A, Fondanèche M-C, Sanal O, Plebani A,
641 Stéphan J-L, Hufnagel M, le Deist F, et al. 2006. Cernunnos, a novel nonhomologous
642 end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell.
643 124(2):287-299. https://doi.org/10.1016/j.cell.2005.12.030
644 Carvaillo J-C, Barouki R, Coumoul X, Audouze K. 2019. Linking Bisphenol S to
645 Adverse Outcome Pathways Using a Combined Text Mining and Systems Biology
646 Approach. Environ Health Perspect. 127(4):47005. https://doi.org/10.1289/EHP4200

647 Chauhan V, Beaton D, Hamada N, Wilkins R, Burtt J, Leblanc J, Cool D, Garnier-Laplace J, Laurier D, Le Y, et al. 2022. Adverse outcome pathway: a path toward better data consolidation and global co-ordination of radiation research. Int J Radiat Biol.:1-10. https://doi.org/10.1080/09553002.2021.2020363

651 Chauhan V, Hamada N, Monceau V, Ebrahimian T, Adam N, Wilkins RC, Sebastian S, 652 Patel ZS, Huff JL, Simonetto C, et al. 2021. Expert consultation is vital for adverse 653 outcome pathway development: a case example of cardiovascular effects of ionizing 654 radiation. Int J Radiat Biol. 97(11):1516-1525. 655 https://doi.org/10.1080/09553002.2021.1969466

656 Chen J-F, Zhang Y, Wilde J, Hansen KC, Lai F, Niswander L. 2014. Microcephaly 657 disease gene Wdr62 regulates mitotic progression of embryonic neural stem cells and 658 brain size. Nat Commun. 5:3885. https://doi.org/10.1038/ncomms4885

659 Coelho AVC, Crovella S. 2017. Microcephaly Prevalence in Infants Born to Zika Virus-Infected Women: A Systematic Review and Meta-Analysis. International Journal of Molecular Sciences. 18(8):1714. https://doi.org/10.3390/ijms18081714

662 Conolly RB, Ankley GT, Cheng W, Mayo ML, Miller DH, Perkins EJ, Villeneuve DL, 663 Watanabe KH. 2017. Quantitative Adverse Outcome Pathways and Their Application to 664 Predictive Toxicology. Environ Sci Technol. 51(8):4661-4672. 665 https://doi.org/10.1021/acs.est.6b06230

666 Constanzo J, Midavaine É, Fouquet J, Lepage M, Descoteaux M, Kirby K, Tremblay L, 667 Masson-Côté L, Geha S, Longpré J-M, et al. 2020. Brain irradiation leads to persistent 668 neuroinflammation and long-term neurocognitive dysfunction in a region-specific 669 manner. Prog Neuropsychopharmacol Biol Psychiatry. 102:109954. 670 https://doi.org/10.1016/j.pnpbp.2020.109954

671 Cosar R, Yurut-Caloglu V, Eskiocak S, Ozen A, Altaner S, Ibis K, Turan N, Denizli B, 672 Uzal C, Saynak M, et al. 2012. Radiation-induced chronic oxidative renal damage can 673 be reduced by amifostine. Med Oncol. 29(2):768-775. https://doi.org/10.1007/s12032- 674 011-9870-7

675 Datta K, Suman S, Kallakury BVS, Fornace AJ. 2012. Exposure to heavy ion radiation 676 induces persistent oxidative stress in mouse intestine. PLoS One. 7(8):e42224. 677 https://doi.org/10.1371/journal.pone.0042224

678 Di Cunto F, Imarisio S, Hirsch E, Broccoli V, Bulfone A, Migheli A, Atzori C, Turco E, 679 Triolo R, Dotto GP, et al. 2000. Defective neurogenesis in citron kinase knockout mice 680 by altered cytokinesis and massive apoptosis. Neuron. 28(1):115-127. 681 https://doi.org/10.1016/s0896-6273(00)00090-8

682 Etienne O, Roque T, Haton C, Boussin FD. 2012. Variation of radiation-sensitivity of 683 neural stem and progenitor cell populations within the developing mouse brain. Int J 684 Radiat Biol. 88(10):694-702. https://doi.org/10.3109/09553002.2012.710927
685 Ghouzzi VE, Bianchi FT, Molineris I, Mounce BC, Berto GE, Rak M, Lebon S, Aubry
686 L, Tocco C, Gai M, et al. 2017. ZIKA virus elicits P53 activation and genotoxic stress
687 in human neural progenitors similar to mutations involved in severe forms of genetic
688 microcephaly and p53. Cell Death Dis. 8(1):e2567.
689 https://doi.org/10.1038/cddis.2016.446

690 Goldstein L. 1930. RADIOGENIC MICROCEPHALY: A SURVEY OF NINETEEN
691 RECORDED CASES, WITH SPECIAL REFERENCE TO OPHTHALMIC DEFECTS.
692 Archives of Neurology & Psychiatry. 24(1):102-115.
693 https://doi.org/10.1001/archneurpsyc.1930.02220130105010

694 Goldstein L, Murphy DP. 1929. Microcephalic idiocy following radium therapy for
695 uterine cancer during pregnancy. American Journal of Obstetrics and Gynecology.
696 18(2):189-195. https://doi.org/10.1016/S0002-9378(29)91057-7

697 Gruber R, Zhou Z, Sukchev M, Joerss T, Frappart P-O, Wang Z-Q. 2011. MCPH1
698 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with
699 mitotic entry through the Chk1-Cdc25 pathway. Nat Cell Biol. 13(11):1325-1334.
700 https://doi.org/10.1038/ncb2342

701 Hagberg H, Mallard C, Ferriero DM, Vannucci SJ, Levison SW, Vexler ZS, Gressens P.
702 2015. The role of inflammation in perinatal brain injury. Nat Rev Neurol. 11(4):192-
703 208. https://doi.org/10.1038/nrneurol.2015.13

704 Hayama S-I, Tsuchiya M, Ochiai K, Nakiri S, Nakanishi S, Ishii N, Kato T, Tanaka A,
705 Konno F, Kawamoto Y, Omi T. 2017. Small head size and delayed body weight growth
706 in wild Japanese monkey fetuses after the Fukushima Daiichi nuclear disaster. Sci Rep.
707 7(1):3528. https://doi.org/10.1038/s41598-017-03866-8

708 Hong Z, Kase Y, Moritake T, Gerelchuluun A, Sun L, Suzuki K, Terunuma T, Yasuoka
709 K, Kumada H, Anzai K, et al. 2013. Lineal energy-based evaluation of oxidative DNA
damage induced by proton beams and X-rays. Int J Radiat Biol. 89(1):36-43.
710 https://doi.org/10.3109/09553002.2012.715791

712 Insolera R, Bazzi H, Shao W, Anderson KV, Shi S-H. 2014. Cortical neurogenesis in
713 the absence of centrioles. Nat Neurosci. 17(11):1528-1535.
714 https://doi.org/10.1038/nn.3831

715 Jaafar L, Podolsky RH, Dynan WS. 2013. Long-term effects of ionizing radiation on
gene expression in a zebrafish model. PLoS One. 8(7):e69445.
716 https://doi.org/10.1371/journal.pone.0069445

718 Jornod F, Jaylet T, Blaha L, Sarigiannis D, Tamisier L, Audouze K. 2021. AOP-
719 helpFinder webserver: a tool for comprehensive analysis of the literature to support
720 adverse outcome pathways development. Bioinformatics: btab750.
721 https://doi.org/10.1093/bioinformatics/btab750

722 Kale A, Piskin Ö, Bas Y, Aydin BG, Can M, Elmas Ö, Büyükuysal Ç. 2018.
723 Neuroprotective effects of Quercetin on radiation-induced brain injury in rats. J Radiat
Res. 59(4):404-410. https://doi.org/10.1093/jrr/rry032
725 Kashiwagi H, Shiraishi K, Sakaguchi K, Nakahama T, Kodama S. 2018. Repair kinetics of DNA double-strand breaks and incidence of apoptosis in mouse neural stem/progenitor cells and their differentiated neurons exposed to ionizing radiation. J Radiat Res. 59(3):261-271. https://doi.org/10.1093/jrr/rrx089

729 Keil JM, Doyle DZ, Qalieh A, Lam MM, Funk OH, Qalieh Y, Shi L, Mohan N, Sorel A, Kwan KY. 2020. Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80. Nat Commun. 11(1):3839. https://doi.org/10.1038/s41467-020-17551-4

733 Kim W, Lee S, Seo D, Kim D, Kim K, Kim E, Kang J, Seong KM, Youn H, Youn B. 2019. Cellular Stress Responses in Radiotherapy. Cells. 8(9):1105. https://doi.org/10.3390/cells8091105

736 Kriegstein A, Alvarez-Buylla A. 2009. The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci. 32:149-184. https://doi.org/10.1146/annurev.neuro.051508.135600

739 Li Jingzheng, Shang Y, Wang L, Zhao B, Sun C, Li Jiali, Liu S, Li C, Tang M, Meng F-L, Zheng P. 2020. Genome integrity and neurogenesis of postnatal hippocampal neural stem/progenitor cells require a unique regulator Filia. Science Advances. 6(44):eaba0682. https://doi.org/10.1126/sciadv.aba0682

743 Lima MC, de Mendonça LR, Rezende AM, Carrera RM, Aníbal-Silva CE, Demers M, D'Aiuto L, Wood J, Chowdari KV, Griffiths M, et al. 2019. The Transcriptional and Protein Profile From Human Infected Neuroprogenitor Cells Is Strongly Correlated to Zika Virus Microcephaly Cytokines Phenotype Evidencing a Persistent Inflammation in the CNS. Front Immunol. 10:1928. https://doi.org/10.3389/fimmu.2019.01928

748 Limoli CL, Giedzinski E, Rola R, Otsuka S, Palmer TD, Fike JR. 2004. Radiation response of neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress. Radiat Res. 161(1):17-27. https://doi.org/10.1667/rr3112

751 Liu H, Jia D, Li A, Chau J, He D, Ruan X, Liu F, Li J, He L, Li B. 2013. p53 Regulates Neural Stem Cell Proliferation and Differentiation via BMP-Smad1 Signaling and Id1. Stem Cells Dev. 22(6):913-927. https://doi.org/10.1089/scd.2012.0370

754 Lu H, Saha J, Beckmann PJ, Hendrickson EA, Davis AJ. 2019. DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response. Nucleic Acids Research. 47(18):9467-9479. https://doi.org/10.1093/nar/gkz694

757 Mao H, McMahon JJ, Tsai Y-H, Wang Z, Silver DL. 2016. Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly. PLoS Genet. 12(9):e1006282. https://doi.org/10.1371/journal.pgen.1006282

761 Marthiens V, Rujano MA, Pennetier C, Tessier S, Paul-Gilloteaux P, Basto R. 2013. Centrosome amplification causes microcephaly. Nat Cell Biol. 15(7):731-740. https://doi.org/10.1038/ncb2746

764 Mfossa A-CM, Verslegers M, Verreet T, Fida H bin, Mysara M, IJcken WFJV, Vos WHD, Moons L, Baatout S, Benotmane MA, et al. 2020. p53 drives premature neuronal
766 differentiation in response to radiation-induced DNA damage during early neurogenesis
767 [Internet]. [accessed 2022 Feb 22]:2020.06.26.171132.
768 https://doi.org/10.1101/2020.06.26.171132

769 Miller RW, Blot WJ. 1972. Small head size after in-utero exposure to atomic radiation.
770 Lancet. 2(7781):784-787. https://doi.org/10.1016/s0140-6736(72)92145-9

771 Moe SJ, Wolf R, Xie L, Landis WG, Kotamäki N, Tollefsen KE. 2021. Quantification
772 of an Adverse Outcome Pathway Network by Bayesian Regression and Bayesian
773 Network Modeling. Integr Environ Assess Manag. 17(1):147-164.
774 https://doi.org/10.1002/ieam.4348

775 Møller AP, Bonisoli-Alquati A, Rudolfsen G, Mousseau TA. 2011. Chernobyl birds
776 have smaller brains. PLoS One. 6(2):e16862.
777 https://doi.org/10.1371/journal.pone.0016862

778 Multhoff G, Molls M, Radons J. 2011. Chronic inflammation in cancer development.
779 Front Immunol. 2:98. https://doi.org/10.3389/fimmu.2011.00098

780 Najafi M, Shirazi A, Motevaseli E, Geraily G, Norouzi F, Heidari M, Rezapoor S. 2017.
781 The melatonin immunomodulatory actions in radiotherapy. Biophys Rev. 9(2):139-148.
782 https://doi.org/10.1007/s12551-017-0256-8

783 Nakagawa K, Aoki Y, Kusama T, Ban N, Nakagawa S, Sasaki Y. 1997. Radiotherapy
during pregnancy: effects on fetuses and neonates. Clin Ther. 19(4):770-777.
785 https://doi.org/10.1016/s0149-2918(97)80101-4

786 Nikitaki Z, Hellweg CE, Georgakilas AG, Ravanat J-L. 2015. Stress-induced DNA
damage biomarkers: applications and limitations. Front Chem. 3:35.
788 https://doi.org/10.3389/fchem.2015.00035

789 Noctor SC, Martínez-Cerdeño V, Ivic L, Kriegstein AR. 2004. Cortical neurons arise in
790 symmetric and asymmetric division zones and migrate through specific phases. Nat
791 Neurosci. 7(2):136-144. https://doi.org/10.1038/nn1172

792 Nowak E, Etienne O, Millet P, Lages CS, Mathieu C, Mouthon M-A, Boussin FD.
793 2006. Radiation-induced H2AX phosphorylation and neural precursor apoptosis in the
794 developing brain of mice. Radiat Res. 165(2):155-164. https://doi.org/10.1667/rr3496.1

795 O'Driscoll M, Jeggo PA. 2008. The role of the DNA damage response pathways in
796 brain development and microcephaly: insight from human disorders. DNA Repair
797 (Amst). 7(7):1039-1050. https://doi.org/10.1016/j.dnarep.2008.03.018

798 OECD. 2018. Handbook supplement to the Guidance Document for developing and
799 assessing Adverse Outcome Pathways [Internet]. https://www.oecd-
800 ilibrary.org/content/paper/5jlv1m9d1g32-en

801 Oh SB, Park HR, Jang YJ, Choi SY, Son TG, Lee J. 2013. Baicalein attenuates
802 impaired hippocampal neurogenesis and the neurocognitive deficits induced by γ-ray
803 radiation. Br J Pharmacol. 168(2):421-431. https://doi.org/10.1111/j.1476-
804 5381.2012.02142.x
805 Orii KE, Lee Y, Kondo N, McKinnon PJ. 2006. Selective utilization of nonhomologous
806 end-joining and homologous recombination DNA repair pathways during nervous
807 system development. Proc Natl Acad Sci U S A. 103(26):10017-10022.
808 https://doi.org/10.1073/pnas.0602436103

809 Ornelas AMM, Pezzuto P, Silveira PP, Melo FO, Ferreira TA, Oliveira-Szejnfeld PS,
810 Leal JI, Amorim MMR, Hamilton S, Rawlinson WD, et al. 2017. Immune activation in
811 amniotic fluid from Zika virus-associated microcephaly. Ann Neurol. 81(1):152-156.
812 https://doi.org/10.1002/ana.24839

813 Osman AM, Sun Y, Burns TC, He L, Kee N, Oliva-Vilarnau N, Alevyzaki A, Zhou K,
814 Louhivuori L, Uhlén P, et al. 2020. Radiation Triggers a Dynamic Sequence of
815 Transient Microglial Alterations in Juvenile Brain. Cell Rep. 31(9):107699.
816 https://doi.org/10.1016/j.celrep.2020.107699

817 Otake M, Schull WJ. 1993. Radiation-related small head sizes among prenatally
818 exposed A-bomb survivors. Int J Radiat Biol. 63(2):255-270.
819 https://doi.org/10.1080/09553009314550341

820 Otake M, Schull WJ. 1998. Radiation-related brain damage and growth retardation
821 among the prenatally exposed atomic bomb survivors. Int J Radiat Biol. 74(2):159-171.
822 https://doi.org/10.1080/095530098141555

823 Passemard S, Kaindl AM, Verloes A. 2013. Chapter 13 - Microcephaly. In: Dulac O,
824 Lassonde M, Sarnat HB, editors. Handbook of Clinical Neurology [Internet]. Vol. 111.
825 [place unknown]: Elsevier; [accessed 2022 Mar 3]; p. 129-141.
826 https://doi.org/10.1016/B978-0-444-52891-9.00013-0

827 Passemard S, Perez F, Gressens P, El Ghouzzi V. 2019. Endoplasmic reticulum and
828 Golgi stress in microcephaly. Cell Stress. 3(12):369-384.
829 https://doi.org/10.15698/cst2019.12.206

830 Phan TP, Maryniak AL, Boatwright CA, Lee J, Atkins A, Tijhuis A, Spierings DC,
831 Bazzi H, Foijer F, Jordan PW, et al. 2021. Centrosome defects cause microcephaly by
832 activating the 53BP1-USP28-TP53 mitotic surveillance pathway. EMBO J.
833 40(1):e106118. https://doi.org/10.15252/embj.2020106118

834 Pilaz L-J, McMahon JJ, Miller EE, Lennox AL, Suzuki A, Salmon E, Silver DL. 2016.
835 Prolonged mitosis of neural progenitors alters cell fate in the developing brain. Neuron.
836 89(1):83-99. https://doi.org/10.1016/j.neuron.2015.12.007

837 Pinson A, Huttner WB. 2021. Neocortex expansion in development and evolution-from
genes to progenitor cell biology. Curr Opin Cell Biol. 73:9-18.
838 https://doi.org/10.1016/j.ceb.2021.04.008
839

840 Plummer G. 1952. Anomalies occurring in children exposed in utero to the atomic
841 bomb in Hiroshima. Pediatrics. 10(6):687-693.

842 Quintens R. 2017. Convergence and divergence between the transcriptional responses to
843 Zika virus infection and prenatal irradiation. Cell Death Dis. 8(3):e2672.
844 https://doi.org/10.1038/cddis.2017.109
845 Quintens R, Verreet T, Janssen A, Neefs M, Leysen L, Michaux A, Verslegers M,
846 Samari N, Pani G, Verheyde J, et al. 2015. Identification of novel radiation-induced
847 p53-dependent transcripts extensively regulated during mouse brain development. Biol
848 Open. 4(3):331-344. https://doi.org/10.1242/bio.20149969

849 Rugard M, Coumoul X, Carvaillo J-C, Barouki R, Audouze K. 2020. Deciphering
850 Adverse Outcome Pathway Network Linked to Bisphenol F Using Text Mining and
851 Systems Toxicology Approaches. Toxicol Sci. 173(1):32-40.
852 https://doi.org/10.1093/toxsci/kfz214

853 Saha S, Woodbine L, Haines J, Coster M, Ricket N, Barazzuol L, Ainsbury E,
854 Sienkiewicz Z, Jeggo P. 2014. Increased apoptosis and DNA double-strand breaks in
855 the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic
856 fields. J R Soc Interface. 11(100):20140783. https://doi.org/10.1098/rsif.2014.0783

857 Salbu B, Teien HC, Lind OC, Tollefsen KE. 2019. Why is the multiple stressor concept
858 of relevance to radioecology? Int J Radiat Biol. 95(7):1015-1024.
859 https://doi.org/10.1080/09553002.2019.1605463

860 Sessa A, Mao C, Hadjantonakis A-K, Klein WH, Broccoli V. 2008. Tbr2 Directs
861 Conversion of Radial Glia into Basal Precursors and Guides Neuronal Amplification by
862 Indirect Neurogenesis in the Developing Neocortex. Neuron. 60(1):56-69.
863 https://doi.org/10.1016/j.neuron.2008.09.028

864 Sharma V, Collins LB, Chen T, Herr N, Takeda S, Sun W, Swenberg JA, Nakamura J.
865 2016. Oxidative stress at low levels can induce clustered DNA lesions leading to NHEJ
866 mediated mutations. Oncotarget. 7(18):25377-25390.
867 https://doi.org/10.18632/oncotarget.8298

868 Shi L, Qalieh A, Lam MM, Keil JM, Kwan KY. 2019. Robust elimination of genome-
869 damaged cells safeguards against brain somatic aneuploidy following Knl1 deletion.
870 Nat Commun. 10(1):2588. https://doi.org/10.1038/s41467-019-10411-w

871 Shimada M, Matsuzaki F, Kato A, Kobayashi J, Matsumoto T, Komatsu K. 2016.
872 Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of
873 Radiation-Induced Microcephaly. PLoS One. 11(7):e0158236.
874 https://doi.org/10.1371/journal.pone.0158236

875 Song Y, Xie L, Lee Y, Tollefsen KE. 2020. De Novo Development of a Quantitative
876 Adverse Outcome Pathway (qAOP) Network for Ultraviolet B (UVB) Radiation Using
877 Targeted Laboratory Tests and Automated Data Mining. Environ Sci Technol.
878 54(20):13147-13156. https://doi.org/10.1021/acs.est.0c03794

879 Tang J, Li Z, Wu Q, Irfan M, Li W, Liu X. 2022. Role of Paralogue of XRCC4 and
880 XLF in DNA Damage Repair and Cancer Development. Front Immunol. 13:852453.
881 https://doi.org/10.3389/fimmu.2022.852453

882 Tappe D, Pérez-Girón JV, Zammarchi L, Rissland J, Ferreira DF, Jaenisch T, Gómez-
883 Medina S, Günther S, Bartoloni A, Muñoz-Fontela C, Schmidt-Chanasit J. 2016.
884 Cytokine kinetics of Zika virus-infected patients from acute to reconvalescent phase.
885 Med Microbiol Immunol. 205(3):269-273. https://doi.org/10.1007/s00430-015-0445-7
886 Verreet T, Rangarajan JR, Quintens R, Verslegers M, Lo AC, Govaerts K, Neefs M,
887 Leysen L, Baatout S, Maes F, et al. 2016. Persistent Impact of In utero Irradiation on
888 Mouse Brain Structure and Function Characterized by MR Imaging and Behavioral
889 Analysis. Front Behav Neurosci. 10:83. https://doi.org/10.3389/fnbeh.2016.00083

890 Verreet T, Verslegers M, Quintens R, Baatout S, Benotmane MA. 2016. Current
891 Evidence for Developmental, Structural, and Functional Brain Defects following
892 Prenatal Radiation Exposure. Neural Plast. 2016:1243527.
893 https://doi.org/10.1155/2016/1243527

894 Villeneuve DL, Crump D, Garcia-Reyero N, Hecker M, Hutchinson TH, LaLone CA,
895 Landesmann B, Lettieri T, Munn S, Nepelska M, et al. 2014. Adverse outcome pathway
896 (AOP) development I: strategies and principles. Toxicol Sci. 142(2):312-320.
897 https://doi.org/10.1093/toxsci/kfu199

898 Wertelecki W, Chambers CD, Yektushok L, Zymak-Zakutnya N, Sosyniuk Z,
899 Lapchenko S, Iektushok B, Akhmedzhanova D, Komov O. 2017. Chornobyl 30 years
900 later: Radiation, pregnancies, and developmental anomalies in Rivne, Ukraine. Eur J
901 Med Genet. 60(1):2-11. https://doi.org/10.1016/j.ejmg.2016.09.019

902 Wertelecki W, Yektushok L, Kuznietsos I, Komov O, Lapchenko S, Akhmedzanova D,
903 Ostapchuk L. 2018. Chornobyl, radiation, neural tube defects, and microcephaly. Eur J
904 Med Genet. 61(9):556-563. https://doi.org/10.1016/j.ejmg.2018.06.005

905 Wertelecki W, Yektushok L, Zymak-Zakutnya N, Wang B, Sosyniuk Z, Lapchenko S,
906 Hobart HH. 2014. Blastopathies and microcephaly in a Chornobyl impacted region of
907 Ukraine. Congenit Anom (Kyoto). 54(3):125-149. https://doi.org/10.1111/cga.12051

908 Woods CG. 2004. Human microcephaly. Current Opinion in Neurobiology. 14(1):112-
909 117. https://doi.org/10.1016/j.conb.2004.01.003

910 Xing L, Wilsch-Bräuninger M, Huttner WB. 2021. How neural stem cells contribute to
911 neocortex development. Biochem Soc Trans. 49(5):1997-2006.
912 https://doi.org/10.1042/BST20200923

913 Yahyapour R, Amini P, Rezapour S, Cheki M, Rezaeyan A, Farhood B, Shabeeb D,
914 Musa AE, Fallah H, Najafi M. 2018. Radiation-induced inflammation and autoimmune
diseases. Military Medical Research. 5(1):9. https://doi.org/10.1186/s40779-018-0156-7

916 Yazlovitskaya EM, Voziyan PA, Manavalan T, Yarbrough WG, Ivanova AV. 2015.
917 Cellular oxidative stress response mediates radiosensitivity in Fus1-deficient mice. Cell
918 Death Dis. 6:e1652. https://doi.org/10.1038/cddis.2014.593

919 Zhang F, Yu J, Yang T, Xu D, Chi Z, Xia Y, Xu Z. 2016. A Novel c-Jun N-terminal
920 Kinase (JNK) Signaling Complex Involved in Neuronal Migration during Brain
921 Development. J Biol Chem. 291(22):11466-11475.
922 https://doi.org/10.1074/jbc.M116.716811

923 Zhou Z-W, Tapias A, Bruhn C, Gruber R, Sukchev M, Wang Z-Q. 2013. DNA damage
924 response in microcephaly development of MCPH1 mouse model. DNA Repair (Amst).
925 12(8):645-655. https://doi.org/10.1016/j.dnarep.2013.04.017
926

927
Figure 1. Description of the preliminary AOP: Ionizing radiation-induced DNA damage leads to microcephaly via apoptosis and premature cell differentiation (AOP 441, https://aopwiki.org/aops/441). MIE (Molecular Initiating Event), Key Events (KE), Adverse Outcome (AO). Key event relationship with high and moderate confidence are depicted respectively by solid and dashed lines.

<figure><img src="image_3.png" /></figure>
Table 1. Description of the Weight of Evidence (WoE) used for the preliminary AOP. MIE (Molecular Initiating Event), Key Events (KE), Adverse Outcome (AO). The event-identities (Event ID) in AOP-Wiki are indicated, as well as the bibliographical references used.

<table><thead><tr><td>Type</td><td>Event ID</td><td>Title</td><td>Detection method</td><td>References</td><td>WoE</td></tr></thead><tbody><tr><td>MIE</td><td>1686</td><td>Direct Deposition of Energy</td><td>Dosimetry</td><td>Chauhan V et al. 2021</td><td>High</td></tr><tr><td>KE</td><td>1194</td><td>Increase, DNA damage</td><td>Immunostaining Western Blot</td><td>Barazzuol and Jego 2016<br>Saha et al. 2014<br>Kashiwagi et al. 2018<br>Nowak et al. 2006<br>Mfossa et al. 2020<br>Bianchi et al. 2017</td><td>High</td></tr><tr><td>KE</td><td>1974</td><td>Activation of TP53</td><td>Immunostaining Western Blot</td><td>Kashiwagi et al. 2018<br>Limoli et al. 2004<br>Bianchi et al. 2017<br>Mfossa et al. 2020</td><td>High</td></tr><tr><td>KE</td><td>1365</td><td>Increase, Apoptosis</td><td>Immunostaining Western Blot<br>TUNNEL assay<br>Pycnotic nucleus</td><td>Shimada et al. 2016<br>Saha et al. 2014<br>Barazzuol and Jego 2016<br>Kashiwagi et al. 2018<br>Nowak et al. 2006<br>Mfossa et al. 2020<br>Bianchi et al. 2017<br>Etienne et al. 2012</td><td>High</td></tr><tr><td>KE</td><td>1979</td><td>Premature cell differentiation</td><td>Immunostaining</td><td>Mfossa et al. 2020<br>Liu et al 2013</td><td>Moderate</td></tr><tr><td>AO</td><td>1978</td><td>Microcephaly</td><td>Head size</td><td>Plummer 1952<br>Miller and Blot 1972<br>Otake and Schull 1998<br>Otake and Schull 1993<br>Shimada et al. 2016<br>Bianchi et al. 2017<br>Mfossa et al. 2020</td><td>High</td></tr></tbody></table>
944 Supplementary Table 1. Description of the potential KE explored in the initial phase of
945 AOP construction. The comment indicates how the potential KE was considered in the
946 preliminary AOP after expert consultation.

947

<table><thead><tr><td>Term</td><td>KE in AOP wiki</td><td>Comment</td></tr></thead><tbody><tr><td>Increased, oxydative stress</td><td>1088</td><td>Insufficient direct evidence</td></tr><tr><td>Oxidative Stress</td><td>1392</td><td>Insufficient direct evidence</td></tr><tr><td>Increase in reactive oxygen and nitrogen species (RONS)</td><td>1632</td><td>Insufficient direct evidence</td></tr><tr><td>Neuroinflamation</td><td>188</td><td>Insufficient direct evidence</td></tr><tr><td>Increase in inflammation</td><td>1633</td><td>Insufficient direct evidence</td></tr><tr><td>Increased DNA double breaks</td><td>1635</td><td>Related to DNA damage</td></tr><tr><td>Cell cycle arrest</td><td>None</td><td>Insufficient direct evidence</td></tr><tr><td>Cell cycle, disrupted</td><td>1505</td><td>Insufficient direct evidence</td></tr><tr><td>Defective cell division</td><td>None</td><td>Insufficient direct evidence</td></tr><tr><td>Defective spindle assembly</td><td>None</td><td>Insufficient direct evidence</td></tr><tr><td>Senescence</td><td>None</td><td>Insufficient direct evidence</td></tr><tr><td>Lipid peroxidation</td><td>1511</td><td>Insufficient direct evidence</td></tr><tr><td>Increase, Lipid peroxidation</td><td>1445</td><td>Insufficient direct evidence</td></tr><tr><td>Protein carbonylation</td><td>None</td><td>Insufficient direct evidence</td></tr><tr><td>Peptide Oxidation</td><td>209</td><td>Insufficient direct evidence</td></tr><tr><td>Mitochondria dysfunction</td><td>1816</td><td>Insufficient direct evidence</td></tr><tr><td>Altered gene expression, P53 dependent apoptosis pathway</td><td>1923</td><td>Difficulty to assess essentiality</td></tr><tr><td>Change in gene expression levels (omics data)</td><td>None</td><td>Difficulty to assess essentiality</td></tr><tr><td>Increased gliosis</td><td>None</td><td>Insufficient direct evidence</td></tr><tr><td>Brain atrophy</td><td>None</td><td>Related to microcephaly</td></tr><tr><td>Reduced brain size</td><td>None</td><td>Related to microcephaly</td></tr><tr><td>Reduced neurogenesis</td><td>None</td><td>Related to premature neuronal differentiation</td></tr><tr><td>General Apoptosis</td><td>1513</td><td>Related to Increase apoptosis</td></tr><tr><td>Neuronal death by apoptosis</td><td>None</td><td>Related to Increase apoptosis</td></tr><tr><td>Premature Neuronal differentiation</td><td>None</td><td>Related to Premature cell differentiation</td></tr><tr><td>Altered vascularization</td><td>None</td><td>Insufficient direct evidence</td></tr><tr><td>Impaired blood-brain transport</td><td>None</td><td>Insufficient direct evidence</td></tr></tbody></table>

948

949

950