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Investigating Cell Type Specific Mechanisms Contributing to Acute Oral Toxicity.

1 Introduction

Acute systemic toxicity after oral, dermal, or inhalation exposure requires that the substance becomes bioavailable at the target site and induces lethality through general toxicity or a specific mechanism. This means that kinetic factors, and mainly absorption, are important determinants of toxicity (EURL ECVAM, 2015). In addition, if the damage involves interference with homeostatic mechanisms at the organ system level, non-exposed tissues and vital organs can also be affected (Gennari et al., 2004; Andrew, 2013).

The assessment of acute systemic toxicity is a core component of the safety assessment of substances in the context of EU and international legislations (Hamm et al., 2017). Information requirements vary depending on the type of substance subject to regulation and the region (EU, 2006, 2008, 2009a,b, 2012). The regulatory landscape in the USA was reviewed during two workshops cosponsored by the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), the People for the Ethical Treatment of Animals (PETA) International Science Consortium Ltd, and the Physicians Committee for Responsible Medicine (PCRM) (Hamm et al., 2017; Clippinger et al., 2018a; Strickland et al., 2018). The relevant information is available at the PETA website (1). In preclinical drug development, however, these studies are no longer required by default to support first clinical trials in humans (Robinson et al., 2008; ICH, 2009; Chapman et al., 2010).

One of the main uses of acute systemic toxicity data is classification and labelling (Seidle et al., 2010; Graepel et al., 2016; Buesen et al., 2016; Strickland et al., 2018). Within the EU, the CLP (Classification, Labelling and Packaging) Regulation (EU, 2008) is used to classify chemicals on the basis of acute oral toxicity into four toxicity categories (categories 1 to 4 of the United Nations Globally Harmonised System of Classification and Labelling (UN GHS)). While CLP does not require animal testing, the classification criteria are based on data derived from animal tests (conducted, for example, under other pieces of legislation), including reduction and refinement methods for the oral, dermal, and inhalation routes (OECD TGs 402, 403, 420, 423, 425, 433, 436). Most of the standard in vivo tests use lethality as the endpoint, even though this has been widely criticized both on animal welfare and scientific grounds (Zbinden and Flury-Roversi, 1981; Hoffmann et al., 2010; Prieto et al., 2013a).

Basal cytotoxicity is certainly a key factor in many prevalent toxicological modes-of-action associated with acute health effects. It covers many general mechanisms of toxicity common to most cell types that can lead to organ failure including, for example, disruption of cell membrane structure or function, inhibition of mitochondrial function, disturbance of protein turnover, and disruption of metabolism and energy production (Gennari et al., 2004; NIH, 2009; Andrew, 2013). This is the reason why the utility of in vitro cytotoxicity assays to predict acute oral toxicity has been extensively investigated (Ekwall, 1999; Halle, 2003; NIH, 2006; Prieto et al., 2013a,b). Recently, Vinken and Blaauboer (2017) proposed the application of an adverse outcome pathway (AOP) framework for basal cytotoxicity consisting of three consecutive steps, i.e., initial cell injury, mitochondrial dysfunction, and cell death. The outcome of the basal cytotoxicity was then suggested by the authors as the first step of a tiered strategy aimed to evaluate the toxicity of new chemical entities. Further, in a second step, more specific types of toxicity could be evaluated.

One of the better known and standardized in vitro methods for basal cytotoxicity is the 3T3 Neutral Red Uptake (NRU) assay (DB-ALM protocol 1392; Stokes et al., 2008). The use of data from the NRU cytotoxicity assay within a Weight-of-Evidence (WoE) assessment is one of the choices for adapting the standard information requirements for acute oral toxicity, as described in the last update of the ECHA's guidance on Information Requirements and Chemical Safety Assessment. This WoE adaptation proposed by ECHA applies primarily to low toxicity substances (i.e., those that are not to be classified for acute toxicity) and it is based on an in-depth analysis of the REACH database (Gissi et al., 2017; ECHA, 2017). Nevertheless, the limitations of the in vitro cytotoxicity assay, such as the lack of metabolic competence of 3T3 cells and difficulty to capture specific mechanisms of action relating to interaction with specific molecular targets in certain tissues, need to be considered when building a WoE case for the purposes of REACH (Buesen et al., 2018; Gissi et al., 2018).

In addition to the assessment of basal cytotoxicity, it is also important to identify cell types and in vitro endpoints that are indicative of cell-type specific toxicities, with a view to incorporating such endpoints into integrated approaches to testing and assessment (IATA), as proposed in the EURL ECVAM strategy to replace, reduce, and refine the use of animals in the assessment of acute mammalian systemic toxicity (EURL ECVAM, 2014). As defined by the OECD, IATA are pragmatic, science-based approaches for chemical hazard or risk characterization that rely on an integrated analysis of existing information in a WoE assessment coupled with the generation of new information, if required (OECD, 2016a). An iterative approach that preferably relies on mechanistic information or available AOPs is followed to answer a defined question in a specific regulatory context, taking into account the acceptable level of uncertainty associated with the decision making (OECD, 2016a; Sachana and Leinala, 2017). The importance of understanding the mechanisms of acute toxicity was further recognized during an international workshop in which a group of experts discussed alternative approaches for identifying acute systemic toxicity (Hamm et al., 2017; Clippinger et al., 2018a). A better theoretical and mechanistic understanding of acute systemic toxicity would be useful to developers of test methods and other predictive tools as well as to validation and regulatory bodies.

Mechanisms involved in cellular failure and susceptible functions compromised in organ failure were discussed at an ECVAM workshop on strategies to replace in vivo acute systemic toxicity testing (Gennari et al., 2004). Several fundamental cellular processes common to many organ systems were identified, including energy production and metabolism (mitochondrial function and glycolysis), transportation of molecules, membrane integrity and secretion of molecules (enzymes, proteins, hormones, neuretransmitters). A number of key events associated with acute human poisoning were further identified in an ICCVAM/ECVAM/ JaCVAM workshop on acute chemical safety testing (NIH, 2009) and it was agreed that mechanistic information could be used to develop more predictive in vitro test methods. A report, commissioned by the US Department of Defense, lists several of the cellular targets or molecular targets that are often associated with the acute lethal or debilitating effects of chemicals. This includes changes in neurotransmission function, altered ion flow, increased permeability of cellular membranes, altered bioenergetics, altered oxygen transport, oxidative stress and reactive oxygen species (ROS) formation, damage to DNA and subcellular systems, and immune-mediated effects (NRC, 2015). Hamm et al. (2017) and Clippinger et al. (2018b) have also reported some of the known mechanisms involved in acute systemic toxicity as part of ongoing activities in the US.

However, despite all the efforts made over the past 20 years in the area of acute systemic toxicity, relevant AOPs, mechanistically informed alternative methods, and IATA for acute systemic toxicity have not been adequately developed. This is partially due to the lack of a complete mechanistic understanding of the key acute toxicity pathways in humans specific for different cell types (e.g., neuronal, cardiac, liver, or kidney).

This study describes the analysis of mechanistic information collected on eight potential organs (i.e., nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system (GI), and immune system) identified as relevant for acute systemic toxicity and using a set of chemicals inducing acute toxicity after oral exposure. This work will support the development of AOPs and IATA in the area of acute systemic toxicity, and will inform the development and application of mechanistically relevant new approach methodologies.

2 Materials and methods

Collection of mechanistic information

Information was collected on the eight potential target organs identified as relevant for acute systemic toxicity during the ECVAM workshop on acute systemic toxicity (Gennari et al., 2004): liver, blood, kidney, cardiovascular system, central and peripheral nervous system (CNS/PNS), lung, immune system, and GI. In safety pharmacology studies, the cardiovascular, respiratory, and central nervous systems are assessed in a core battery since they are considered vital organs or systems, the functions of which are acutely critical for life (ICH, 2000).

In order to approach the ambitious task of mapping mechanisms specific for these potential target organs, a three-step approach was taken to identify the potential pathways of target organ toxicity.

1. Based on a literature review using *target organ* and *acute toxicity* and *mechanism* as key words, commonly recognized pathways of toxicity were identified for each target organ/system. Information was derived from published literature, toxicology handbooks, short descriptions of reference compounds used in the EU FP6 project ACuteTox (3), and internet databases (HSDB (4), INCHEM (5), PubChem (6), PubMed (7), Scopus (8), Google Scholar (9)). The pathways were then organized and visualized according to the target organ/system, the cell type, the effect, and the mechanism. In this context, the effect refers to any adverse reaction that could be observed or measured (in vivo) and the mechanism refers to the molecular or cellular process that is interrupted by chemical stressors and leads to the observed adverse effect.

2. In the second phase, the "completeness" of the theoretical pathways of toxicity that were developed in phase 1 was probed. In order to do so, we consulted the in-house database and selected chemicals that were shown to be acutely toxic. For these chemicals, a thorough literature search was conducted to identify the target organ and mechanism of toxicity, searching first for *chemical* AND *acute toxicity* and *mechanism*, followed by *chemical* AND *target organ*. These mechanisms were then added into the generated "maps" if they were not already present. Chemicals for which both in vivo acute oral toxicity data and in vitro cytotoxicity data were available were selected. On the basis of reference in vivo oral [LD.sub.50] data and of the 2000 mg/kg body weight threshold introduced by the CLP Regulation, all compounds with an acute [LD.sub.50] mean value below or equal to 2000 mg/kg were identified as acutely toxic, whereas those with an acute oral [LD.sub.50] mean value above 2000 mg/kg were identified as non-acutely toxic. Only the chemicals that fell into our group of toxic chemicals were considered in this second phase.

3. In a third phase, chemicals from the group of non-toxic chemicals that, nevertheless, had been assigned a harmonized classification (Annex VI of EU CLP Regulation) were identified and selected.

The mechanisms collected and shown in this report are not intended to be exhaustive.

Selection of chemicals with in vivo [LD.sub.50] values and in vitro cytotoxicity data

In the in-house database 178 test chemicals had oral [LD.sub.50] values (10) that were collected from publicly available databases (e.g., ChemIDplus, IUCLID, RTECS, and HSDB), Merck index, EU Risk Assessment Reports, Sax's Dangerous Properties of Industrial Materials, and the published literature. According to the calculated mean [LD.sub.50] values, 112 test chemicals were assigned to an EU CLP acute oral toxicity category and 66 remain as non-toxic (i.e., no category assigned because the [LD.sub.50] was higher than 2000 mg/kg). Eleven out of the 66 non-classified chemicals had an official acute oral classification.

In vitro cytotoxicity data were available for 177 test chemicals that had been screened in the following international projects: NICEATM/ECVAM validation study (NIH, 2006), the EU FP6 project ACuteTox (Prieto et al., 2013a), and the ECVAM validation study (Prieto et al., 2013b). The list of chemicals used in each study is available through the JRC Chemical Lists of Information System (CheList (11)).

When the two sets were compared, in vitro cytotoxicity data were not available for two compounds, formaldehyde and carbon tetrachloride, and in vivo oral [LD.sub.50] data were not found for benz(a)anthracene. Therefore, the final common set contained 176 chemicals.

3 Results

3.1 Overall analysis of mechanistic maps

The mechanistic information collected following the three-step strategy was visualized in maps according to the eight organs/ systems. The layout and structure across organs and systems was harmonized and analyzed as shown below. Three maps were created per target organ/system. A first map illustrated the mechanisms found based on information collected from literature (step 1 under methods). The second map was an updated version based on the information collected from the in-house list of selected compounds (steps 2 and 3 under methods). The final harmonized version of each organ/system was shown by the third map (Fig. 1-8).

Information on mechanisms of toxicity was collected for 114 out of the 123 oral acutely toxic chemicals (see Methods). In terms of target organ/systems, the overall analysis summarized in Figure 9 shows that, according to the information found, the nervous and cardiovascular systems are the most frequent targets (67 and 39 chemicals, respectively) followed by liver, kidney, lung, gastrointestinal system, blood and immune system (31, 30, 24, 18, 11, and 3 chemicals, respectively). Twenty-six chemicals appear to target single organs, in particular the nervous systems (12 chemicals). Seventy-five chemicals affect more than one organ/system and thirteen chemicals affect all organs (non-specific target organ effects) (Fig. 10). Indirect effects were reported for 9 chemicals with multi-organ/system effects: 6 on the lung, 1 on the kidney, and 4 on the cardiovascular system (Fig. 9).

General cytotoxicity mechanisms were cited for 72 chemicals and target organ/system specific effects for 40 chemicals (11 chemicals acting on a single organ/system and 29 on multiple targets) (see Tab. 1). For pentachlorobenzene and tetramethylthiuram monosulfide, the specific mechanism of acute toxicity was not found.

Tables 2-9 provide an overview of the specific target organ/ system mechanisms leading to acute toxicity according to the information collected from the literature. The list of mechanisms shown is neither exhaustive nor definitive.

Referring to organ specific mechanisms of toxicity, interference with neurotransmitters and/or neurotransmission and impairment of propagation of electrical activity are among the main reported mechanisms for chemicals that target the nervous system. In particular, many chemicals interfere at the level of receptors and ion channel function.

Chemicals that target the cardiovascular system often interfere with ion balance/signaling/membrane potential of the cell and with intracellular signaling mechanisms.

For many of the chemicals that damage the liver after an acute insult, mechanisms such as depletion of free radical scavengers, ROS production, lipid peroxidation (grouped under oxidative stress induced inflammation), and necrosis were reported.

Alterations in kidney tubule cell structure (accumulation in proximal tubular cells, loss of tubular epithelial barrier, and/or tight junctions), alterations in tubule cell metabolism (interference with ion balance), tubular obstruction (impaired [Na.sup.+] and water reabsorption, distal cast formation, crystal deposition), and alterations in cell viability (necrosis) are among the most reported mechanisms leading to acute renal failure.

The in vivo classification for acute oral toxicity (i.e., the assigned CLP acute oral toxicity categories based on the collected mean oral [LD.sub.50] values) of the chemicals acting via specific mechanisms of toxicity at organ/system level was evaluated in view of the information found in the literature for each chemical. Table 10 summarizes the outcome of this analysis, confirming that the nervous and cardiovascular systems are the most frequent targets for chemicals inducing acute oral toxicity.

3.2 Analysis of mechanistic information and in vitro 3T3 NRU cytotoxicity results

A total of 97 chemicals, for which in vivo and in vitro cytotoxicity data were available, have been identified with target organ/ system specific effects, of which 91 were predicted as acutely toxic by the 3T3 NRU cytotoxicity assay ([LD.sub.50] [less than or equal to] 2000 mg/kg). Table 11 summarizes the prediction of the acute oral toxicity (EU CLP toxicity categories) by the in vitro cytotoxicity assay for these chemicals.

Figure 11 complements Table 11 by adding the collected mechanistic information. It also shows the percentage of chemicals identified in vitro as acutely toxic acting through cell type specific mechanisms of toxicity and via general cytotoxic mechanisms when the acute oral toxicity category was correctly predicted (50 chemicals), under-predicted (32 chemicals), and over-predicted (9 chemicals) by the in vitro cytotoxicity assay.

An overview of the information collected with regard to specific target organ/system and general cytotoxicity for the chemicals that are correctly assigned to the CLP acute oral toxicity category, under-predicted and over-predicted by the in vitro cytotoxicity assay, respectively, is provided in the supplementary information (Tab. S1, S2, and S3 (12)).

Among the 50 correctly predicted chemicals, 29 act through some general mechanisms of cytotoxicity (58%) and 21 only via cell type specific mechanisms of toxicity (42%).

Among the 32 under-predicted chemicals, 20 act through a general mechanism of cytotoxicity (63%) and only 12 via cell type specific mechanisms of toxicity (38%).

Among the 9 chemicals with toxicity category over-predicted by the cytotoxicity assay, 6 act through a general mechanism of cytotoxicity (67%) and 3 only via specific mechanisms of toxicity (33%).

Among the 6 chemicals falsely predicted as non-classified (Fig. 12), two act through some mechanism of general cytotoxicity (33%) and four act only via cell type specific mechanisms of toxicity (67%), as shown in the supplementary information (Tab. S412).

3.2.1 Acute oral toxicity category 1

From the compounds assigned in vivo to the acute oral toxicity category 1 (fatal if swallowed), three (i.e., brucine, disulfoton, and physostigmine) target the nervous system and act via specific mechanisms (e.g., inhibition of cholinesterase, antagonism of glycine receptor). Among the remaining compounds, 1-phenyl-2-thiourea has the lung as target organ and needs bio-activation, and triethylenemelamine affects the immune system. General mechanisms of cytotoxicity have also been reported for these two compounds. For brucine, necrosis was found as the mechanism responsible for kidney tubular cell damage. When in vivo and in vitro mean values are compared, all compounds are misclassified by the in vitro cytotoxicity assay. Triethylene melamine is under-classified by one toxicity category and the other four compounds by 3 toxicity categories.

3.2.2 Acute oral toxicity category 2

Among the compounds assigned in vivo to the acute oral toxicity category 2 (fatal if swallowed), only colchicine is correctly predicted by the cytotoxicity assay. Colchicine inhibits microtubule formation and, thus, effectively inhibits mitosis, which is a general mechanism of toxicity. This mechanism of toxicity is also reported as the one responsible for the toxicity at the level of the nervous system and the liver. Digoxin and aconitine, which were predicted as false negatives by the cytotoxicity assay, act via specific mechanisms of toxicity such as interference with transporter enzymes (e.g., [Na.sup.+]-[K.sup.+]-ATPase) and calcium channels. Digoxin targets the cardiovascular system while aconitine acts on both the nervous and the cardiovascular system (Qiu et al., 2008; Chan, 2009; Prassas et al., 2011; Sun et al., 2014).

The compounds assigned to the toxicity category 2 in vivo but under-predicted in vitro act mainly via specific target organ toxicity mechanisms. For D-amphetamine, parathion, and strychnine, the central nervous system is the main target and specific mechanisms of toxicity were identified (i.e., inhibition of acetylcholinesterase, antagonism of glycine receptor, stimulation of the release of norepinephrine and dopamine). The kidney is the target for ochratoxin A, a well-known nephrotoxic agent acting on tubular cells (Ozbek, 2012). General cytotoxicity is also reported for ochratoxin A, and necrosis has been found as the mechanism underlying strychnine effects on kidney tubular cell damage. Epinephrine hydrogen tartrate targets the cardiovascular system, while warfarin targets both the cardiovascular system and blood cells (Hanley, 2004; Klaassen, 2001).

3.2.3 Acute oral toxicity category 3

General mechanisms of toxicity were also described for the chemicals correctly assigned in vitro to the acute oral toxicity category 3 (toxic if swallowed). Only for ethyl chloroacetate no general mechanisms of toxicity were reported, although it is a moderate irritant via the oral route. For the compounds under-predicted in vitro by only one toxicity category, mechanisms of general cytotoxicity were reported. For sodium salt of chloroacetic acid and pentachlorophenol also the mechanisms identified at target organ level were general mechanisms of toxicity such as interference with mitochondrial function (CNS), membrane disruption and/or interference with macromolecules, depletion of free radical scavenger (such as glutathione, catalase) content in liver tissue, and compromised mitochondrial respiration of tubular cells (kidney). Dichlorvos and theophylline act through general mechanisms of cytotoxicity at CNS level, and also via specific mechanisms of toxicity. Seventy-three per cent of these under-predicted compounds are linked to the CNS as the target system (i.e., GABAA receptor agonist, blockade of the GABAA-receptor coupled sodium channel, interference with the normal flux of Na+ and K+ ions across the axon membrane during neuronal signaling, antagonism of N-methyl-D-aspartate (NMDA) receptors and inhibition of serotonin/norepinephrine reuptake, agonist of nicotinic cholinergic receptors, neurotransmitter clearance from synaptic cleft). Verapamil, barium chloride, and theophylline act at the level of the heart by different mechanisms (blocking the calcium channel and binding to the cytosolic surface of the channel; interference with potassium channels, interference with intracellular signaling mechanisms, such as enzymatic activity, e.g., phosphodiesterases and protein kinases). Fat accumulation in hepatocytes was also described as the liver specific mechanism of toxicity of barium chloride (Ananda et al., 2013).

3.2.4 Acute oral toxicity category 4

General mechanisms of toxicity are reported for 49% of the compounds correctly assigned to the acute oral toxicity category 4 (harmful if swallowed). For several of these compounds, general cytotoxicity was identified at the target organ/level alone or in addition to other specific mechanisms of toxicity (e.g., acetylsalicylic acid uncouples mitochondrial oxidative phosphorylation and also inhibits Krebs cycle dehydrogenases at CNS level (Ekwall et al., 1998); valproic acid alters the activity of the GABA neurotransmitter by increasing the inhibitory activity of GABA through inhibition of GABA degradation, inhibition of GABA transaminobutyrate, increased GABA synthesis, decreased turnover and inhibition of the GABA reuptake by the glia and synaptic mechanisms. It also interferes with cellular metabolic processes, interacts with membrane ion channels (Sztajnkrycer, 2002; Chateauvieux et al., 2010), and induces oxidative stress by compromising the antioxidant status of the neuronal tissue (Chaudhary and Parvez, 2012); caffeine in the CNS competitively antagonizes adenosine receptors, inhibits phosphodiesterase, stimulates catecholamine release, and increases free calcium and intracellular cAMP (Fredholm et al., 1999); orphenadrine chloride competitively antagonizes acetylcholine binding at the neuroreceptor sites and induces necrosis in liver (Sangster et al., 1978; Ekwall et al., 1998)). The nervous and the cardiovascular system appeared as targets for 70% and 54% of the harmful compounds, respectively. Among the compounds acting via specific mechanisms of toxicity, 65% (13/20) targeted both the nervous and the cardiovascular system.

Four harmful compounds were falsely predicted in vitro as non-acutely toxic. Of those, isoniazid and paraldehyde act via specific mechanisms such as interference at the level of CNS receptors and ion channel function, depletion of GABA (isoniazid), impairment of the propagation of electrical activity in the CNS (paraldehyde), and inflammation of the GI mucosa (paraldehyde) (Gilman, 1985; Carpentier et al., 1992). The harmful effects of ethylene glycol mainly result from the accumulation of its more toxic metabolites (Hess et al., 2004). Diethylene glycol is metabolized to 2-hydroxyethoxyacetaldehyde by alcohol dehydrogenase oxidation, then to 2-hydroxyacetic acid (HEAA) by aldehyde dehydrogenase. HEAA causes acidosis, renal failure, and neurologic dysfunction. It is thought that the parent compound is toxic as well (Schep et al., 2009). The formation of toxic metabolites will be missed in the in vitro cell system due to the lack of metabolic competence of the 3T3 cells. This could explain, at least in part, the misclassification by the in vitro approach.

Many of the harmful compounds are extensively or rapidly metabolized in the liver and toxic metabolites were reported for five compounds (quinidine sulfate dehydrate (Kim and Benowitz, 1990), chloroform (HSDB, ACuteTox project; Hodgson, 2004), chloral hydrate (Beland, 1999; Pershad et al., 1999; Dogan-Duyar et al., 2010), sodium valproate (ACuteTox project; Sztajnkrycer, 2002), and malathion (ACuteTox project; Simoneschi et al., 2014)).

4 Discussion

The exercise reported here served several purposes. First of all, the mechanistic information collected was visually summarized, allowing direct comparison across target organs/systems. Secondly, by organizing acute toxic effects by their mechanism and cell type(s), we could start to associate known acutely toxic compounds with the different mechanisms. Finally, organizing information in this manner should facilitate the development of AOPs and IATA, for example by identifying properties that are requisites of in vitro testing systems for specific target organ toxicity testing.

This work also aimed to identify specific (complementary) mechanisms of acute toxicity that are perhaps not covered by the validated 3T3 NRU cytotoxicity assay. Therefore, in our analysis we tried to address: (i) whether chemicals can be identified and classified based on either positive or negative specific effects on target organ(s) (according to the 2000 mg/kg threshold) using the 3T3 NRU assay; (ii) which organs are the most frequent targets; and (iii) whether the triggered pathways of toxicity are conserved across organs.

In the overall analysis, of the 97 chemicals identified with target organ specific effects, 94% (91/97) were predicted as acutely toxic by the in vitro cytotoxicity assay and 6% (6/97) as non-toxic. When comparing the positive (i.e., acutely toxic) and negative (i.e., non-acutely toxic) in vitro predictions with those of the in vivo study, it turned out that all six negatives were false predictions (false negatives), while 55% of the positive predictions were correctly predicted, according to a CLP acute toxicity category, 35% under-predicted and 10% over-predicted. When evaluating the performance of any alternative approach for the purposes of regulatory classification, it is also necessary to consider the uncertainty associated with both the in vivo and the in vitro data. Actually, the analysis of consistency in classification published by Hoffmann et al. (2010) showed that conventional in vivo acute oral toxicity tests are intrinsically imprecise themselves and, about 44% of the substances would ambiguously occur within the limits of two adjacent classification categories (with at least 90% probability). A discussion of in vivo and in vitro data variability is outside the scope of this paper. Therefore, for the purpose of the mechanistic analysis shown and discussed here, the assignment of the compounds to the CLP acute oral toxicity categories was made based on the collected mean values (in vivo and in vitro). Another major source of uncertainty, not analyzed here due to lack of information, is the role of ADME (absorption, distribution, metabolism, and excretion) in determining the acute toxicity category. ADME has also been identified as a source of uncertainty in many OECD IATA case studies that are based on new approach methodologies. Several regulatory bodies have published guidance on the identification, characterization, and reporting of uncertainty (SCHEER, 2018).

A closer look at the chemicals acting through the specific target organs has not revealed a clear pattern with regard to which specific mechanisms of target organ toxicity are representative of compounds in the different CLP acute oral toxicity categories. For instance, approximately the same percentage of compounds acting through mechanisms of neurotoxicity was found in each acute oral toxicity category (i.e., 55% of the highly toxic chemicals allocated to toxicity categories 1 and 2, 50% of the toxic chemicals in category 3, and 56% of the harmful chemicals in category 4). A similar situation holds true for chemicals that act through mechanisms of cardiovascular toxicity, which were allocated to toxicity categories 2, 3, and 4 (33%, 19%, and 40%, respectively). Mechanisms of nephrotoxicity were also found for chemicals in all toxicity categories. Mechanisms of liver, lung, and blood toxicity were described for a small percentage of the highly toxic compounds (8%-21%). Based on these results, it can be concluded that mechanisms of toxicity specific for each organ can be triggered by compounds that belong to the different CLP acute oral toxicity categories. This is not surprising since the CLP categorization is based on potency, which can result from both toxicokinetic and toxicodynamic factors.

From the information collected and the analysis presented it can be concluded that general cytotoxicity is an important determinant of acute systemic toxicity. Overall, the majority of the analyzed chemicals (63%) causing acute lethal toxicity act via some general (rather than organ specific) mechanisms of toxicity. The nervous and the cardiovascular systems are the most frequent targets, with changes in neurotransmission and altered ion flow being important mechanisms often associated with acute neurotoxicity and cardiotoxicity, respectively.

It is well recognized that the use of basal cytotoxicity alone to determine the acute toxicity of a chemical may not always be enough and, furthermore, it depends on the chemical's kinetic behavior and/or its specific mechanisms of toxicity. These features may need to be considered in order to correctly estimate the in vitro concentration causing toxicity that could be compared with the concentration that the target cells in vivo would be exposed to (EU FP6 project ACuteTox). In silico tools such as the Virtual Cell Based Assay (VCBA) can be used to simulate the distribution of the chemicals in the in vitro system (Zaldivar Comenges et al., 2017). By comparing the simulated and the nominal [IC.sub.50] concentrations of the dissolved chemical, the influence of the in vitro kinetics on the cytotoxicity result may be anticipated. In addition, in vivo kinetics is an important determinant of acute systemic toxicity that requires further investigation (Graepel et al., 2017; Duarte Lopes Mascarenhas Proenqa et al., 2017). In silico tools such as Physiologically Based Kinetic (PBK) models (Paini et al., 2017) can be used to simulate the kinetics and distribution of chemicals in vivo.

Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. Therefore, in addition to kinetic considerations, in vitro misclassifications could be also linked to the number of acute oral toxicity categories under CLP and the associated [LD.sub.50] ranges, which are not based on a particular mechanistic rationale. Indeed, the outcome of the classification analysis carried out in the context of the EU FP6 project ACuteTox indicated that it is challenging to make a clear distinction between acute oral toxicity categories 1, 2, and 3 based on in vitro concentration-response data (Kinsner-Ovaskainen et al., 2013) and, therefore, three levels of toxicity (i.e., level 1: combination of categories 1 to 3, level 2: category 4, and level 3: non-classified) were proposed (Prieto et al., 2013a). A similar grouping was also considered by Norlen et al. (2012) in an investigation of the predictive performances of five alternative approaches for the assessment of acute oral toxicity. Overall, the value of the CLP classification into four acute oral toxicity categories could be challenged.

Building on all the collected/generated information it would be worth trying to develop an alternative way of classifying chemicals for acute oral toxicity based mainly on cytotoxicity and kinetic information, and complemented, if needed, with relevant organ specific mechanisms of toxicity. Based on the mechanistic knowledge discussed in this paper, we propose to integrate in vitro assays anchored to the most frequent mechanisms of acute toxicity specific for each organ (CNS, heart, liver, and kidney) into an IATA. An IATA can include defined approaches, i.e., formalized decision-making approaches that apply fixed data interpretation procedures to data generated with a defined set of information sources (OECD, 2016b). In this regard, an in vitro cytotoxicity assay would be used together with specific target tissue toxicity mechanisms tested by assays permitting evaluation of neurotoxicity (as the most sensitive), followed by cardiotoxicity, hepatotoxicity, and kidney toxicity. Such a battery of tests should be designed to allow assessment of the compounds based on their cytotoxicity (e.g., based on the 3T3 NRU assay) and organ specific mechanisms. In the broader context of IATA, these in vitro mechanistic data should be integrated with additional sources of information (QSAR, read-across, in chemico, human data, in vivo data, etc.) including, where appropriate, exposure and ADME information.

The development of AOPs relevant to acute neurotoxicity, cardiotoxicity, hepatotoxicity, etc., is already ongoing. However, as indicated, they are at different stages of development (Tab. S512). It is worth noting that some of the relevant AOPs are not specific to acute toxicity but nevertheless include key events that are relevant to acute exposure effects. Interestingly, some of the chemicals identified in these AOPs as triggers of molecular initiating events overlap with the chemicals reviewed in this report. The mechanistic information provided in this paper should inform the development of AOPs relevant to acute systemic toxicity, as well as AOP-informed IATA. The further development of AOPs and IATA should focus on the major target organs identified, i.e., the CNS, heart, liver, and kidney.

Conflict of interest

The authors declare that they have no conflict of interest to disclose.

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Pilar Prieto, Rabea Graepel, Kirsten Gerloff, Lara Lamon, Magdalini Sachana, Francesca Pistollato, Laura Gribaldo, Anna Bal-Price and Andrew Worth

EU Commission Joint Research Centre (JRC), Ispra, Italy

Received May 18, 2018; Accepted July 6, 2018; Epub July 12, 2018; [c] The Authors, 2018.

doi: 10.14573/altex.1805181

Correspondence: Pilar Prieto, PhD, European Commission, Joint Research Centre,

Directorate F--Health, Consumers and Reference Materials, Chemical Safety and Alternative Methods Unit,

EURL ECVAM, Ispra, Italy

(pilar.prieto-peraita@ec.europa.eu)

Caption: Fig. 1: Target organ blood--visualization of mechanisms leading to acute systemic toxicity CO, carbon monoxide

Caption: Fig. 2: Target organ liver--visualization of mechanisms leading to acute systemic toxicity ROS, reactive oxygen species

Caption: Fig. 3: Target organ lung--visualization of mechanisms leading to acute systemic toxicity Th2, T helper type 2 cells; ROS, reactive oxygen species; CCR3, C-C motif chemokine receptor 3; ICAM-1, Intercellular Adhesion Molecule 1; CNS, central nervous system

Caption: Fig. 4: Cardiovascular system--visualization of mechanisms leading to acute systemic toxicity

GPCRs, G protein-coupled receptors; CNS, central nervous system; PNS, peripheral nervous system; ROS, reactive oxygen species; NPY, neuropeptide Y; P2X, purinergic receptors; M2, muscarinic acetylcholine receptor; VIP, vasoactive intestinal peptide

Caption: Fig. 5: Nervous system--visualization of mechanisms leading to acute systemic toxicity

ROS, reactive oxygen species; BBB, blood brain barrier; 5-HT, 5-hydroxytryptamine; NA, noradrenaline; GABA, gamma-aminobutyric acid; NMDA, N-methyl-D-aspartate; AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; D2, dopamine

Caption: Fig. 6: Immune system--visualization of mechanisms leading to acute systemic toxicity

NFAT, nuclear factor of activated T-cells; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells

Caption: Fig. 7: Gastrointestinal system--visualization of mechanisms leading to acute systemic toxicity

Caption: Fig. 8: Target organ kidney--visualization of mechanisms leading to acute systemic toxicity

ROS, reactive oxygen species
Tab. 1: Chemicals acting through general cytotoxic mechanisms and/
or specific mechanisms of toxicity

General cytotoxic mechanisms                  Specific mechanism
                                              of toxicity

(4-Ammonio-m-           Endosulfan            ([+ or -])-
tolyl)ethyl                                   Propranolol
(2-hydroxyethyl)                              hydrochloride
ammonium sulfate

1,2,3,4-                Ethoxyquin            ([+ or -])-Verapamil
Tetrachlorobenzene                            hydrochloride

1,2,4-                  Ferrous sulfate       1-Naphthylamine
Trichlorobenzene

1,2-Dichlorobenzene     Formaldehyde          1-Phenyl-3-
                                              pyrazolidone

17[alpha]-              Glutethimide          2,4,6-Tris
Ethynyloestradiol                             (dimethylaminomethyl)
                                              phenol

1-Phenyl-2-thiourea     Haloperidol           5,5-
                                              Diphenylhydantoin

2,4-                    Hexachlorophene       Acetophenone
Dichlorophenoxyacetic
acid

4-Aminofolic acid       Isoniazid             Ammonium chloride

5-Fluorouracil          Lindane               Atropine sulfate
                                              monohydrate

Acetaldehyde            Maleic acid           Codeine

Acetylsalicylic         Malononitrile         D-Amphetamine
acid

Aconitine               Maprotiline           Diazepam

Acrolein                Mercury II chloride   Diethylene glycol

Acrylamide              Nicotine              Digoxin

Amitriptilyne           Ochratoxin A          Diphenhydramine
hydrochloride                                 hydrochloride

Arsenic trioxide        Octyl 3,4,5-          Disopyramide
                        trihydroxybenzoate

Barium chloride         Orphenadrine          Disulfoton
                        hydrochloride

Benzaldehyde            Paraquat dichloride   Epinephrine hydrogen
                                              tartrate

Brucine                 p-Benzoquinone        Ethyl chloroacetate

Busulfan                Pentachlorophenol     Ethylene glycol

Cadmium (III)           Phenanthrene          Fenpropathrin
chloride

Caffeine                Phenol                Glufosinate-
                                              ammonium

Carbon                  Potassium cyanide     Lithium carbonate
tetrachloride

Chloral hydrate         Sodium arsenite       Lithium sulfate

Chloroform              Sodium cyanate        Malathion

Chloroquine             Sodium lauryl         Meprobamate
bis(phosphate)          sulfate

Chlorpromazine          Sodium oxalate        Methadone
                                              hydrochloride

cis-                    Sodium salt of        N-isopropyl-N'-
Diammineplatinum        chloroacetic acid     phenyl-p-
(II) dichloride                               phenylenediamine

Colchicine              Sodium selenate       Paraldehyde

Copper sulfate          Sodium valproate      Parathion

Cupric sulfate          Strychnine            Phenobarbital
pentahydrate

Cyclohexamide           Tert-butyl            Physostigmine
                        hydroperoxide

Cyclosporin A           Thallium sulfate      Procainamide
                                              hydrochloride

Diallyl phthalate       Theophylline          Quinidine sulfate
                                              dehydrate

Dichlorvos              Triethylenemelamine   Resorcinol

Diquat dibromide        Valproic acid         Rifampicin

                                              Sodium pentobarbital

                                              Thioridazine
                                              hydrochloride

                                              Triphenyltin
                                              hydroxide

                                              Warfarin

Tab. 2: Specific mechanisms of acute blood toxicity

Mechanisms             Example of chemicals   References

Decrease oxygen carrying capacity

* Interference with    N-isopropyl-N'-        Williamson et al.,
hemoglobin             phenyl-p-              1981
                       phenylenediamine

* Oxidation of the     Resorcinol             NJ RTK, 2010
oxygen carrying iron   1-Naphthylamine        NJ RTK, 2004
molecule to
[Fe.sup.3+]

Clotting factor exhaustion

* Interference with    Warfarin               Hanley, 2004
clotting factor
production

Lysis of cells

* Binding to cell      Rifampicin             POISINDEX[R] System
(antigen) triggers                            (a); Manika et al.,
immune-mediated                               2013
destruction
                       Quinidine sulfate      Freedman et al.,
                                              1956

* Direct destruction   Copper sulfate         Franchitto et al.,
of cells, e.g., via                           2008
oxidative damage to
cell wall

(a) https://www.micromedexsolutions.com/home/dispatch
(accessed 09.07.2018). Login required.

Tab. 3: Specific mechanisms of acute liver toxicity

Mechanisms              Example of chemicals   References

Disturbance of          17[alpha]-Ethynyl      Wan and O'Brien,
normal bile acid        estradiol              2014; Davis et al.,
secretion                                      1978

* Changes in            Chlorpromazine         Jennings et al.,
membrane                                       2014
permeability of
hepatocytes or
biliary canaliculi

Fat accumulation in     Barium chloride        Ananda et al., 2013
hepatocytes

Membrane disruption     Isoniazid              Saukkonen et al.,
and/or interference                            2006;
with macromolecules

                                               Boelsterli and Lee,
                                               2014

Inflammation induced
by oxidative stress

* Lipid                 Carbon                 El-Hadary and
peroxidation            tetrachloride          Ramadan Hassanien,
                                               2016

Tab. 4: Specific mechanisms of acute lung toxicity

Mechanisms                  Example of         References
                            chemicals

Pulmonary endothelium       1-Phenyl-2-        Scott et al.,
damage                      thiourea           1990; Henderson
                                               et al., 2004

Increase capillary          Dichlorvos         Li et al., 1989
permeability

Muscarinic action           Dichlorvos         Li et al., 1989

Destruction of Type I       Cadmium chloride   INCHEM, 2017
epithelial cells

Inflammation                Chloroform         de Oliveira et
                                               al., 2015

Induced broncho- and        Tert-butyl         Olafsdottir et
vasoconstriction mediated   hydroperoxide      al., 1991
by thromboxane

Intra-alveolar hemorrhage   Paraquat           Dinis-Oliveira,
                                               2008

Deposits of calcium         Ethylene glycol    Pomara et al.,
oxalate crystals in lung                       2008;
parenchyma                                     Leth and
                                               Gregersen, 2005

Irritation to respiratory   Acrolein           Bein and Leikauf,
tract                                          2011

Lung accumulation through   Paraquat           Dinis-Oliveira,
the polyamine uptake                           2008
system

Tab. 5: Specific mechanisms of acute cardiovascular toxicity

Mechanisms             Example of chemicals   References

Interference with ion balance/signaling/membrane potential of cell

* Mimic                Digoxin                Nicolas et al.,
substrate/block                               2015;
transporter enzymes                           Prassas et al., 2011
such as [Na.sup.+]-
[K.sup.+]-ATPase       Thallium sulphate      Riyaz et al., 2013

* Interference with    Thallium sulphate      Riyaz et al., 2013
sodium and/or
potassium channels

                       Barium chloride        Bhoelan et al., 2014

                       5,5-                   Ekwall et al., 1998
                       Diphenylhydantoine

                       Quinidine sulphate     Kim and Benowitz,
                       dehydrate              1990

                       Disopyramide           Kim and Benowitz,
                                              1990

                       Procainamide           Kim and Benowitz,
                       hydrochloride          1990

                       Amitriptyline          Woolf et al., 2007
                       hydrochloride

* Interference with    Aconitine              Sun et al., 2014
[Ca.sup.2+] channels

* QT interval          Thioridazine           Beach et al., 2013
prolongation           hydrochloride

                       Haloperidol            Raudenska et al.,
                                              2013; Henderson et
                                              al., 1991

* Calcium channel      Verapamil              Nicolas et al.,
blocker and binding                           2015; Meister et
to the cytosolic                              al., 2010
surface of the
channel

* Stabilization of     Chloroquine            Ekwall et al., 1998
cell membrane          bis(phosphate)
leading to reduced
excitation and
conduction

* Mimic/block          Atropine sulphate      Ekwall et al., 1998
parasympathetic
activity

* Prevention of the    Amitriptyline          Dollery, 1991;
reuptake of heart      hydrochloride          Ekwall et al., 1998
noradrenaline

* Pronounced           Propranolol            Kerns et al., 1997
negative
chronotropic and
inotropic effect and
a quinidine-like
effect

Interference with intracellular signaling mechanisms

* Interference with    Caffeine               Ekwall et al., 1998
adenosine receptors

Increased vasoconstriction

* Activation of        Epinephrine hydrogen   Zhang et al., 2011
[beta]1-adrenergic     tartrate
receptors,
[beta]2-adrenergic
receptors in blood
vessels

Increased capillary    Warfarin               Hanley, 2004
fragility

Tab. 6: Specific mechanisms of acute neurotoxicity

Mechanisms                  Example of          References
                            chemicals

Interference with neurotransmitters/neurotransmission

* Inhibition of glutamine   Glufosinate         Llufs et al.,
synthetase and glutamate    ammonium            2008
decarboxylase

* Inhibition of the         Chloral hydrate     Kreuter et al.,
dopamine transporter                            2004; Sabeti et
                                                al., 2003

* Slowing down              D-amphetamine       Fitzgerald and
catecholamine metabolism                        Bronstein, 2013
by inhibiting monoamine
oxidase

Neurotransmitter release into synaptic cleft

* Stimulation of            Potassium           Patel et al.,
glutamate release which     cyanide             1993
can activate glutamate
receptors to initiate
excitotoxic processes

* Stimulation of the        D-amphetamine       Fitzgerald and
release of norepinephrine                       Bronstein, 2013
and dopamine from stores
in adrenergic nerve
terminals

* Attenuation of            Chloral hydrate     Kreuter et al.,
glutamate release and                           2004
reduction of activation
of glutamate receptors

Neurotransmitter clearance from synaptic cleft

* Inhibition of             Dichlorvos          Binukumar and
acetylcholinesterase and                        Gill, 2010;
accumulation of                                 EXETOXNET,
acetylcholine                                   1999; Sachana
                                                et al., 2001

                            Physostigmine       Gilman, 1985

                            Disulfoton          ATSDR, 1995

                            Parathion           Casarett and
                                                Doull, 2001

* Increased acetylcholine   Phenol              Liao and Oehme,
release at the                                  1980
neuromuscular junction

* Blockage of the           Amitriptilyne       Dollery, 1991;
neuronal reuptake of        hydrochloride       Ekwall et al.,
norepinephrine,                                 1998
serotonin, and dopamine

* Selective                 Maprotiline         Jan et al.,
norepinephrine re-uptake                        2013; Baumann
blockade                                        and Maitre,
                                                1979

* Depletion of              Isoniazid           Casarett and
gamma-aminobutyric acid                         Doull, 2001
(GABA)

* Increase of GABA by       Sodium              Sztajnkrycer,
indirect mechanisms         valproate           2002;
involving inhibition of                         Chateauvieux et
the enzyme succinate                            al., 2010
semialdehyde
dehydrogenase (SSA-DH) in
the GABA shunt

Interference at level of    Phenobarbital       Jana et al.,
receptor                                        2014

                            Theophylline        Nakada et al.,
                                                1983

* Blockage of the action    Atropine            Ekwall et al.,
of acetylcholine at         sulfate             1998
muscarinic receptors        monohydrate

* Competitive antagonism    Caffeine            Fredholm et
of cellular adenosine                           al., 1999
receptors

* Antagonist at the         Brucine             Teske et al.,
glycine receptor                                2011

                            Strychnine          Teske et al.,
                                                2011

* Blocking the release of   Codeine             NCIt, 2018;
inhibitory                                      Takahama and
neurotransmitters such as                       Shirasaki, 2007
GABA and acetylcholine

* Down-regulation of GABA   Diazepam            Casarett and
receptors                                       Doull, 2001

* Antagonizing chloride     Endosulfan          Jang et al.,
ion transport in GABA                           2016
receptors

* Interaction with GABAA    Meprobamate         Rho et al.,
receptors in a                                  1997
barbiturate-like fashion

* Inhibition of NMDA        Meprobamate         Rho et al.,
receptors                                       1997

* Blockade of the           Lindane             POISINDEX[R]
GABA-receptor coupled                           System (a)
sodium channel

* GABAA receptor agonist    Sodium              Dollery, 1991
                            pentobarbital

* Inhibition of the         Valproic acid       TOXNET, 2015
reuptake of GABA into the                       (b);
glia and nerve endings                          POISINDEX[R]
                                                System (a)

* Interference at the       Chloroform          Dick, 2006;
level of GABAA receptors                        Greenblatt and
                                                Meng, 2001

                            Phenobarbital       Jana et al.,
                                                2014

                            Valproic acid       Sztajnkrycer,
                                                2002;
                                                Chateauvieux et
                                                al., 2010

* Anticholinergic effects   Quinidine           Kim and
                            sulfate             Benowitz, 1990
                            dehydrate
                            Disopyramide

* Competitive antagonism    Orphenadrine        POISINDEX[R]
of acetylcholine at the     hydrochloride       Systema; Rejdak
neuroreceptor sites                             et al., 2011

* Blockade of the           Diphenhydramine     Pragst et al.,
H1-receptors                hydrochloride       2006

* Direct stimulation of     D-amphetamine       Fitzgerald and
[alpha]- and                                    Bronstein, 2013
[beta]-adrenergic
receptors

* Glutamate receptor        Glufosinate         Matsumura et
activation                  ammonium            al., 2001

* Dopamine receptor         Chlorpromazine      Haddad and
antagonism                                      Winchester,
                                                1990

* Blockage of dopamine D2   Thioridazine        POISINDEX[R]
receptor                    hydrochloride       Systema

* Competitive blockade of   Haloperidol         Raudenska et
postsynaptic dopamine                           al., 2013
(D2) receptors

* NMDA antagonism and       Methadone           Zorn and Fudin,
inhibition of                                   2011; Jamero et
serotonin/norepinephrine                        al., 2011
reuptake

* Agonist at nicotinic      Nicotine            Williams and
cholinergic receptors                           Robinson, 1984
Impaired propagation of
electrical activity

* Interaction with          5,5-                Ekwall et al.,
membrane ion channels       Diphenylhydantoin   1998
([Na.sup.+], [K.sup.+],
[Cl.sup.-], [Ca.sup.2+])    Aconitine           Chan, 2009;
                                                Peng et al.,
                                                2009

                            Fenpropathrin       Xiong et al.,
                                                2016; Spencer
                                                et al., 2001

* Interference with         Thallium            Osorio-Rico et
transporter enzymes (e.g.   sulfate             al., 2017;
[Na.sup.+]-[K.sup.+]-                           Ekwall et al.,
ATPase) (mimic                                  1998; Casarett
substrate/block)                                and Doull, 2001

                            Aconitine           Peng et al.,
                                                2009

* Interference with the     Lindane             Vucevic et al.,
normal flux of [Na.sup.+]                       2009
and [K.sup.+] ions across
the axon membrane as
nerve impulses pass

Peripheral neuropathy/ Polyneuropathy

Myelinopathy

* Intramyelinic edema       Hexachlorophene     Persson et al.,
                                                1978; Casarett
                                                and Doull's,
                                                2001

Axonopathy

* Blocking neurofilament    Acrylamide          Le Quesne,
transport via cross                             1985; LoPachin
linking of neurofilaments                       et al., 2003

* Neurofilament filled      Acrylamide          Le Quesne,
swelling of proximal axon                       1985; LoPachin
                                                et al., 2003

* Inhibition of             Colchicine          Gooneratne et
microtubule formation via                       al., 2014;
binding to tubulin                              Finkelstein et
                                                al., 2010

(a) http://www.thomsonhc.com

(b) https://toxnet.nlm.nih.gov/cgi-bin/sis/search/
a?dbs+hsdb:@term+@DOCNO+3582

Tab. 7: Specific mechanisms of acute immune toxicity

Mechanisms             Example of      References
                       chemicals

* Degenerative         Ochratoxin A    Al-Anati and
changes in                             Petzinger, 2006
combination with
slow replacement of
affected immune
cells due to
inhibition of
protein synthesis

* Decrease in whole    Triethylene     Bickham et al., 1994
blood cell counts      melamine
or subpopulations
                       Triphenyl tin   Vos et al., 1984
                       hydroxide

* Changes in bone      Triethylene     Bickham et al., 1994
marrow cell            melamine
proliferation

Tab. 8: Specific mechanisms of acute gastrointestinal toxicity

Mechanisms             Example of chemicals   References

Epithelial cell        Colchicine             Iacobuzio-Donahue et
damage                                        al., 2001

* Corrosion/           Ferrous sulfate        Yuen and Gossman,
irritation                                    2018
of the mucosa

* Interference with    Barium chloride        Bhoelan et al., 2014
potassium channels

* Enzyme activation    Theophylline           Barnes, 2013

Inflammation of the    5-Fluorouracil         Boussios et al.,
mucosa                                        2012

Formation of           Formaldehyde           Wood, 2014; Pandey
metabolite (formic                            et al., 2000; Eells
acid) at the place                            et al., 1981
of contact

Tab. 9: Specific mechanisms of acute kidney toxicity

Mechanisms             Example of          References
                       chemicals

Vasoconstriction

* Arteriolar           Codeine             Pokorny and
vasoconstriction                           Saunders, 1994
indirect effect by
rhabdomyolysis         Endosulfan          Jang et al., 2016

                       Brucine             Teske et al., 2011;
                                           Achappa et al., 2012

                       Diphenhydramine     Pragst et al., 2006

* Deficiency           Acetylsalicylic     Ferenbach and
vasodilators (PG2)     acid                Bonventre, 2016

* Endothelial damage   CsA                 Bonventre, 2014
with increase in
vasoconstrictors

* Interaction with     Lithium             Bonventre, 2003
renal V2-vasopressin
receptor

Glumerulonephritis     Lithium             Naughton, 2008

Interstitial           Acetylsalicylic     Naughton, 2008
nephritis              acid

Alterations in tubule cell structure

* Accumulation in      Cisplatin           Bulacio and Torres,
cells of proximal                          2013; Kuhlmann et
tubular cells                              al., 1997

                       Cadmium chloride    Ozbek, 2012

                       Mercury chloride    Bonventre, 2003;
                                           Zhou et al., 2008

* Loss of tubular      Ochratoxin A        Gennari et al., 2004
epithelial barrier
and/or tight
junctions

Alterations in tubule cell metabolism

* Interference with    Ammonium chloride   McEvoy, 2006
ion balance

Tubular obstruction

* Impaired             Cisplatin           Safirstein, 2004
[Na.sup.+] and water
reabsorption

* Distal cast          Diethylene glycol   Fowles et al., 2017
formation
                       Ethylene glycol     Fowles et al., 2017;
                                           Hess et al., 2004;
                                           Huhn and Rosenberg,
                                           1995; Pomara et al.,
                                           2008

* Crystal deposition   Sodium oxalate      Pawar and Vyawahare,
and tubular                                2017
obstruction

Generation of          Cisplatin           Bonventre, 2003
inflammatory and
vasoactive mediators

Alterations in cell viability

* Necrosis of          Mercury chloride    Zhou et al., 2008
tubular epithelium
                       4-ammonio-m-        EPA TSCATS (a)
                       tolyl)ethyl
                       (2-hydroxyethyl)
                       ammonium
                       sulfate

                       Cadmium chloride    Bonventre, 2003

                       Brucine             Liu et al., 2015

(a) https://bit.ly/2QkpbQ5

Tab. 10: Contribution of specific target organ/system mechanisms of
toxicity to the in vivo acute oral toxic category of chemicals

Organ specific mechanisms       (a) CLP Cat. 1   (a) CLP Cat. 2
of acute toxicity               (5 chemicals)    (15 chemicals)

Neurotoxicity                   3 (60%)          8 (53%)
Cardiovascular toxicity         0                5 (33%)
Liver toxicity                  0                1 (7%)
Kidney toxicity                 1 (20%)          4 (27%)
Lung toxicity                   1 (20%)          0
Gastrointestinal toxicity       0                2 (13%)
Blood toxicity                  0                2 (13%)
Immune toxicity                 1 (20%)          1 (7%)

Organ specific mechanisms       (a) CLP Cat. 3    (a) CLP Cat. 4
of acute toxicity               (26 chemicals)    (52 chemicals)

Neurotoxicity                   13 (50%)          29 (56%)
Cardiovascular toxicity         5 (19%)           21 (40%)
Liver toxicity                  4 (15%)           11 (21%)
Kidney toxicity                 4 (15%)           10 (19%)
Lung toxicity                   7 (27%)           5 (10%)
Gastrointestinal toxicity       5 (19%)           4 (8%)
Blood toxicity                  0                 4 (8%)
Immune toxicity                 1 (4%)            0

(a) Cat. 1: [less than or equal to] 5 mg/kg; Cat. 2: > 5 mg/kg,
[less than or equal to] 50 mg/kg; Cat. 3: > 50 mg/kg, [less than or
equal to] 300 mg/kg; Cat. 4: > 300 mg/kg, [less than or equal to]
2000 mg/kg

Tab. 11: Summary of prediction of EU CLP toxicity
categories in vivo and in vitro for the set of chemicals
classified for acute oral toxicity

Shadow cells indicate concordant predictions. EU CLP:
EU regulation on classification, labelling and packaging of
substances and mixtures; Cat: acute oral toxicity category; Cat. 1:
rat oral [LD.sub.50] [less than or equal to] 5 mg/kg; Cat. 2: 5mg/kg
< rat oral [LD.sub.50] [less than or equal to] 50 mg/kg; Cat. 3: 50
mg/kg < rat oral [LD.sub.50] [less than or equal to] 300 mg/kg; Cat.
4: 300 mg/kg < rat oral [LD.sub.50] [less than or equal to] 2000
mg/kg

3T3 NRU predicted    Reference in vivo oral [LD.sub.5]0 (mg/kg)
toxicity (mg/kg)
                     Cat. 1   Cat. 2   Cat. 3   Cat. 4

Cat. 1               0        0        0        0
Cat. 2               1        1        1        0
Cat. 3               0        6        9        8
Cat. 4               4        6        15       40

Fig. 9: Frequency of target organs/systems effects after acute
oral insult

The number on top of each bar represents the number of chemicals
affecting a particular organ/system. GI, gastrointestinal system

                             Number of chemicals

                  Indirect   Single organ/   Multi-organ/
                  effects    system direct   system direct
                             effects         effects

Blood                         3               8               11

Lung               6          2              16               24

Liver                         2              29               31

Kidney             1          3              26               30

Nervous System               12              55               67

Cardiovascular     4          3              32               39
system

Immune system                 1               2                3

GI system                                    18               18

Note: Table made from bar graph.

Fig. 10: Chemicals with specific target organs/systems effects
(single and multiple) and non-specific effects

Indirectly acting chemicals are included under multi-target
organ/system.

                                     Number of chemicals

Multi-target organ/system                    75
Single target organ/system                   26
Non-specific target organ effects            13

Note: Table made from bar graph.

Fig. 11: Specific target organ/system toxicity and general
cytotoxicity reported for the 91 chemicals predicted by the 3T3 NRU
cytotoxicity assay as positive chemicals (i.e., [LD.sub.50] [less
than or equal to] 2000 mg/kg)

Bars in each group represent from left to right: nervous system,
cardiovascular system, liver, kidney, lung, gastrointestinal
system, blood, immune system, general cytotoxicity, only specific
target organ/systems. Cat., acute oral toxicity category

% Chemicals

Cat. correctly   66%  48%  36%  28%  20%  16%  10%   0%  50%  20%
predicted--50
chemicals

Cat. under-      72%  31%  19%  28%  28%  19%   9%   6%  50%  25%
predicted--32
chemicals

Cat. over-       33%  22%  22%  11%  11%  22%  33%  11%  56%  40%
predicted--9
chemicals

Note: Table made from bar graph.

Fig. 12: Specific target organ/system toxicity and general
cytotoxicity reported for the 6 chemicals falsely predicted as
negatives by the 3T3 NRU cytotoxicity assay

GI, gastrointestinal system

% Chemicals

Nervous System             83%
Cardiovascular system      50%
Liver                      17%
Kidney                     33%
Lung                       33%
GI                         17%
Blood                       0%
Immune system               0%
General cytotoxicity       17%
Only target organ/system   67%

Note: Table made from bar graph.
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Author:Prieto, Pilar; Graepel, Rabea; Gerloff, Kirsten; Lamon, Lara; Sachana, Magdalini; Pistollato, France
Publication:ALTEX: Alternatives to Animal Experimentation
Article Type:Technical report
Date:Jan 1, 2019
Words:13860
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