SNAPSHOT

Key Event Component

Domain of Applicability

Key Event Description

Disruption of the thyroid hormone system is increasingly being recognized as an important toxicity pathway, as it can cause many adverse outcomes. Thyroid hormones do not only play an important role in the adult individual, but they are also critical during embryonic development. Thyroid hormones (THs) play an important role in a wide range of biological processes in vertebrates including growth, development, reproduction, cardiac function, thermoregulation, response to injury, tissue repair and homeostasis. Numerous chemicals are known to disturb thyroid function, for example by inhibiting thyroperoxidase (TPO) or deiodinase (DIO), upregulating excretion pathways or modifying gene expression. The two major thyroid hormones are triiodothyronine (T3) and thyroxine (T4), both iodinated derivatives of tyrosine. The synthesis of the thyroid hormones is a process that involves several steps. Thyroglobulin, the thyroid hormone precursor, is produced by the thyroid epithelial cells and transported to the lumen via exocytosis. Then thyroperoxidase (TPO) plays an essential role in the production of mainly T4. The prohormone T4 is then released in the circulation under the influence of thyroid stimulating hormone (TSH), in order to be transported to the various tissues, including the liver, the kidneys and the heart. Most TH actions depend on the binding of T3 to its nuclear receptors. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable to convert T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3 is the preferred substrate for DIO1 (Hennemann G, Visser TJ 1997). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate. DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively

How it is Measured or Detected

At this time, there are no approved OECD or EPA guideline protocols for measurement of DIO inhibition. Deiodination is the major pathway regulating T3 bioavailability in mammalian tissues. In vitro assays can be used to examine inhibition of deiodinase 1 (DIO1) activity upon exposure to thyroid disrupting compounds.

Several methods for deiodinase activity measurements are available. A first in vitro assay measures deiodinase activities by quantifying the radioactive iodine release from iodine-labelled substrates, depending on the preferred substrates of the isoforms of deiodinases. Another assay uses a chromatography-based method coupled to mass spectroscopy to measure products of thyroxin resulting from deiodinase type-1 activity (Butt et al., 2010). A colorimetric method (Renko et al., 2012), the Sandell-Kolthoff method, that measures the release of iodine from T4 is also available. Each of these assays requires a source of deiodinase which can be obtained for example using unexposed pig liver tissue (available from slaughterhouses) or rat liver tissue. Hornung et al. (2018) on the other hand used an adenovirus expression system to produce the DIO1 enzyme and developed an assay for nonradioactive measurement of iodide released using the Sandell-Kolthoff method in a 96-well plate format. This assay was then used to screen the ToxCast Phase 1 chemical library. The specific synthesis of DIO1 through the adenovirus expression system provides an important advantage over other methods where activity of the different deiodinase isoforms needs to be distinguished in other ways, such as based on differences in enzyme kinetics.

References

Butt, C.M., Wang, D., Stapleton, H.M. 2011. Halogenated phenolic contaminants inhibit the in vitro activity of the thyroid-regulating deiodinases in human liver. Toxicological sciences 124: 339-347.

Hornung, M.W., Korte, J.J., Olker, J.H., Denny, J.S., Knutsen, C., Hartig, P.C., Cardon, M.C., Degitz, S.J., 2018. Screening the ToxCast Phase 1 Chemical Library for Inhibition of Deiodinase Type 1 Activity. Toxicological Sciences 162, 570-581.

Renko, K., Hoefie, C.S., Hiller, F., Schomburg, L., Köhrle, J. 2012. Identification of Iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay. Endocrinology, 153: 2506-2513.

Visser, T.J., Van Overmeeren, E., Fekkes, D., Docter, R., Hennemann, G. 1979. Inhibition of iodothyronine 5'-deiodinase by thioureylenes: structure-activity relationship. FEBS Letters, 103, 2.

Key Event Component

Domain of Applicability

Key Event Description

There are two biological active thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), and a few inactive iodothyronines (rT3, 3,5-T2), which are all derived from the modification of tyrosine molecules (Hulbert, 2000). However, the plasma concentrations of the other iodothyronines are significantly lower than those of T3 and T4. The different iodothyronines are formed by the sequential outer or inner ring monodeiodination of T4 by the deiodinating enzymes, Dio1, Dio2, and Dio3 (Gereben et al., 2008). Deiodinase structure is considered to be unique, as THs are the only molecules in the body that incorporate iodide.

The circulatory system serves as the major transport and delivery system for THs from synthesis in the gland to delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In humans, the major transport proteins are TBG (thyroxine binding globulin), TTR (transthyretin) and albumin. The percent bound to these proteins in adult humans is about 75, 15 and 10 percent, respectively (Schussler 2000). Unbound (free) hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. In serum, it is the free form of the hormone that is active.

There are major species differences in the predominant binding proteins and their affinities for THs (see section below on Taxonomic applicability). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis (Zoeller et al., 2007).

It is notable that the changes measured in the TH concentration reflect mainly the changes in the serum transport proteins rather than changes in the thyroid status. These thyroid-binding proteins serve as hormonal store which ensure their even and constant distribution in the different tissues, while they protect the most sensitive ones in the case of severe changes in thyroid availability, like in thyroidectomies (Obregon et al., 1981). Until recently, it was believed that all of the effects of TH were mediated by the binding of T3 to the thyroid nuclear receptors (TRa and TRb), a notion which is now questionable due to the increasing evidence that support the non-genomic action of TH (Davis et al., 2010, Moeller et al., 2006). Many non-nuclear TH binding sites have been identified to date and they usually lead to rapid cellular response in TH-effects (Bassett et al., 2003), but the specific pathways that are activated in this regard need to be elucidated.

The production of THs in the thyroid gland and the circulation levels in the bloodstream are self-controlled by an efficiently regulated feedback mechanism across the Hypothalamus-Pituitary-Thyroid (HPT) axis. One of the most unique characteristics of TH is their ability to regulate their own concentration, not only in the plasma level, but also in the individual cell level, to maintain their homeostasis. This is succeed by the efficient regulatory mechanism of the thyroid hormone axis which consists of the following: (1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH), (2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary, (3) hormonal transport by the plasma binding proteins, (4) cellular uptake mechanisms in the cell level, (5) intracellular control of TH concentration by the deiodinating mechanism (6) transcriptional function of the nuclear thyroid hormone receptor and (7) in the fetus, the transplacental passage of T4 and T3 (Cheng et al., 2010).

In regards to the brain, the TH concentration involves also an additional level of regulation, namely the hormonal transport through the Blood Brain Barrier (BBB) (Williams, 2008). The TRH and the TSH are actually regulating the production of pro-hormone T4 and in a lesser extent of T3, which is the biologically active TH. The rest of the required amount of T3 is produced by outer ring deiodination of T4 by the deiodinating enzymes D1 and D2 (Bianco et al., 2006), a process which takes place mainly in liver and kidneys but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009). Both hormones exert their action in almost all tissues of mammals and they are acting intracellularly, and thus the uptake of T3 and T4 by the target cells is a crucial step of the overall pathway. The trans-membrane transport of TH is performed mainly through transporters that differ depending on the cell type (Hennemann et al., 2001; Friesema et al., 2005; Visser et al., 2008). Many transporter proteins have been identified up to date but the monocarboxylate transporters (Mct8, Mct10) and the anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH (Jansen et al., 2005).

T3 and T4 have significant effects on normal development, neural differentiation, growth rate and metabolism (Yen, 2001; Brent, 2012; Williams, 2008), with the most prominent ones to occur during the fetal development and early childhood. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs. The thyroidal actions though are not only restricted to mammals, as their high significance has been identified also for other vertebrates, with the most well-studied to be the amphibian metamorphosis (Furlow and Neff, 2006). The importance of the thyroid-regulated pathways becomes more apparent in iodine deficient areas of the world, where a higher rate of cretinism and growth retardation has been observed and linked to decreased TH levels (Gilbert et al., 2012). Another very common cause of severe hypothyroidism in human is the congenital hypothyroidism, but the manifestation of these effects is only detectable in the lack of adequate treatment and is mainly related to neurological impairment and growth retardation (Glinoer, 2001), emphasizing the role of TH in neurodevelopment in all above cases. In adults, the thyroid-related effects are mainly linked to metabolic activities, such as deficiencies in oxygen consumption, and in the metabolism of the vitamin, proteins, lipids and carbohydrates, but these defects are subtle and reversible (Oetting and Yen, 2007). Blood tests to detect the amount of thyroid hormone (T4) and thyroid stimulating hormone (TSH) are routinely done for newborn babies for the diagnosis of congenital hypothyroidism at the earliest stage possible.

How it is Measured or Detected

T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone are considered more direct indicators of T4 and T3 activities in the body. The majority of T3 and T4 measurements are made using either RIA or ELISA kits. In animal studies, total T3 and T4 are typically measured as the concentrations of free hormone are very low and difficult to detect. Historically, the most widely used method in toxicology is RIA. The method is routinely used in rodent endocrine and toxicity studies. The ELISA method has become more routine in rodent studies. The ELISA method is a commonly used as a human clinical test method. Least common is analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, though methods employing HLPC and mass spectrometry (DeVito et al., 1999; Miller et al., 2009).

Any of these measurements should be evaluated for fit-for-purpose, relationship to the actual endpoint of interest, repeatability, and reproducibility. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by a an indirect methodology, whereas analytical determination is the most direct measurement available. All of these methods, particularly RIA, are repeatable and reproducible.

References

• Bartalena L, Robbins J.Thyroid hormone transport proteins.Clin Lab Med. 1993 Sep;13(3):583-98.
• Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.
• Bianco AC, Kim BW. (2006). Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest. 116: 2571–2579.
• Brent GA. (2012). Mechanisms of thyroid hormone action. J Clin Invest. 122: 3035-3043.
• Cheng SY, Leonard JL, Davis PJ. (2010).Molecular aspects of thyroid hormone actions. Endocr Rev. 31:139–170.
• Davis PJ, Zhou M, Davis FB, Lansing L, Mousa SA, Lin HY. (2010). Mini-review: Cell surface receptor for thyroid hormone and nongenomic regulation of ion fluxes in excitable cells. Physiol Behav. 99:237–239.
• DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, *Meier C, Miller R, Tietge J, Tyl R. (1999). Screening methods for thyroid hormone disruptors. Environ Health Perspect. 107:407-415.
• Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitamins? Proc Soc Exp Biol Med. 214:302-317.
• Friesema EC, Jansen J, Milici C, Visser TJ. (2005). Thyroid hormone transporters. Vitam Horm. 70: 137–167.
• Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47.
• Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Simonides WS, Zeöld A, Bianco AC. (2008). Cellular and molecular basis of deiodinase-regulated thyroid hormone signalling. Endocr Rev. 29:898–938.
• Gilbert ME, Rovet J, Chen Z, Koibuchi N. (2012).Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology. 33: 842-852.
• Glinoer D. (2001).Potential consequences of maternal hypothyroidism on the offspring: evidence and implications. Horm Res. 55:109-114.
• Hennemann G, Docter R, Friesema EC, de Jong M, Krenning EP, Visser TJ. (2001). Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev. 22:451-476.
• Heyland A, Hodin J. (2004). Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of non-feeding development. Evolution. 58: 524-538.
• Heyland A, Moroz LL. (2005). Cross-kingdom hormonal signaling: an insight from thyroid hormone functions in marine larvae. J Exp Biol. 208:4355-4361.
• Hulbert A J. (2000). Thyroid hormones and their effects: A new perspective. Biol Rev. 75: 519-631.
• Jansen J, Friesema EC, Milici C, Visser TJ. (2005). Thyroid hormone transporters in health and disease. Thyroid. 15: 757-768.
• Larsen PR. (2009).Type 2 iodothyronine deiodinase in human skeletal muscle: new insights into its physiological role and regulation. J Clin Endocrinol Metab. 94:1893-1895.
• Manzon RG, Youson JH. (1997). The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.). Gen Comp Endocrinol. 106:211-220.
• Miller MD, Crofton KM, Rice DC, Zoeller RT. (2009).Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 117:1033-1041.
• Moeller LC, Dumitrescu AM, Seo H, Refetoff S. (2006). Thyroid hormone mediated changes in gene expression can be initiated by cytosolic action of the thyroid hormone receptor β through the phosphatidylinositol 3-kinase pathway. NRS. 4:1-4.
• Obregon MJ, Mallol J, Escobar del Rey F, Morreale de Escobar G. (1981). Presence of l-thyroxine and 3,5,3-triiodo-l-thyronine in tissues from thyroidectomised rats. Endocrinology 109:908-913.
• Oetting A, Yen PM. (2007). New insights into thyroid hormone action. Best Pract Res Clin Endocrinol Metab. 21:193–208.
• Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149.
• Visser WE, Friesema EC, Jansen J, Visser TJ. (2008). Thyroid hormone transport in and out of cells. Trends Endocrinol Metab. 19:50-56.
• Williams GR. (2008). Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 20:784–794.
• Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression.FEBS J. 2009 Oct;276(19):5357-66.
• Yaoita Y, Brown DD. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev. 4:1917-1924.
• Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142.
• Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53

Key Event Component

Domain of Applicability

Key Event Description

The swim bladder of bony fish is evolutionary homologous to the lung (Zheng et al., 2011). The teleost swim bladder is a gas-filled structure that consists of two chambers, the posterior and anterior chamber. In zebrafish, the posterior chamber inflates around 96 h post fertilization (hpf) which is 2 days post hatch, and the anterior chamber inflates around 21 dpf. In fathead minnow, the posterior and anterior chamber inflate around 6 and 14 dpf respectively.

The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007). Dumbarton et al. (2010) showed that the anterior chamber of zebrafish has particularly closely packed and highly organized bundles of muscle fibres, suggesting that contraction of these muscles would reduce swim bladder volume. While it had previously been suggested that the posterior chamber had a more important role as a hydrostatic organ, this implies high importance of the anterior chamber for buoyancy. The anterior chamber has an additional role in hearing (Bang et al., 2002). Weberian ossicles (the Weberian apparatus) connect the anterior chamber to the inner ear resulting in an amplification of sound waves. Reduced inflation of the anterior chamber may manifest itself as either a complete failure to inflate the chamber or reduced size of the chamber. Reduced size is often associated with a deviating morphology.

How it is Measured or Detected

In several fish species, inflation of the anterior chamber can be observed using a stereomicroscope because the larvae are still transparent during the larval stage. This is for example true for zebrafish and fathead minnow. Anterior chamber size can then be measured based on photographs with a calibrator.

References

Zheng, W., Wang, Z., Collins, J.E., Andrews, R.M., Stemple, D., Gong, Z. 2011. Comparative transcriptome analyses indicate molecular homology of zebrafish swim bladder and mammalian lung. PLoS One 6, http://dx.doi.org/10.1371/

Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007. Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985, http://dx.doi.org/10.1002/jmor.

Dumbarton, T.C., Stoyek, M., Croll, R.P., Smith, F.M., 2010. Adrenergic control of swimbladder deflation in the zebrafish (Danio rerio). J. Exp. Biol. 213,2536–2546, http://dx.doi.org/10.1242/jeb.039792.

Bang, P.I., Yelick, P.C., Malicko, J.J., Sewell, W.F. 2002. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. Journal of Neuroscience Methods. 118, 177-187.

Key Event Component

Domain of Applicability

Key Event Description

Hearing refers to the ability to perceive sound vibrations propagated as pressure changes through a medium such as air or water. Reduced hearing in the context of this key event can refer to reduction in the perceived volume of a sound relative to the amplitude of sound waves. Reduced hearing may also refer to a reduced range of frequencies that can be perceived.

How it is Measured or Detected

Hearing is generally measured behaviorally or electrophysiologically.

• Common behavioral tests involve transmission of pure tones of defined amplitude and frequency using and audiometer or PC and using a behavioral response (e.g., clicking a button; startle response) to determine whether the tone is perceived.

Electrophysiological tests:

• Auditory brainstem response (ABR): Uses electrodes placed on the head to detect auditory evoked potentials from background electrical activity in the brain.

Hearing tests in Fish:

• Through the mid-late 1980s conditioning and behavioral tests were most commonly employed in testing fish hearing. Methods reviewed by Fay (1988)
• A high throughput behavioral test for detecting auditory response in fish has been described (Bang et al. 2002).
• Invasive electrophysiological methods involving surgical insertion of electrodes into the auditory nerves have been employed.
• Non-invasive recording of Auditory Evoked Potentials (AEPs; synonymous with ABRs) are now the most common approach for measuring hearing in fish. AEPs can be recorded via electrodes attached cutaneously to the head (see review by Ladich and Fay, 2013).

References

• Fay RR (1988) Hearing in vertebrates: a psychophysics databook. Hill-Fay Associates, Winnetka, Ill
• Ladich F, Fay RR. Auditory evoked potential audiometry in fish. Reviews in Fish Biology and Fisheries. 2013;23(3):317-364. doi:10.1007/s11160-012-9297-z.
• Bang PI, Yelick PC, Malicki JJ, Sewell WF. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. J Neurosci Methods. 2002 Aug 30;118(2):177-87. PubMed PMID: 12204308.

Key Event Component

Domain of Applicability

Key Event Description

Adequate swimming performance in fish is essential for behaviour such as foraging, predator avoidance and reproduction.

How it is Measured or Detected

For fish larvae, automated observation and tracking systems are commercially available and increasingly used for measuring swimming performance including distance travelled, duration of movements, swimming speed, etc. This kind of measurements is often included in publications describing effects of chemicals in zebrafish larvae (Hagenaars et al., 2014; Stinckens et al., 2016; Vergauwen et al., 2015).

References

Hagenaars, A., Stinckens, E., Vergauwen, L., Bervoets, L., Knapen, D., 2014. PFOSaffects posterior swim bladder chamber inflation and swimming performanceof zebrafish larvae. Aquat. Toxicol. 157, 225–235.

Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.

Vergauwen, Lucia; Nørgaard Schmidt, Stine; Maho, Walid; Stickens, Evelyn; Hagenaars, An; Blust, Ronny; Mayer, Philipp; Covaci, Adrian; Knapen, Dries. 2014. A high throughput passive dosing format for the Fish Embryo Acute Toxicity test. Chemosphere. 139: 9-17.

Key Event Component

Domain of Applicability

Key Event Description

Young of year refers to young animals (usually fish) produced in one reproductive year, which have not yet reached one year of age. Small fish, hatched from eggs spawned in the current year, are considered young of year.

Young of year survival directly impacts population structure, growth and fitness. Maintenance of sustainable fish and wildlife populations is an accepted regulatory goal upon which risk assessments and risk management decisions are based.

How it is Measured or Detected

Young of year survival can be measured:

• in the lab by recording survival during prolonged exposure experiments
• in dedicated mesocosms, or in drainable ponds
• in the field, for example by determining age structure after one capture, or by capture-tag-recapture efforts

Key Event Component

Domain of Applicability

Key Event Description

Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is an accepted regulatory goal upon which risk assessments and risk management decisions are based.

How it is Measured or Detected

Population trajectories, either hypothetical or site specific, can be estimated via population modeling based on measurements of vital rates or reasonable surrogates measured in laboratory studies. As an example, Miller and Ankley 2004 used measures of cumulative fecundity from laboratory studies with repeat spawning fish species to predict population-level consequences of continuous exposure.

Regulatory Significance of the AO

Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.

References

• Miller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17ß-trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.

List of Adjacent Key Event Relationships