Functional assessments in repeat-dose toxicity studies: the art of the possible

Abstract

Clinical and nonclinical safety liabilities remain a major cause of adverse drug reactions, candidate drug attrition, delays during development, labelling restrictions, non-approval, and product withdrawal. Many of the toxicities are functional in nature and/or in origin. Whereas pharmacological responses tend to be fairly rapid in onset, and are therefore detectable after a single dose, some diminish on repeated dosing, whereas others increase in magnitude and therefore can be missed or underestimated in single-dose safety pharmacology studies. Functional measurements can be incorporated into repeat-dose toxicity studies, either routinely or on an ad hoc basis. Drivers for this are both scientific (see above), and regulatory (e.g., ICH S6, S7, S9). There are inherent challenges in achieving this: the availability of suitable technical and scientific expertise in the test facility; unsuitable laboratory conditions; use of simultaneous (as opposed to staggered) dosing; requirement for toxicokinetic sampling; unsuitability of certain techniques (e.g., use of anaesthesia; surgical implantation; food restriction); equipment availability at close proximity; sensitivity of the methods to detect small, clinically relevant, changes. Nonetheless, ‘fit-for-purpose’ data can still be acquired without requiring additional animals. Examples include assessment of behaviour, sensorimotor, visual, and autonomic functions, ambulatory ECG and blood pressure, echocardiography, respiratory, gastrointestinal, renal and hepatic functions. This is entirely achievable if functional measurements are relatively unobtrusive, both with respect to the animals and to the toxicology study itself. Careful pharmacological validation of any methods used, and establishing their detection sensitivity, is vital to ensure the credibility of generated data.

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1. Drug-related safety liabilities: a challenge for drug development

Safety liabilities (toxicities and adverse effects in nonclinical species, and adverse drug reactions (ADRs) in humans), remain a major cause of drug attrition during preclinical and clinical development, and of withdrawal of medicines from the market, accounting for approximately one third of all drug discontinuations.^1–5 Short of terminating drug projects, safety liabilities also cause delays during development, intensify labelling restrictions, and negatively impact patient compliance and physician preference, leading ultimately to reduced product success. Toxicities associated with the cardiovascular and hepatic systems account for the majority of drug attrition, withdrawal and ADRs, followed to a lesser degree by toxicities associated with the nervous, renal and gastrointestinal systems.^6–12 Phase I clinical trials in healthy volunteers are reasonably safe,¹³ partly reflecting effective preclinical testing strategies to eliminate high-risk safety liabilities. More subtle but potentially project-derailing toxicities often emerge only when drugs are administered for long periods of time to large patient populations. Often these liabilities are not detected either preclinically or in early clinical trials. A retrospective industry analysis published in 2000¹⁴ suggested that, together, the rodent and non-rodent safety evaluations of up to 1 month in duration enabled prediction of 71% of the toxicities subsequently seen in man. It also highlighted the limitation of existing approaches in ensuring the development of safe medicines. Side effects can occur after acute (i.e., single dose administration) or repeated dosing and can be functional and/or structural in nature. In fact, many of the recent high-profile examples of drug-induced toxicities in humans were functional in nature or in origin, e.g., human ether-à-go-go related gene (hERG) channel block by a variety of drugs associated with risk of torsade de pointes;¹⁵ cyclo-oxygenase-2 (COX-2) inhibitors and their association with modest blood pressure elevation and myocardial infarction risk,¹⁶ anti-obesity drugs and cardiovascular risks,¹⁷ phosphodiesterase-5 (PDE-5) inhibitors and hearing deficits,¹⁸ anti-diabetic drugs (e.g., taspoglutide) and nausea and vomiting.¹⁹ In a time of increased public scrutiny, escalating industry costs, and finite resources at regulatory agencies, the need for developing safer medicines is more important than ever. This review aims to provide evidence and illustrations on how the value of in vivo toxicology studies can be enhanced by including the measurement of functional endpoints.

By ‘functional endpoints’ we mean behavioural, physiological and biochemical measurements of function, as opposed to pathological changes in morphology or biomarkers thereof (including blood-borne biomarkers). (Nor do we mean ‘functional’ in the sense of gene expression.) So, although we would welcome the use of methods to detect pathological changes during the in-life phase, including blood-borne biochemical signals (e.g., RNA; proteins; enzymes; cytokines and other inflammatory markers; etc.), and ‘omics’, this is outside the scope of this article, and the reader is referred to recent reviews on these topics.^20–27

In addition to adding an extra dimension to the information content of toxicology studies, the inclusion of functional endpoints would also have a 3Rs benefit,²⁸ particularly if this could be achieved without the use of additional animals and without significantly increasing the welfare burden on individual animals involved in these studies. In particular, it would assist with reducing the numbers of animals used, firstly by minimising or obviating the need for standalone repeat-dose investigative studies addressing a specific functional endpoint, and secondly, by providing clearer information to prevent compounds with problematic safety profiles progressing into further, extensive nonclinical in vivo regulatory toxicology evaluations, only to be stopped eventually because of adverse effects.

On the face of it, there might appear to be 3Rs gains from replacing standalone single-dose safety pharmacology studies with measurements in repeat-dose toxicity studies. However, we would argue that this is only valid if the design and data quality can be replicated entirely within a repeat-dose toxicity study, on the first day of dosing. This is problematic, as will become clear later.

2. Principal aims of repeat-dose toxicity studies

Toxicology studies are conducted at any point from early drug discovery (to assess adverse effects related to the intended molecular target and/or chemical series) through to late-stage development (i.e., regulatory studies to support clinical trials and marketing). The primary aim of regulatory repeat-dose toxicity studies in animals is to identify the potential hazards of new chemical entities (NCEs). This is achieved by evaluating target organ toxicities and the exposure levels at which these occur, thereby enabling detection of potential safety hazards, and safety risk assessment, before human exposure.

The standard duration of the regulatory toxicology studies run prior to human exposure is 28 days of dosing (termed ‘one-month studies’), which permits dosing of the same duration in humans;^29–32 they are required in a rodent and a non-rodent species, and are conducted according to the principles of Good Laboratory Practice (GLP³¹). These one month studies are preceded by maximal tolerated dose/dose range-finding (MTD/DRF) studies. The MTD phase involves dose escalation and adjustment until the maximal tolerated dose is established for a single administration. The DRF phase involves administering the MTD (alongside lower doses and a vehicle control group), usually for 7 or 14 days. The MTD/DRF studies provide a degree of confidence that a one-month study can proceed safely and successfully using these doses throughout.^30,33

Longer-term toxicology studies (greater than 1 month in duration) are conducted in parallel to early clinical trials to enable the dosing of patients over longer periods.^29,30–32 In addition to these general toxicology studies, specialised repeat-dose toxicity studies are also required. These include rodent carcinogenicity studies running for up to 2 years, which are aimed at identifying the oncogenic potential of new medicines, reproductive toxicology studies to assess the effects of drugs administered during pregnancy on embryonic and postnatal development, and juvenile toxicity studies, which are required before administration of drugs to infants.^31,34–39

The primary endpoints of general toxicology studies are pathological ones: histopathological examination of a standard set of tissues post-mortem. In addition, during the in-life phase of the study, blood samples are taken for assessment of effects of the test compound on haematology and plasma chemistry, and for measurement of plasma exposure to the test compound (‘toxicokinetics’), urine is collected for urinalysis, and ophthalmoscopy is performed. The only physiological assessments made routinely are of body weight, food consumption (per cage, in the case of group-housed rodents), urine biochemistry, and (in non-rodent species), electrocardiogram (ECG) (Table 1). A key outcome of the studies is the establishment of a ‘no observed adverse effect level’ (NOAEL⁴⁰).

Table 1 In-life measurements and blood sampling on main study animals during a repeat dose toxicity study^a

	Rodent	Nonrodent
a Modified from Keller & Banks (2006).^29 These measurements are also continued beyond the dosing phase in recovery groups.
Toxicokinetic sampling	Usually on satellite groups (microsampling may change this), usually multiple sampling on Day 1 and at end of dosing phase	Usually multiple sampling on Day 1 and at end of dosing phase
Clinical observations	At least twice-daily	At least twice-daily
Body weight	At least weekly (usually twice-weekly)	At least weekly
Food consumption	Usually weekly	Daily
Water consumption	Usually weekly	Not routinely measured
Ophthalmoscopy	Pre-study, end of dosing phase	Pre-study, end of dosing phase
ECG	Not routine	Pre-study, end of dosing phase
Haematology and clinical chemistry	At least once during dosing phase	Pre-study, at least once during dosing phase
Urinalysis	Metabolism cage, at least once during dosing phase	Metabolism cage or bladder catheterization, pre-study and at least once during dosing phase

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Regulatory general toxicology studies are usually run according to well-defined study designs, standard operating procedures, and GLP. In such a highly regulated environment it is difficult to change the status quo, leading to a relatively conservative mindset within the pharmaceutical toxicology community. As a result, the basic design of the one-month rodent and non-rodent toxicology studies have remained largely unchanged since the 1970s.

3. Drivers for change

These are of two types: scientific and regulatory. Together, these drivers have stimulated interest in the inclusion of functional endpoints in repeat-dose toxicity studies on conventional pharmaceuticals (i.e., non-biologics), as reflected by the growing number of workshops/symposia incorporated into national and international conferences on toxicology and safety pharmacology (Table 2), and an increase in the number of poster presentations related to this topic at annual meetings of the Safety Pharmacology Society (SPS) over recent years.⁴¹

Table 2 Growing interest in incorporation of functional endpoints into repeat-dose toxicity studies

Year	Society	Activity
2004	Safety Pharmacology Society, annual meeting	1 invited presentation
2008	Safety Pharmacology Society, annual meeting	6 invited presentations
2010	Society of Toxicology, annual meeting	Dedicated sessions; posters
2010	British Toxicology Society, spring meeting	1 invited presentation
2010	American College of Toxicology, annual meeting	Half-day workshop
2011	Eurotox annual meeting	Half-day workshop
2011	Safety Pharmacology Society, annual meeting	Continuing education course
2012	Safety Pharmacology Society	Membership survey
2012–13	Safety Pharmacology Society	Webinar series
2012	Safety Pharmacology Society, annual meeting	Continuing education course
2012	Safety Pharmacology Society, annual meeting	Plenary session

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3a. Scientific drivers

Arguments for change primarily arose from within the toxicology community, led by Gerhard Zbinden from the late 1970s onwards^42–44 (Fig. 1) and subsequently echoed by others.^45–47 In fact, recommendations to include neurobehavioural assessments within toxicity studies date back even earlier,^48,49 and would appear to have resonated with government agencies responsible for regulating the chemical and agrochemical industrial toxicology communities, but evidently not with their pharmaceutical counterparts. This lead to innovation in neurobehavioural assessments in chronic exposure toxicology studies in the chemical and agrochemical industries,^50,51 with consortium initiatives leading to international standardisation,⁵² but there have been no equivalent initiatives in the pharmaceutical industry.

Fig. 1 The title says it all. To a pharmacologist, reliance on pathology endpoints is akin to describing what happened in a football match from the state of the pitch! (Image reprinted by permission from Macmillan Publishers Ltd: Proceedings of 9th International Congress of Pharmacology, vol. 1, pp. 43–49, copyright 1984.)

Another scientific argument for evaluating functional effects on repeat-dosing is the phenomenon of delayed-onset effects that may be missed if just assessing the effects of a single administration. Mechanisms for delayed effects include both pharmacokinetic ones (e.g. progressive accumulation in the body or in a particular organ, especially heart or brain), drug metabolism (formation of a pharmacologically active metabolite), and changes in cellular biochemistry (receptor downregulation; upregulation; inhibition of ion channel trafficking, e.g. hERG; inhibition of axonal transport; etc.). Although the first two mechanisms would be expected to manifest themselves within the 24 h timeframe of a single-administration safety pharmacology study, there are exceptions, including the delayed onset, progressive effect of amiodarone on QT interval due to myocardial accumulation over days and weeks.⁵³ An in-house example of a QT effect increasing over repeated dosing is shown in Fig. 2.

Fig. 2 Example of drug-induced QTc prolongation during a 28-day toxicology study in beagle dogs. ECGs were recording in freely moving dogs using a noninvasive telemetry system. Open symbols: vehicle-treated, n = 5; filled symbols: compound-treated group, n = 4–6. Note the overall stability of QTc over the one-month recording period in the vehicle control group. *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle control group. (In-house data.)

Probably the majority of functional effects are not associated with any pathological correlates. On the other hand, some slowly-developing functional changes can precede the onset of pathological damage revealed by standard histopathological techniques, or occur at lower exposures, and can therefore provide a clinically translatable biomarker to inform and limit dose escalation in clinical trials, and may also provide potential earlier endpoints in terms of animal welfare. Examples include preclinical assessment of visual acuity for agents with a risk of retinal toxicity.⁵⁴

Some functional changes are accurately coupled to the time course of the pathology, and thereby mirror the pathological changes without the requirement for termination of animals for histopathology at different time points. An example is given in Fig. 3, whereby the development of muscle pathology (fibrodysplasia) in rats is reflected by their deterioration in performing a task of motor coordination (see also Nodop Mazurek et al., 2011⁵⁵).

Fig. 3 Deterioration of performance in rats in an accelerating rotarod task during repeated dosing of a compound associated with skeletal muscle toxicity. Open symbols: vehicle-treated, n = 10; filled symbols: compound-treated group, n = 10. A deficit in rotarod performance was detected before the appearance of clinical signs. It also mirrored the development of pathology. *P < 0.05; **P < 0.01 vs. vehicle control group. (In-house data.)

Finally, some pathological changes occur as a consequence of functional changes, for example specific cardiac lesions in dogs,^56,57 and monkeys,⁵⁸ developing as a result of sustained episodes of drug-induced hypotension and/or tachycardia.

3b. Regulatory drivers

In regulatory toxicology in the pharmaceutical industry (as is also the case in the chemical and agrochemical industries), the primary drivers for change have always been regulatory requirements, either implicit (in guidelines) or implied. The obvious reason for this is that it is difficult to demonstrate the value of inclusion of additional tests when other companies are not doing them and the regulatory authorities are not looking for them. Arguments such as predicting adverse functional effects in humans, either to anticipate them preclinically and therefore facilitate management of the issues, or even to stop further development of the compound preclinically, are often over-ruled. Similarly, CROs are unlikely to develop methodologies for inclusion in repeat-dose toxicity studies unless there is a demand from study sponsors.

The 1997 ICH Guidance document (S6) on preclinical safety assessment of biopharmaceutical products,^59,60 indicated that safety pharmacology endpoints could be assessed during toxicology studies, rather than requiring standalone studies. At the time, biopharmaceutical products only represented a small fraction of the output of the pharmaceutical industry into clinical development, thereby providing only a minor push in this direction. However, as the preclinical development of biopharmaceutical products began to grow, the integration of safety pharmacology endpoints into regulatory repeat-dose toxicity studies on these therapeutic agents has become more commonplace. More recently, FDA guidance on Exploratory IND studies (i.e., clinical trials of limited exposure on NCEs for which there is no therapeutic intent) also encouraged the integration of functional measurements into toxicology studies,⁶¹ thus reducing the necessity for standalone safety pharmacology studies. However, as this is a minority activity within drug development, the impact on the culture of running toxicology studies has been minimal.

Another influence has been the recently revised ICH Guidance document (S9) on oncology products,⁶² which also largely removed the regulatory requirement for standalone safety pharmacology studies where such products were destined for evaluation in patients with end-stage cancer. Nonetheless, the guidance encouraged a science-driven approach, so that anticipated or known safety liabilities should be considered and addressed accordingly; for example anticipated cardiotoxicity associated with tyrosine kinase inhibitors.^63–65 In addition, for logistical reasons, safety pharmacology assessments are often incorporated into inhalation toxicology studies in rodent and non-rodent species, rather than performing separate studies. These regulatory drivers have largely offered an ‘instead of’ (cost-saving) rather than an ‘as well as’ (value-adding) approach, which does give some cause for concern, as will be addressed later. Nonetheless, at least they provide opportunities for toxicologists and safety pharmacologists to work together to achieve meaningful functional observations and measurements within the constraints of repeat-dose toxicity studies.

The only regulatory driver thus far for more extensive, higher quality functional assessments in repeat-dose toxicity studies in the pharmaceutical industry has been in the area of QT prolongation and torsade de pointes (see below). Although there has been less focus on other areas (haemodynamics, neurobehavioural, respiratory, renal and gastrointestinal), nonetheless there are growing concerns that subtle functional changes may lead to undesirable and unacceptable safety risks to patients (e.g., increased incidence of cardiovascular adverse events or elevated cardiovascular risk factors with drugs used to treat coronary heart disease, diabetes, obesity, arthritis, attention-deficit hyperactivity disorder, and psychiatric disorders).^16,66–71 Such liabilities only revealed themselves after chronic therapy, which therefore suggests missed opportunities to be addressed, where feasible, in repeat-dose toxicology studies.

In addition, certain specialised regulatory requirements include the incorporation of specific behavioural measurements. These include regulatory guidelines on post-natal development, requiring behavioural testing of first-generation offspring exposed to NCEs during gestation,^35,38 and juvenile toxicity studies, where neonatal and/or juvenile animals are dosed and assessed using behavioural tests.^36,37 The behavioural tests include a functional observational battery (FOB), locomotor activity, motor co-ordination (e.g., rotarod) and learning and memory tests. Yet although pharmaceutical toxicologists routinely apply such behavioural assessments for these specialised purposes, there is no inclination to include them in general toxicity studies (on sexually mature animals). This is a curious paradox in approach.

4. Challenges and opportunities on the road to change

The primary aim of a repeat-dose toxicity study is to expose animals to different dose levels of a test compound over a prolonged period, assess in-life indicators of systemic toxicity (Table 1), and finally, evaluate histological changes post-mortem. Any additional functional measurements should not and must not interfere with these aims or affect their outcome. In addition, the study design and laboratory conditions may not be optimal in terms of obtaining high-quality functional data. However, whereas the ideal aim is accurate measurement, sometimes we may have to settle for ‘quantitative detection’.

Comparing and contrasting toxicology and safety pharmacology studies gives some insight into potential operational differences (Table 3). Achieving integration of meaningful functional assessments into toxicology studies requires certain compromises and adjustments. For example, depending on the study type and functional measurements, it may require a staggered start date (to enable measurements across all the animals in the main study group, e.g., spread over two consecutive days), staggered dosing on the day of functional assessments (to enable measurements at the T_max time point in each individual animal), and so on.

Table 3 Differences in emphasis and operational paradigms between general toxicology and safety pharmacology studies

	General toxicology	Safety pharmacology^72
a Type A: dose-dependent; predictable from primary, secondary and safety pharmacology; Type B: idiosyncratic response, not predictable, not dose-related; Type C: long-term adaptive changes; Type D: delayed effects, e.g. carcinogenicity, teratogenicity; Type E: rebound effects following discontinuation of therapy (see ref. 72). AUC: area under curve; TK: toxicokinetics; ANOVA: analysis of variance.
GLP	Yes, inviolable for regulatory studies; not required for DRF/MTD studies	Yes (for core battery studies), although some software may not be fully GLP-compliant; also, non-core battery studies need only to be conducted to GLP ‘to the greatest extent feasible’
Primary adverse effect type predicted^a	Types C–E (B)^71	Type A^71
Primary endpoints	Gross clinical signs; histopathology. Studies scheduled to accommodate necropsy slots	Functional responses/effects. No necropsy to consider
Dosing regimen	Chronic, repeat-dose; animals dosed all in one session (usually a.m.)	Single dose (usually); dosing staggered to accommodate functional measurements
Dose or exposure level	Lowest dose tested can be much larger than the anticipated ‘therapeutic’ dose. Highest dose usually the MTD or maximum dose of (usually) 1 g kg^−1	Lowest dose tested is around the ‘therapeutic’ dose level; highest dose may be the single-dose MTD
Cardinal exposure parameter	AUC; TK sampling takes priority	C max TK sample taken after key functional measurements
Sex of animals	Males and females	Usually males
Age of animals	Sexually mature animals used	Sexually mature nonrodents used, but behavioural studies usually require young rats
Strain of animals	Usually restricted to standard strains	May sometimes require non-standard strains (e.g. pigmented rats)
Training/habituation	Variable	Some functional measurements may require pre-training and/or habituation of animals
Facilities	Often busy, noisy environments. May be difficult to accommodate bulky equipment or to adjust lighting levels etc.	Functional measurements require a quiet room; usually conducted in custom-designed facilities with lighting controls etc.
Basis for risk assessment	NOEL, NOAEL	Margins
Statistical analysis	Trend tests; group sizes adequate to detect histopathological effects	Within-animal pair-wise comparisons of means or ANOVA compared to vehicle controls; studies powered to detect the functional effect
Study design	Established discipline	Continuously evolving discipline
Staff education/expertise	Toxicology; biochemistry; molecular biology; veterinary	Pharmacology; physiology

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Ideally, areas assigned for collecting functional data should be located remote from corridor noise (e.g., trundling of cage racks etc.; loud conversations), and not in an anteroom where other activities are occurring, and/or which technical staff have to walk through to access study animals. Entry to this dedicated quiet area during data collection should be restricted to staff involved in the measurements, with noise kept to a minimum in adjacent areas. Staff requiring access to the other animals on the study should do so without disturbing the functional observations/measurements. There should be sufficient space to accommodate bulky test equipment, positioned safely and ergonomically. It is also desirable that the lighting control has local (manual) override.

An important scientific issue is the phenomenon of tolerance to the effects of repeat-dosing. This can occur with many classes of drug, and in various organ systems. As Haefely (1986)⁷³ elegantly put it: “Some form of adaptive syndrome is the inevitable consequence of the reciprocal interaction between most or all classes of drugs and the organism”. This poses something of a problem. The first day of dosing (Day 1) of a repeat-dose toxicity study is generally the busiest one for the staff, it is already fully-loaded with repeated toxicokinetic blood sampling at specific time points, and the procedure rooms are relatively crowded with the toxicology technicians, study director, Quality Assurance auditor (in GLP studies), and even the sponsor's monitoring scientist (in the case of a study run at a CRO). This is really a day to avoid for quiet and careful measurement of physiological and behavioural parameters. But to opt for (say) Day 2 instead may miss the peak effect of first administration, which may have faded or changed entirely on second administration. This is not an issue when functional measurements in repeat-dose toxicity studies are ‘in addition to’ single-dose safety pharmacology studies, but it could be when they are run ‘instead of’.

5. Criteria for suitable methodology

Ideally, methods used to assess functional endpoints in repeat-dose toxicity studies should be noninvasive, non-stressful, not require anaesthesia, and not require food restriction. Otherwise, satellite groups should be used, or even a separate, repeat-dose safety pharmacology study performed. Equipment needs to be portable and relatively non-bulky: ideally, the equipment should be moved to where the study animals are housed and not the other way round. Alternatively the equipment can be an integral part of the facilities, and purposely dedicated to measurements (e.g., noninvasive telemetry hardware). Measurements should be conducted by experienced and trained scientists and technicians fully au fait with how to obtain good functional measurements, and in collecting, analysing and interpreting data. This is easier to achieve if safety pharmacology and general toxicology departments are in close proximity, with the safety pharmacology staff incorporated within the GLP Compliance Programme. The design of the study should be discussed with a statistician ahead of the study where possible, particularly with respect to statistical power and any requirement for pre-study measurements. Toxicology studies may be underpowered relative to standalone safety pharmacology studies to detect some functional changes and are likely to include equal numbers of males and females. For example, sensitivity of detection of changes in QT interval using conventional (manual) ECG in toxicology studies in dogs is lower than in telemetered dog safety pharmacology studies.^74–76 However, the introduction of noninvasive telemetry systems has enabled a significant improvement in the sensitivity of toxicology studies in relation to detection of QTc changes. Ideally, data recordings should be at the same time(s) post-dose for each animal, at roughly the same time of day throughout the study, and should (where technically possible) collect data over a 24 h period. Techniques used for long-term monitoring need to be suitable for use in group-housed (or pair-housed) animals; certainly in the EU, social housing will become a welfare requirement.⁷⁷ Finally, apart from certain non-compliant data acquisition software systems, there is no reason why GLP compliance cannot be claimed for this aspect of the study.

Examples of suitable and less suitable methods for application to main study animals in repeat-dose toxicity studies are given in Tables 4 and 5, respectively.

Table 4 Examples of functional assessments suitable for incorporation into repeat-dose toxicology studies^a

Organ system/function	Technique/method	Species
a The techniques listed are generally either noninvasive or minimally invasive, and cause minimal impact on the animal and on the conduct and design of the toxicology study. Where possible, references have been selected which discuss the method in the context of safety assessment (safety pharmacology or toxicology) or cover multiple species or techniques.
Cardiovascular system
ECG interval durations, amplitude and morphology	Conventional ‘snapshot’ recordings in restrained animals	Dog; monkey; minipig^75,79–84
ECG interval durations, amplitude and morphology	Surface electrodes using noninvasive telemetry in freely moving animals	Dog; monkey; minipig^85–88
Left ventricular function	Echocardiography	Dog; rat; minipig^89–94
Arterial blood pressure	Tail-cuff (restrained)	Dog; rat^95,96
Arterial blood pressure	Ambulatory tail-cuff (telemetry)	Dog^88,97
Nervous system
Global neurobehavioural assessment	Functional observational battery or Irwin test	Rat; mouse; dog; monkey^52,98–108
Ambulatory activity (home cage)	RFID microchip transponder	Rat; mouse^109
Ambulatory activity (novel arena)	Locomotor activity (photocell beam breaks or videotracking)	Rat; mouse^100,104,108,110
Motor coordination	Accelerating rotarod; beam walking; gait analysis	Rat^55,104,111
Cognitive function	Avoidance paradigms (brief footshock stimulus on a single occasion)	Rat; mouse^104,112,113
Auditory function	Brainstem auditory evoked response	Dog^114
Auditory function	Pre-pulse modulation of startle reflex; startle stimulus-response curves	Rat; mouse^54,104,115–118
Visual acuity	Optomotor reflex	Rat; mouse^54,119
Iris control	Pupil diameter; pupillary reflex response to light stimulus	Rat; dog^120
Nociception	Tail flick latency	Rat; mouse^100,104
Neuromuscular	Grip strength	Rat^100
Salivation	Absorption into pre-weighed gauze	Dog^121
Respiratory system
Respiration rate, inspiratory and expiratory times, tidal volume, minute volume, peak inspiratory and expiratory flows	Whole-body plethysmography	Rat; mouse^122
(Ditto)	Inductive plethysmography	Dog; monkey^87,97,123,124
Renal system
Water intake, urine volume, urinary excretion of key electrolytes (Na^+, K^+, Cl^−). Estimated GFR and fractional excretion of electrolytes	Urine collection in metabolic cages	Rat^29,125
Gastrointestinal system
General assessment	Faeces weight, consistency, appearance	Rat; Dog^126
Gastric emptying time; intestinal transit time; intestinal pressures	Telemetry capsule (e.g., SmartPill™; Bravo™)	Dog^127,128
Hepatic system
Bile acid analysis	Recoverable, swallowed thread	Dog^129
General metabolic functions
Rectal temperature	Rectal temperature probe (thermocouple or thermistor)	Rat; mouse; dog^130
Interscapular temperature	RFID microchip transponder	Rat; mouse^131–133
Glycaemic control	Glucose tolerance test (serial blood microsampling)	Rat; mouse; dog; monkey; minipig^134–139
Mitochondrial function	Blood glucose and lactate	Rat; mouse; dog; monkey; minipig^140–143
Oxygen consumption	Whole-body indirect calorimetry	Rat; mouse^144

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Table 5 Examples of functional assessments less suited for incorporation into repeat-dose toxicology studies on the main study animals^a

Organ system/function	Technique/method	Species	Reason for reduced suitability	Comments
a The techniques listed generally require either surgical implantation pre-study, or restraint, or general anaesthesia, or extensive pre-study training, or restricted feeding (for food reward operant paradigms), and therefore impact on the animal and on the conduct and design of the toxicology study. They are generally more suited to standalone repeat-dose safety pharmacology studies. Where possible, references have been selected which discuss the method in the context of safety assessment (safety pharmacology or toxicology) or cover multiple species or techniques.
Cardiovascular system
ECG; left ventricular function; arterial blood pressure	Surgically implanted telemetry	Dog; monkey; minipig^145	Expensive; surgery required; pathology associated with surgery sites and/or detached emboli	May be cost-effective/ethically acceptable in studies of >1 month duration
Nervous system
Retinal function	Electroretinogram	Rat; mouse; dog; monkey^146	Anaesthesia required; corneal abrasion may occur; low throughput; requires pigmented rats	Preferable as standalone study or on satellite animals
Nerve conduction velocity	Recordings from caudal nerve	Rat^100,147	Anaesthesia required	Preferable as standalone study or on satellite animals
Cognitive function	Morris watermaze	Rat; mouse^112,113	Swimming-induced stress; preferable to use pigmented animals; bulky test equipment; low throughput	Preferable as standalone study or on satellite animals
Cognitive function	Food-baited mazes	Rat; mouse^112,113	Requires daily food restriction to provide motivation to work for food reward; bulky test equipment; low throughput	Preferable as standalone study or on satellite animals
Cognitive function	Operant paradigms	Rat; mouse^112,113	Requires daily food restriction to provide motivation to work for food reward; large rack of wired-up test chambers; requires extensive pre-training	Preferable as standalone study or on satellite animals
Respiratory system
Respiration rate, inspiratory and expiratory times, tidal volume, minute volume, peak inspiratory and expiratory flows	Face mask	Dog^148	Requires habituation	Preferable as standalone study or on satellite animals
(Ditto)	Head-out plethysmography	Rat; mouse^149–152	Requires habituation; restraint is stressful	Preferable as standalone study or on satellite animals
Flow-volume plots, lung resistance, dynamic compliance	Forced manoeuvres	Rat^149–151,153	Requires terminal anaesthesia (tracheal cannulation); low throughput	Requires standalone study
Renal system
GFR	Clearance of intravenously administered marker	Rat; mouse; dog; monkey; minipig^154–156	Requires administration of an ‘inert’ marker compound; serial blood sampling; plasma analysis	Marker may not be toxicologically inert; microsampling may suffice for rodents
Gastrointestinal system
Gastric acid secretion	Pyloric catheterization (Shay model)	Rat^157	Surgery required; pathology associated with surgery sites	Rarely used in safety studies
Gastric emptying	Gamma scintigraphy	Rat; mouse; dog; monkey; minipig^158	Requires anaesthesia	Anaesthetic may affect gastric emptying time; radioactivity
Gastric emptying	PK-based model	Dog^159	Requires administration of two additional compounds; serial blood sampling; plasma analysis	PK marker compounds may not be toxicologically inert/may be drug–drug interactions
Gastrointestinal transit	Charcoal meal (time to appearance in faeces)	Mouse; rat^160	Possible adsorption of test compound onto the administered charcoal
Hepatic system
Bile collection	Bile duct cannulation	Rat; dog; monkey^161–163	Surgery required; pathology associated with surgery sites; possible effects on drug metabolism and excretion	Use declining
General metabolic functions
Core temperature	Intraperitoneal telemetry implant	Rat; mouse^164	Surgery required; pathology associated with surgery sites	Preferable as standalone study or on satellite animals

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6. Application to specific organ functions

The five organ functions assessed in safety pharmacology are the cardiovascular system, the nervous system, the respiratory system, the gastrointestinal system and the renal system. To these we would add the liver, as well as general systemic metabolic functions, as both are amenable to collection of at least some biochemical functional data. Examples of techniques used in these studies are given below, illustrated with data where available from our in-house studies as well as from published literature.

6a. Cardiovascular system

(i) ECG intervals and morphology. Concerned by the increasing number of marketed drugs linked to torsade de pointes, a rare but potentially lethal ventricular arrhythmia, in 1996 the European Committee on Safety of Medicinal Products (CPMP) issued a ‘Points to Consider’ document outlining guidance on detecting this liability preclinically and clinically.⁷⁸ Prior to release of this document, whereas some pharmaceutical companies conducted carefully designed safety pharmacology studies to assess effects on ventricular repolarisation, by measuring changes in the QT interval, others did not, relying on snapshot measurements of QT interval taken from dogs (or monkeys) in repeat-dose toxicity studies. Often these measurements were suboptimal, in terms of signal quality, baseline variability, elevated heart rate, statistical power, and improper use of correction factors.⁷⁴ The CPMP document had a profound effect on the standards applied to the task of detecting effects on ventricular repolarisation in dogs, and considerable advances have subsequently been made in this area.^165,166 It is important that these standards are not allowed to slip in the pursuit of efficiency in incorporating functional endpoints into repeat-dose toxicity studies, leading to history repeating itself.

Acquiring ‘snapshot’ ECG recordings by surface ECG electrodes requires the use of calm animals, both to reduce movement artefacts and to provide optimal conditions for detecting drug-induced QT prolongation, namely, low resting heart rates. This is because of the phenomenon of ‘reverse-use dependence’ (or ‘reverse-rate dependence’) in interaction with the hERG K⁺ ion channel, whereby the channel is more susceptible to drug-induced blockade during the resting state (i.e., during diastole).¹⁶⁷ Low resting heart rates are achieved by habituation of the animals to the recording procedure. In the case of dogs, some authors recommend recording ECG from dogs in a lateral recumbent position,⁷⁹ whereas lead II ECG recordings adequate for measurement of intervals can be obtained from dogs in a standing position, either lightly restrained by a technician or in a sling.⁷⁴ In the case of cynomolgus monkeys, they are either restrained in a chair, manually restrained in dorsal recumbency, or sedated, to enable recording of lead II ECG.^81–84 Even with habituation, physical restraint elevates heart rate in monkeys, which can make it difficult to accurately measure the end of the T-wave if it merges into the subsequent P-wave,¹⁶⁸ and tachycardia reduces the likelihood of detecting drug-induced QT prolongation (see above). In minipigs, ECG recordings are taken during sling restraint.¹⁶⁹ Such measurements are made pre-study, pre-dose, and at pre-determined time points post-dose. Nowadays, data acquisition software is used to capture runs of ECG waveforms, and automatically place markers on the waveforms to measure PR interval, QRS duration, and QT interval. However, experienced cardiovascular safety scientists (physiologists, pharmacologists, or veterinarians) are required to check that the markers have been placed correctly by the software and to identify abnormal ECG morphology and arrhythmias.¹⁷⁰

In recent years, jacketed, noninvasive, ambulatory telemetry systems have become commercially available for use in dogs and primates.^85–88 These enable continuous recordings of lead II ECG from surface electrodes for at least 24 hours in undisturbed, freely-moving animals in their home environment, when awake or asleep. The same requirements for access to cardiovascular expertise apply as for conventional ECG recordings. In addition, a detailed knowledge and understanding of the daily routines and activities within the animal housing unit (e.g., feeding times; presence of technicians; husbandry activities; etc.) is required in order to design appropriate studies, and make intelligent interpretations of the data. Data quality obtained from jacketed telemetry in beagle dogs is superior to that obtained conventionally, including achieving lower resting heart rates (Fig. 4) and there is more of it. This approach enables determination of onset, time course, duration, magnitude and reversibility of effects as well as dose-dependence and PK–PD relationships. Plots of QT against RR interval circumvent the inaccuracies associated with QT correction factors, and can reveal drug-induced shifts across the spectrum of heart rates.¹⁴⁵ In contrast to detecting effects on the QT interval, sampling during naturally-occurring episodes of elevated heart rates facilitates detection of drug-induced QRS prolongation by sodium channel blockers,¹⁷¹ as in contrast to the hERG potassium channel, sodium channel block is use-dependent (i.e., rate-dependent).¹⁶⁷ Detection of cardiac arrhythmias is more likely over a 24 h sampling period than with snapshot ECGs, as is the detection of pre-existing waveform abnormalities pre-study.^81,172,173 The impact on resources of using a jacketed telemetry system is minimal (Fig. 5), although it is acknowledged that an initial significant capital investment is required. When used to complement standalone, single-dose safety pharmacology studies in dogs with surgically implanted telemetry devices, noninvasive ECG measurements can sometimes reveal effects that intensify on repeated dosing (Fig. 2).

Fig. 4 Pre-study values of heart rate and QTcV obtained in dogs by surgically implanted telemetry (‘the ‘gold standard’; n = 129 recordings from 61 studies), conventional ‘snapshot’ ECG measurements (with the dog restrained in a sling; n = 760 recordings from 29 studies), and by jacketed external telemetry (n = 211 recordings from 19 studies). Data are means ± standard deviation. Note the elevated heart rates in the ‘snapshot’ ECG dataset, and the slightly larger standard deviations for QTcV and heart rate using this method. (In-house data.)

Fig. 5 Time estimates for pre-study habituation of dogs, acquisition of ECG data, data analysis (ECG morphology and intervals), and data expert review, using ‘single snapshot’ conventional manual ECG measurements (with the dog restrained in a sling), ‘double snapshot’ ECG measurements collected the same way, or jacketed external telemetry. (In-house data.)

(ii) Left ventricular function. The use of echocardiography in unanaesthetised dogs is a well-established technique.⁸⁹ Left ventricular function can be monitored using echocardiography in unsedated, pre-trained, sling-restrained (or laterally recumbent) dogs^90–93 and minipigs.⁹⁴ Echocardiography can also be used in cynomolgus monkeys, but they require sedation¹⁷⁴ or anaesthesia.¹⁷⁵ In beagle dogs, good concordance is observed between left ventricular ejection fraction (LVEF) derived by echocardiography, and left ventricular dP/dt_max measured in the same animals by implanted telemetry.^92,93

(iii) Arterial blood pressure. In rodents, indirect (i.e., noninvasive) methods of blood pressure measurement utilise a tail-cuff method. However, they can only reliably measure systolic blood pressure and heart rate, require pre-warming and restraint to obtain the measurements, and even after pre-study habituation to these recording conditions, the values of systolic blood pressure and heart rate obtained are elevated compared to unrestrained animals with implanted telemetry devices.⁹⁵ Recent improvements in tail-cuff blood pressure methodology are a promising development,⁹⁶ but even so, such a method requires highly skilled operators and a dedicated, quiet environment. Given that regulatory repeat-dose toxicity studies are carried out in two species, it would seem sensible to run cardiovascular assessments in the larger, non-rodent species, although cardiovascular measurements in rodents may be informative in non-regulatory, DRF studies, especially given the small group sizes used in non-rodent DRF studies. The tail-cuff blood pressure method itself is also well established in dogs,^176,177 and the recent introduction of high-definition oscillometry (HDO) enables capture of the blood pressure waveform, with accurate measurements of systolic, diastolic and mean arterial blood pressures.¹⁷⁸ HDO has also been used in cynomolgus monkeys.^{177,179–182}

Ambulatory tail-cuff methods are now available for dogs, combining simultaneous ECG and blood pressure measurements in the same animals by noninvasive telemetry.^88,97,182 An alternative approach involves a minimally invasive measurement of blood pressure in dogs or cynomolgus monkeys via a catheter implanted in a relatively superficial small artery, e.g., using a miniature (rodent) telemetry transmitter. Signals are registered either with the dog in a sling, or by using a signal booster housed in a collar, or in a jacket (e.g., ECG external telemetry jacket),¹⁸³ to enable continuous 24 hour recordings in freely-moving animals.

6b. Nervous system

Adverse effects on the central and peripheral nervous system (including special senses) are commonly encountered throughout clinical development and post-marketing approval, and make a significant contribution to drug attrition, labelling, and patient compliance.^10,11 Standard approaches to detect drug-induced neurotoxicity (functional and pathological) are limited to a single-dose neurobehavioural assessment as part of the safety pharmacology core battery,¹¹³ and 3–4 coronal sections through the entire neuraxis for histopathology.¹⁸⁴ Reliable blood-borne biomarkers of drug-induced neurotoxicity do not currently exist. This is therefore an area of relative neglect in preclinical toxicology evaluation, and so functional assessments in repeat-dose toxicity studies are worth considering. Technically, there is nothing preventing routine inclusion of global neurobehavioural assessments (by a trained observer) and automated activity monitoring in repeat-dose toxicity studies, and evaluation of specific neurobehavioural systems could be incorporated on a case-by-case basis.

(i) Global neurobehavioural assessment. Due to the extreme diversity and complexity of nervous system functions, it is not possible to devise a convenient in vivo assessment that incorporates all of them. Therefore, in safety pharmacology the first-tier test used is a neurobehavioural assessment, either in the format of the FOB or Irwin test.^{50,52,98,101,108,113} Similar assessments have been devised for dogs^103,105,106 and monkeys.^107,185,186

None of the individual components within the FOB or Irwin test are definitive for the variable being measured, in that they are relatively crude and the tests incorporated are a compromise between detection sensitivity and practicality (speed). Some potential adverse effects on the nervous system are not addressed at all, including cognitive functions and special senses. Where there is specific cause for concern arising from the knowledge of a compound (e.g., primary pharmacology, secondary pharmacological profile, chemical or pharmacological class, disease indication, competitor compounds, literature), appropriate, singular tests can be incorporated into a repeat-dose toxicity study on a case-by-case basis, to address the issue. Examples of suitable tests are listed in Table 4.

(ii) Automated activity analysis. Activity of rodents can be assessed in two ways: home cage activity, or activity in a novel enclosure (spontaneous locomotor activity). These are quite different measures. The latter involves a conflict between the motivation to explore, and raised anxiety in unfamiliar surroundings. It can be monitored using either arrays of photocell beams (with a second set positioned to detect rearing), or by videotracking.^{108,110,187,188} Ambulatory activity and rearing in a novel environment is initially high and gradually decays over 30 minutes or so, as the animal explores and habituates to its surroundings, eventually spending the majority of time either stationary or grooming.¹¹³ Subsequent exposure to the same (or similar) test arena results in inter-trial habituation, with reduced activity after the first trial, limiting its suitability for longitudinal testing in a repeat-dose toxicity study. Home cage activity does not have the conflict or habituation components, providing a reproducible 24 h cycle of activity for each individual. Both types of activity are affected by time of day, lighting level, extraneous noise, and the presence of human observers. A sedative effect of a test compound would be difficult to detect on home cage activity of rodents during the light phase, as this is already very low. Nonetheless, in a repeat-dose toxicity study, the most convenient format to use would be home cage activity. Until fairly recently, this has required single-housing of animals,¹⁸⁹ or surgical implantation of telemetry devices,¹⁹⁰ neither of which is ideal for a repeat-dose toxicity study.

However, technology is emerging for measuring individual physiological parameters and behaviours in group-housed rodents in their home cage, without resorting to surgical implantation of telemetry transmitters. Techniques exist for measuring individual ambulatory activity in group-housed rodents in their home cage, using a radiofrequency identity microchip transponder (‘RFID chip’) implanted by subcutaneous injection, detected by a floor plate reader placed under the cage (TSE ‘TraffiCage’).¹⁰⁹ It is also possible to monitor individual food and water consumption in group-housed rodents in the same way.¹⁹¹ An initiative is also currently underway under the ‘CRACK-IT’ scheme, funded by the UK National Centre for the 3Rs, to monitor individual ambulatory activity and body temperature, and to detect convulsions, in group-housed rodents in repeat-dose toxicity studies.¹⁹² Such a methodology could also be useful in detecting changes in activity and body temperature occurring during withdrawal from treatment with drugs carrying a physical dependence liability,¹⁹³ if applied to the recovery groups in a rat one-month study.

Activity of dogs¹⁹⁴ and monkeys^195,196 can be conveniently measured in their home enclosures using an accelerometer placed in a collar, or as an integral part of a noninvasive telemetry system (e.g., EMKA Technologies; VivoMetrics LifeShirt®; DSI JET™). Recently, quantitative videotracking of group-housed cynomolgus monkeys has been achieved using differently coloured jackets.¹⁹⁷

All such methods are suitable for inclusion in repeat-dose toxicity studies.

(iii) Tests on specific neurobehavioural systems. Examples of ‘singular tests’ incorporated into repeat-dose toxicity studies in our laboratories include evaluation of the risk of (a) visual dysfunction, assessed using an optomotor method in rodents;^54,119 (b) mydriasis and impaired pupillary light reflex, assessed by measuring pupil diameter in rodents and dogs;¹⁰¹ (c) increased or decreased salivation in dogs, measured using a pre-weighed gauze swab placed in the jowl (Fig. 6) (see also Bagheri et al., 1992¹²¹); (d) impaired muscle function, assessed by accelerating rotarod performance in rats (Fig. 3) (see also Nodop Mazurek et al., 2001⁵⁵); (e) peripheral neuropathy, assessed by the same method in rats, or using a beam walking task.¹⁹⁸ Each of these assessments significantly contributed to decision-making in the projects, either in terms of compound progression, design of Phase I clinical trials or development of risk management and mitigation plans.

Fig. 6 Excessive salivation detected on Day 2 (first recording occasion) but not on subsequent occasions post-dose in beagle dogs. Salivation was quantified by placing a pre-weighed gauze swab inside a jowl for 20 s, which was then removed and re-weighed. Open symbols: vehicle-treated, n = 12; filled symbols: high dose group, n = 12. **P < 0.01 vs. vehicle control group.

6c. Respiratory system

In safety pharmacology, the respiratory system is one of three classified by ICH S7A as ‘immediately vital for life’, and assessment of effects of candidate drugs on rate and depth of breathing is mandated by the ICH S7A guidelines.¹⁹⁹ These parameters can easily be acquired from rodents (usually rats) using whole-body plethysmography, a noninvasive, non-restraint method that measures small changes in chamber pressure as a consequence of inspiration and expiration.¹⁵⁰ Standalone safety pharmacology studies using whole-body plethysmography typically involve a 60 to 90 minute settling period prior to compound administration to ensure a steady respiratory signal and to reduce any artefacts in the signal due to ambulatory movement, sniffing, grooming or rearing behaviours. Post-dose recording usually continues for up to 6 hours. The technique requires air flow through the chambers (either via a plumbed-in air supply to the room, or from compressed air cylinders) and must employ appropriate temperature and humidity compensation to ensure an accurate determination of tidal volume.²⁰⁰ Application of this technique into repeat-dose studies is feasible²⁰¹ but, depending on the number of chambers recorded from simultaneously, will generally require a staggered start date for the study to accommodate 4 hour recordings from 32 rats (vehicle plus three dose levels), split across (say) 2 consecutive days. For early toxicology studies (e.g., DRF) a condensed protocol can be used, with recording around the T_max only and comparing just the high dose and vehicle control group, enabling detection of potential respiratory ‘flags’ for a particular compound with all the animals tested within a single 2–3 h session. For this, rats are placed in the chambers for 1 h, with the first 45 minutes as habituation, and the final 15 minutes for recordings, timed to be around the T_max.²⁰²

Other technological advances enable measurement of respiratory parameters in dogs using a non-restraint, jacketed telemetry method. This technique is known as respiratory inductive plethysmography²⁰³ and involves the placement of flexible belts around the thorax and abdomen, enabling continuous measurement of respiratory parameters for up to 24 hours.¹²³ This noninvasive telemetry technique also enables simultaneous measurement of cardiovascular parameters⁹⁷ and can also be adapted to non-human primates,^87,124 making it an attractive proposition for inclusion in repeat-dose toxicity studies.

6d. Gastrointestinal system

The majority of drug candidates are administered orally, and therefore the gastrointestinal tract (particularly the stomach) is exposed to a far higher concentration of drug than any other organ system. As a result of this, the incidence of gastrointestinal side effects is relatively high throughout clinical development and post-marketing.¹⁰

Useful data can be obtained inexpensively simply by analysing faecal pellets in rodents, in terms of number, consistency and colour. This is ideally best performed when animals are singly housed in metabolic cages (as is usual practice towards the end of a repeat-dose investigation to assess renal function) or in whole-body plethysmography chambers.¹²⁶ Though not a quantitative approach, large changes in gastrointestinal function should be detectable, which would be valuable in an early toxicology study (e.g., DRF study).

A simple, non-lethal, charcoal meal method, which measures the rate of gastrointestinal transit, has been described for use in mice and is suitable for inclusion in toxicology studies.¹⁶⁰ Food is withdrawn 3 hours pre-dose, and mice are dosed with test compound. An aqueous suspension of 5% charcoal is administered by oral gavage, and food is restored 1 h later. Animals are observed at 5 min intervals until the first appearance of charcoal-containing faeces, which is taken as the gastrointestinal transit time. Potential issues with this method include the variability of the data, and adsorption of some drugs onto the charcoal. An elegant method has been described in rats using a small (0.85 × 1.5 mm), coated, magnetic dipole administered by oral gavage. The rat is then placed in a restraining tube on a sensor matrix which tracks the progression of the magnet through the gastrointestinal tract during a series of 20 minute recordings (Motilis MTS-1²⁰⁴).

Dogs have been used extensively as an animal model of gastric and intestinal function in humans in the published literature, but to a much lesser extent in toxicology. The non-surgical methods used have included gamma scintigraphy, radiographic imaging using radio-opaque material, tracer studies using breath analysis, administration of a telemetric capsule, and pharmacokinetic measurement of drug markers in plasma.^158,159 Of these, telemetric capsules are probably the most suitable for use in toxicology studies (Table 4). SmartPill™ technology, marketed for human use, has been used in dogs to assess adverse effects on gastrointestinal function.^127,205,206 This is a small transmitter which is dosed orally to dogs and measures temperature, pressure and pH. Another similar transmitter (Bravo™; Medtronics Inc.) has been described for use in dogs, which merely measures pH.¹²⁸ Both these measure gastric emptying time, from the sudden increase in pH when exiting the stomach into the duodenum. Measurement of intestinal transit requires the measurement of temperature or pressure, to detect excretion in the faeces. A current problem with capsule methods in the dog is that the relatively large capsule size prolongs the time it takes to exit the stomach into the duodenum, thereby reporting abnormally long gastric emptying times; another, more practical issue is re-ingestion by the dog (or by its companion) post-defaecation.

Other approaches are suitable only for specific components of gastrointestinal function. For the gastric emptying component per se, an alternative to capsules has been described recently, using ultrasonography in unsedated, gently restrained beagle dogs. This merely requires measurements of the amplitude and frequency of antral contractions, collected in 3 minute samples at 30 minute intervals.²⁰⁷ For measuring gastric pH, samples of stomach fluid can be aspirated using a catheter within (say) a gavage tube in dogs, and the pH measured with a pH meter.²⁰⁸

6e. Renal system

The kidneys are crucial for a variety of homeostatic functions that are susceptible to the effects of drugs. These include the regulation of body electrolyte levels, maintenance of acid–base balance, long-term regulation of blood pressure, removal of waste products and a significant endocrine function.²⁰⁹ Many of the homeostatic mechanisms that involve the kidney are complex and are also influenced by other non-renal factors. Consequently, introducing meaningful reliable functional endpoints for the majority of these functions into regulatory toxicity studies is a challenge. However urine, the kidney's principal excretory product, can easily be collected either from spontaneously-voiding, freely-moving rodents using metabolism cages (‘metabolic cages’ or ‘metabowls’) over a range of time scales up to 24 h, or in larger non-rodents, as catheterised bladder “spot” urine samples.^{20,29,125,210,211}

(i) Estimates of general excretory function. A number of parameters are currently included in most repeat-dose toxicity studies and these can give an indication of the general excretory function of the kidneys. These include urea and creatinine in the plasma, and the measurement of specific gravity or osmolarity of the urine. These are widely accepted, translatable, and the caveats around interpretation of each are widely recognised.²⁰ Cystatin C has been proposed as an additional plasma marker for renal function and it has gained traction in human medicine.²¹² In nonclinical species its predictive value appears similar to that of creatinine.²¹³ In urine, quantitative measures of urinary glucose and total protein (often normalised to creatinine concentration) can give an indication of the reabsorbative function of the renal tubule, although these must be interpreted in a holistic manner in conjunction with other findings and the recognised mechanism of action of any candidate drug. The rationale for the glucose measurement is that over 99% of filtered glucose is reabsorbed by the proximal tubule; although this process is saturable (by hyperglycaemia), if glycosuria occurs during normoglycaemia this may indicate functional impairment in the proximal tubule.²⁰

Additionally, other analytes such as the electrolytes, and pH, can be measured in urine with a view to detect perturbations. However interpretation of any changes remains problematic with no reliable association between functional observations and underlying pathologies in most instances. In most cases, alterations in excretion can be viewed as a normal response to maintain internal homeostasis.

(ii) Glomerular filtration rate (GFR). A long-standing and broadly-accepted measure of general renal function is the measurement of GFR, the rate at which fluid is filtered by the kidneys. GFR can be calculated using a wide number of techniques that employ variants of the equation:

GFR in humans is estimated by measuring the plasma clearance of an administered inert marker compound (e.g., inulin) that is excreted exclusively by the kidneys, and does not undergo further tubular reabsorption or secretion after glomerular filtration.²⁰ Intravenously administered clearance markers have been used in dogs (e.g., iohexol¹⁵⁵) and rats (e.g., inulin;¹⁵⁴ iodixanol¹⁵⁶). However, their use in regulatory toxicology studies could be criticised on the grounds of hypothetical concerns around their innate toxicity or drug–drug interactions.

Alternatively, in toxicology studies GFR can be estimated by measuring creatinine clearance rate, which is the volume of blood plasma that is cleared of creatinine per unit time.²⁰ Creatinine is produced naturally by muscle, and concentrations in blood remain broadly at steady state. It is freely filtered by the glomerulus, but because of additional active secretion by the peritubular capillaries, creatinine clearance slightly overestimates actual GFR. On the face of it, it would appear that it should be relatively straightforward to include estimates of GFR into repeat-dose toxicity studies. However, to ensure accurate estimations the error in measurement of any one of the individual components of the equation used must be rigorously minimised. Firstly, for urine flow (urine volume/time), all animals must have accurate measurements of the time spent on collection. Secondly the plasma concentrations of creatinine and electrolytes etc. should ideally reflect the mean concentration experienced during the collection period. This implies that blood samples should be taken immediately prior to and after the collection period to determine a mean, or possibly as an extrapolation from a single blood sample taken mid-way through collection. Clearly these stipulations, from a practical perspective, impede the integration of this assessment into toxicology studies.

It should also be noted that any changes in renal function may often be only temporary and reversible, with no long-term consequence for the animal. Not only does the kidney have a high functional reserve (approximately 75%), it is capable of profound regeneration and recovery as well as marked up-regulation of the function of individual nephrons.

6f. Liver

The liver has a wide number of specific functions that include amino acid and protein synthesis (including albumin and various coagulation factors) and degradation (i.e. haemoglobin), several roles in carbohydrate and lipid metabolism, bile production, hormone production (IGF-1, thrombopoietin, angiotensin), storage of multiple substances (i.e. iron, copper and Vitamins A, D and B12) as well as the detoxification (or toxification) of exogenous substances including drugs.^214,215 For the vast majority of drugs, the liver is central to their metabolic transformation prior to excretion. Multiple assessments of liver function are already included in one month toxicity studies on a routine basis and have been for many years, although often these are overlooked. These include urea, bilirubin, cholesterol, albumin, fibrinogen, glucose, total protein, alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), and triglycerides. However, despite this, late stage attrition due to liver safety concerns remains a major cause of compound failure.^8,10,11 The reasons for this failure to detect hepatotoxicity preclinically include issues such as a lack of specificity for liver function of the analytes (e.g. albumin, total bilirubin),^215,216 the large functional reserve in the liver,²¹⁶ and the often idiosyncratic nature of hepatotoxicity that only emerges with testing in large patient populations with diverse phenotypes.

With the exception of bilirubin, changes in liver function markers sampled as part of repeat-dose toxicity studies are not seen as primarily indicative of altered hepatic function, due to numerous caveats.²¹⁶ Other aspects of altered liver function can only be studied post-mortem; for example, detection of cytochrome P450 enzyme induction is currently assessed by enzyme-linked immunosorbent assay (ELISA) on liver microsomes prepared from fresh liver tissue taken at necropsy. Whilst acknowledging that it is not (currently) possible to study the entire gamut of liver functions noninvasively in vivo, we should still look for opportunities where they exist. A specific example is in assessing effects on bile acid composition.

(i) Bile acids and bile acid profiling. The bile acids are a family of cholesterol-derived anionic acids that undergo highly efficient enterohepatic circulation. Primary bile acids are formed in the hepatocytes and excreted (conjugated) in the bile. Following gall-bladder contractions the bile acids enter the duodenum via the bile duct, whereupon they can be deconjugated or degraded. This is followed by either reabsorption into the portal circulation or excretion in the stool. In health, nearly all bile acids that enter the portal system are removed from the circulation by the liver. Consequently, levels in the general circulation are low. Therefore, in fasted animals, increases in bile acids can be associated with decreased functional hepatic mass, reduced portal blood flow to the liver, or choleostasis. Assays for total bile acids are widely available and well validated and can be easily included into toxicology studies. They are used infrequently in clinical trials as they are not perceived to have value over existing assessments of hepatic function, although we would argue that in rodent and dog studies of a limited duration, such a sensitive test for hepatic function has a place.

Additionally, enhanced techniques have been described to quantify bile acid species using mass spectrometry.^217,218 Here bile, either collected at necropsy or from plasma samples, is profiled. Primary bile acid profiles could act as early indicators of toxicity in drug induced liver injury, and the profile may vary in a predictable manner following hepatic oxidative stress.²¹⁹ A noninvasive sampling method for intestinal bile secretions has recently described in dogs,¹²⁹ based on the Entero-Test method used as a diagnostic tool in humans.²²⁰ The method involves the dog swallowing a thread attached to its neck collar; after a suitable period to enable passage of the thread tip into the duodenum, the thread is subsequently withdrawn and analysed.¹²⁹ This would be suitable for repeat-dose toxicity studies, and is a 3Rs refinement of the bile duct cannulation method of bile collection, which also impacts on liver function (e.g., down-regulation of CYP3A2¹⁶³), and on the biliary excretion and metabolism of some drugs by gut microflora.¹⁶²

6g. Body temperature: general metabolic functions

One physiological variable of fundamental importance is body temperature. This is not measured in toxicology studies, but may be inferred, correctly or incorrectly, by clinical observations such as ‘piloerection’ and ‘cold extremities’. Approximately 26% of non-CNS targeted test compounds were found to cause a small (∼1 °C) decrease in rectal temperature in the rat in safety pharmacology studies at the MTD dose level,¹⁰¹ which is thought to be an adaptive response to ingestion of a toxic agent (reducing cellular toxicity by reducing body temperature^130,164). However, larger decreases in temperature may reflect either a more profound toxicity, a specific pharmacological effect on central thermoregulatory control, impaired metabolic heat production, vasodilatory heat loss, or a physiological response to hypoglycaemia²²¹ or hypoxia (anapyrexia).²²² In addition, mice (unlike rats) undergo bouts of torpor when food is unavailable (e.g., during protocol-imposed overnight fasts), whereby they enter a hypometabolic state and a lowered core temperature.²²³ This can also be driven by pharmacological and toxicological mechanisms.²²³ Conversely, increases in temperature in rodent or non-rodent species may reveal other toxicological mechanisms, including skeletal muscle toxicity or an immunological response.²²⁴ Collection of temperature data in repeat-dose toxicity studies is therefore extremely informative.

The use of a rectal thermocouple carries a potential risk of physical damage to the rectal wall, transfer of any gastrointestinal infections between individuals, and contamination of vehicle control animals with traces of test compound from treated animals. In addition, core temperature increases as a result of handling,²²⁵ and rectal temperature underestimates true core temperature, for which measurement from the colon is required in the rat (i.e., 6–8 cm past the anal sphincter²²⁵).

A completely noninvasive approach is achievable using infrared thermography. Modern thermal imaging cameras (such as the FLIR ThermacCAM™ SC640) offer dual visual and thermal image capture, with fine image resolution, and can capture accurate regional surface temperatures from freely-moving rodents, such as the temperature gradient from base to tip of the tail.^132,133,226 However, to measure core temperature the angle of view has to be aligned with the axis of the ear canal (which is equivalent to core temperature at normal laboratory housing temperatures), and skin temperature measurement requires shaving (and re-shaving) of fur. This might be required, for example, to reveal the skin overlying the interscapular brown adipose tissue (iBAT), a major source of adaptive non-shivering thermogenesis in rodents.²²⁵ Thermal imaging is invaluable for certain bespoke, problem-solving studies, but it requires a skilled operator, and data extraction is relatively time consuming, so it is less suited for routine use.

As mentioned previously, RFID transponders exist which measure temperature and are small enough to be injected subcutaneously in rats and mice.^131–133 Temperature is measured with a hand-held wand receiver from freely-moving animals, without contact (e.g., through the wall of the home cage). When injected subcutaneously into the interscapular region, the RFID chip is measuring temperature above the iBAT. At normal laboratory housing temperatures, this temperature is slightly higher than rectal temperature in the rat,¹³² rectal temperature being slightly lower than core temperature in this species.²²⁵ Drug-induced changes in thermoregulation generally result in the iBAT temperature mirroring changes in rectal temperature.¹³³ Although some drugs will lower core temperature by vasodilatory heat loss, with the iBAT temperature being maintained and therefore not reporting hypothermia, for most purposes in toxicology the iBAT temperature does reflect what is happening to core temperature. We are starting to use subcutaneous RFID temperature chips more routinely in rat DRF studies in our laboratories. The RFID chips can be sterilized and re-used, and are therefore relatively inexpensive. Whichever method of body temperature measurement is used, it is crucial to record and report ambient temperature in the housing room/procedure room.

One of many causes of hypothermia is mitochondrial toxicity. Benchtop analytical techniques are improving all the time, and requiring smaller blood volumes (i.e., microsampling). For example, glucose can be analysed from a drop of blood using a handheld monitor (e.g., Accu-chek®; AlphaTRAK®)¹⁴² and lactate monitors such as The EDGE™ and Lactate Pro only require 3–5 μL of blood, lending themselves to following the time course of drug-induced effects (e.g., metabolic acidosis) in rodents. Other benchtop systems such as iSTAT™ and epoc™ require a larger volume of blood (∼100 μL), but can measure a range of metabolic intermediates, electrolytes, blood gases, and pH, and have been used in rats¹⁴⁰ as well as larger species.^141,143 Assessment of glycaemic control per se is generally done using an oral or intravenous glucose tolerance test, which has been used across the toxicology species.^134–139 Another useful measurement, when there is cause for concern, is oxygen consumption using whole-body indirect calorimetry, which reflects overall metabolic rate.¹⁴⁴ This can be measured by placing rodents in small chambers for, say, three hours (one hour habituation, 2 hours’ recording), using a system such as Oxymax.²²⁷

7. Proposals for routine and ad hoc inclusion of functional endpoints in repeat-dose toxicity studies

Where functional measurements are included in repeat-dose toxicity studies in place of standalone safety pharmacology studies, we may have to attempt to conduct the measurements on Day 2 of dosing, and on at least one other occasion. In doing so we are conceding that any adverse effects detected may have been of a larger magnitude on Day 1, but that this is a trade-off to avoid adding to the complexity of Day 1. In addition, on the days of functional measurements the dosing should be staggered to enable the functional assessments to be made at approximately the same time point(s) post-dose in all the animals tested. Finally, the study start–finish dates may need to be staggered to accommodate the functional assessments on all the animals to be tested on Day 2 of dosing (and on the same study days for subsequent recordings). The logistical problems involved in doing this, particularly in trying to accommodate FOB/Irwin assessments and whole-body plethysmography recordings into a single rodent toxicology study, should not be underestimated.²²⁸

Where functional measurements are included in addition to standalone safety pharmacology studies, more flexibility can be afforded to the measurements, thereby minimising alterations to the conventional toxicology study design. The functional measurements should therefore be designed to have minimum impact on the conventional repeat-dose toxicity study protocol, and minimum impact on the study animals. In early non-GLP toxicity evaluations (e.g., the MTD/DRF studies), where the objective of any functional assessments is ‘hazard detection’ rather than ‘risk assessment’, we can afford to scale-down the measurements from the standard designs in safety pharmacology studies, and also perhaps just assess the high-dose group and the vehicle controls. This would be sufficient to detect large effects, providing an early warning of significant effects ahead of the regulatory GLP toxicology/safety pharmacology studies, thereby enabling sufficient time for proper evaluation in an interim, bespoke study, or in the one month studies. In either scenario, multiple measurements can be conducted on the same animals, either on the same or different days within the study.

We would propose the following recommendations. For early (non-GLP) toxicology studies in rodents (e.g., DRF studies), consider a single (∼2 h) session of functional assessments including FOB/Irwin, whole-body plethysmography, and assessment of faecal pellets, on any suitable day during the study. This can be restricted to the vehicle and high-dose groups, as it is merely to act as a flag to trigger definitive investigative studies if required. Such follow-up investigations could even be as part of the regulatory one-month studies. For DRF studies in nonrodents, consider including jacketed ECG telemetry when employing group sizes of 2 males + 2 females per treatment group (data from group sizes smaller than this are of questionable value). For one-month regulatory toxicology (GLP) studies, consider routine inclusion of jacketed telemetry recordings of ECG, and respiratory rate and depth, in the nonrodent studies on all candidate drugs. For compounds targeting the CNS, or penetrating the CNS, consider inclusion of a FOB/Irwin assessment pre-study, in week 1, and in week 4 of the rodent study. Also consider inclusion of bespoke, singular assessments of specific behaviours/autonomic functions in either the rodent or nonrodent studies, if previously flagged.

8. Future possibilities

8a. Implanted telemetry?

Although, ideally, techniques for acquiring functional data in toxicology studies should avoid the use of surgical implantation, and anaesthesia, there may be arguments for doing this in studies of greater than one month's duration. For example, recent guidance on the development of drugs to treat type II diabetes²²⁹ has recommended the evaluation of cardiovascular risks in Phase II and III clinical trials. Known risk factors for cardiovascular-related mortality are the potentially detrimental effects of small but chronic elevations in arterial blood pressure²³⁰ or heart rate,²³¹ and so drug-induced changes of this type would be undesirable. Whilst drug-induced elevations of blood pressure and/or heart rate may be detectable using noninvasive tail-cuff telemetry in dogs, more accurate data for decision-making would be obtained using implanted telemetry devices. Whereas this might be deemed uneconomic for a one-month toxicology study, it may be economically and ethically valid in (say) a 6-month non-rodent toxicology study on a candidate drug for type II diabetes. The duration of study also matches the guaranteed battery life of the implants (usually 6 months). An alternative view is that we could deploy this technique in the one-month study where there is cause for concern, so as to stop a compound prior to further investment of time, resources, finances and animals. Although either approach would be expensive, it is less expensive than uncovering a potentially unacceptable hypertensive effect during Phase III clinical trials. One way of reducing both the welfare impact and the cost would be to use mouse-size blood pressure telemetry implants in a non-rodent species, using a signal booster in a collar or jacket,¹⁸³ as mentioned earlier (‘minimally-invasive telemetry’).

8b. Imaging?

Similarly, whereas the use of general anaesthesia to enable imaging may raise objections in a one-month toxicology study in rodents, such methodology could be envisaged in a study of longer duration. This would enable any of a range of imaging techniques, including magnetic resonance imaging (MRI; to detect changes in size and structure); magnetic resonance spectroscopy (MRS; to detect biochemical changes associated with drug-induced toxicity in various organs), and functional magnetic resonance imaging (fMRI; e.g., to detect changes in neuronal activity in the CNS).^232,233 Yet the majority of these techniques require immobilisation (e.g., by anaesthesia), and administration of contrast agents or radioactive markers,^234–236 which will clearly limit their application in repeat-dose studies. However, the use of non-chemical immobilisation techniques such as snuggle wraps in conjunction with techniques such as Doppler flow ultrasonography or arterial spin labelling MRI, that do not require the administration of contrast agents or radioactivity, offer intriguing possibilities that may bridge to standard toxicology studies.

For early detection and longitudinal monitoring of renal toxicity, a range of advanced imaging techniques using ultrasonography (doppler, microbubble, targeted microbubble), single-photon emission computed tomography (SPECT), and MRI have been developed. These offer the possibility to investigate in real time, noninvasively, changes in GFR, renal blood flow and renal pathology. Cardiac and pulmonary toxicity have been followed longitudinally using ¹⁸F-fluorodeoxyglucose positron emission tomography (FDG-PET) in rats,²³⁷ but this method can be applied to most organ systems. For imaging of hepatotoxicity, methods exist to measure changes in rodent hepatic volume longitudinally using MRI;²³⁸ hepatic fat content (steatosis) using MRS; visualise choleostasis using contrast MRI;²³⁹ and measure free radical generation in real time using free L-band electron spin resonance (ESR) spectroscopy.²⁴⁰In vivo imaging of retinal cell layers is obtainable using optical coherence tomography (OCT), which has now achieved a level of resolution suitable for use in rodents.²⁴¹

8c. Breath analysis?

Another technique which is becoming achievable in rodents due to improved analytical methodology is breath analysis. This has been used to detect renal failure in rats, albeit by direct sampling from the trachea under anaesthesia;²⁴² at some point in the future we may expect to be able to detect a range of exhaled biomarkers of toxicity sampled from rodents in small chambers (e.g., during whole-body plethysmography recordings). In the meantime, the use of blood microsampling will suffice as it is minimally invasive.³²

9. Summary and conclusions

The inclusion of functional endpoints in toxicology studies can be viewed as part of an overall strategy to reduce candidate drug attrition due to toxicity. This would include target safety risk assessment at the outset of a drug discovery programme, the early application of in silico approaches (structure–activity modelling; pathophysiological modelling; bioinformatics),^243–245 automated in vitro screening across target organs, subcellular components and ‘adverse’ molecular targets prior to candidate drug selection,^245–248 increasing the ‘information content’ of early in vivo toxicity studies (including the deployment of toxicity biomarkers and ‘omics’ in both toxicity studies and efficacy studies),^27,249 and better understanding of preclinical–clinical translation,^14,15,250 all contributing to more informed and consistent decision-making.^{7,8,12,251–253}

An augmentation of long-term toxicity studies by including functional assessments would enable unprecedented longitudinal investigation of adverse effects of candidate drugs prior to investment in the expensive phases of clinical development (Phases II and III). After decades of missed opportunities to do this, the option to assess safety pharmacology studies (i.e., functional) endpoints in certain circumstances (e.g., biologics; terminal cancer agents; exploratory INDs) has encouraged toxicology departments to incorporate physiological and behavioural measurements into the regulatory toxicology studies. If this can be done for these special cases (as well as for developmental and juvenile toxicity studies), then surely it could be done for mainstream drug discovery projects, either routinely or on a case-by-case basis. The more we know about a new chemical entity before it enters clinical development, the better prepared we are for what lies ahead. Perhaps what is needed is a change in mindset: is the role of toxicology in the pharmaceutical industry merely to clear regulatory hurdles to enable entry into clinical development, or is it to try to minimise expensive failures during it?

Acknowledgements

We would like to thank numerous colleagues in the Safety Pharmacology Department, Toxicology Sciences and Laboratory Animal Sciences at AstraZeneca for helping to put many of these ideas into action.

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Footnote

† Present address: Sanofi R&D, Rue Pr Joseph Blayac, 34184 Montpellier Cedex 04, France.

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