Bulgarian Journal of Veterinary Medicine, 2016 ONLINE FIRST ISSN 1311-1477; DOI: 10.15547/bjvm.983

Review

TOXICITY STUDIES OF AND CHEMICALS IN ANIMALS: AN OVERVIEW

S. A. SAGANUWAN Department of Veterinary Physiology, and Biochemistry, College of Veterinary Medicine, University of Agriculture, Makurdi, Benue State, Nigeria

Summary Saganuwan, S. A., 2016. studies of drugs and chemicals in animals: An overview. Bulg. J. Vet. Med. (online first).

Toxicity study is the investigation of either short or long-term toxic effects of a or chemical on animals. The toxicity is dose-dependent as asserted by Paracelsus over 500 years ago. However, short-term toxic effect is determined using median (LD50) first introduced by Trevan in 1927 and revised many times. Presently there is a growing preponderance of rejection of scientific papers on study, simply because of the belief that in the current hazard and safety as- sessment of drugs and chemicals, LD50 values are no longer used. In view of this, literature search was carried out with a view to investigating the relevance of LD50 in development and assessment of drugs and chemicals. The findings revealed that in the past, many animals had been used for LD50 determination. OECD has reduced the number of test animals to 5–15 and presently it is further re- duced to 2–6. Acute toxicity study is being carried out in medicinal research and in the study of patent medicine. Although the application of LD50 has been drastically reduced, it is still applied and accepted in some parts of the world. Moreover, animals on which LD50 tests are conducted, should be allowed to die to see the end effect of the test drug or chemical because euthanisia of test animals may mask some toxicity signs of the test agents. Therefore, toxicity study of drugs and chemicals is a sci- entific process necessary for discovery and development of drugs as well as identification of potential . Key words: , Paracelsus, therapeutic agent, , toxicity

INTRODUCTION

Attempts have been made to see whether polycyclic hydrocarbons or the number of the chemical structure of the molecule chlorine atoms in chlorinated hydrocar- itself may serve as a predictor of toxic or bons. Such classification had some suc- carcinogenic effects (Golberg, 1983; cess but is far from perfect (Goddard et Rosenkranz et al., 1999). Such predictions al., 1996). Nevertheless, Paracelsus as- are often based on particular active sites, serted that toxicity effects of toxicants and such as the number of aromatic rings in therapeutic agents are dose-dependent Toxicity studies of drugs and chemicals in animals: An overview

(Castleman, 2002). In that regard, Trevan Turnbull, 2000). The random stopping introduced the concept of the median le- rule in UDP improves the overall ability thal dose (LD50) in 1927. Median lethal of the test to respond to varying underly- dose is the amount of test agent that can ing conditions, but also causes the re- causes death in 50% of test animals. There ported level of confidence and the actual are different methods used to estimate level of confidence to differ somewhat median lethal dose (Karber, 1931; Reed & (Botham, 2003). Of all the principles of Muench, 1938; Miller & Tainter, 1944; the three alternatives (fixed dose; acute Litchfield & Wilcoxon, 1949; Lorke, toxic class and up-and-down), up-and- 1983; ASTM, 1987; Dixon, 1991; Sa- down is the most suitable, because it gives ganuwan, 2011; Saganuwan et al., 2011; end-point-evidence toxicity and obeys Saganuwan & Onyeyili, 2016). Earlier stopping criteria to limit number of ani- methods adopted for the estimation of mals used, using only 2–6 animals with median lethal dose used many animals possible 1–3 deaths (Shiryaev & (40–100), but for up-and-down procedure, Spokoing, 2000). 4–15 animals can be used (OECD, 1987; 2000a,b; 2001a,b,c; Stitzel, 2000; HISTORICAL BASIS OF TOXICITY UNECE, 2003). Saganuwan (2014) re- STUDIES vised the up-and-down procedure (UDP) to the use of 2–6 animals that may likely Roman herbalists were as likely to be kil- give 1–3 reversals. If there is no death lers as healers. As a result, throughout the above 15,000 mg/kg body weight, the test Roman Empire herbalists became ob- agent is practically safe and that serves as sessed with the identification of herbal the limit boundary. The revised method and the development of saves time, reserves large numbers of known as theriaca (in Greek meaning animals that may be wasted and ignores remedies for venomous bites). The ancient complex mathematical manipulation in- world’s most -paranoid ruler was volved (Saganuwan, 2014). The method is Mithridates Eupalor (120–60 B.C.), king faster and its findings are relatively com- of Pontus, on the Black Sea. He took in- parable to the findings with other methods creasing doses of poisons to develop a (Saganuwan, 2012). For all the methods, tolerance and make himself immune. In 66 the study outcome is likely to be influ- B.C., Romans attacked him, he attempted enced by the choice of starting dose to poison himself but he has become tol- level(s) relative to the true LD50 value, erant and finally ordered a slave to stab especially in the case of shallow slopes him to death to escape embarrassment (Gribaldo et al., 2005). Therefore, infor- from enemies (Castleman, 2002). The mation on toxic effects seen only at dose bubonic plague caused by Yersinia pestis levels close to a lethal dose will not be and spread by rat flea, Xenopsylla cheopis obtained (Van der Heuvel et al., 1990). probably began in Asia in the year 1330, However, the dangerous dose for humans spreading to Norht Africa, Europe and the may be 1–100% more or less of the de- Middle East. This epidemic caused 25 to termined LD50 (Dreisbach & Robertson, 33% drop in population of Europe bet- 1987). Because small numbers of animals ween 1330 and 1350. Programmes were were used, the actual level of confidence launched against the Jews of Europe, who is generally not exact (Jennison & were thought responsible for the epi-

2 BJVM, ××, No × S. A. Saganuwan demic, even though the pope pointed out Paracelsus to introduce many potentially the ludicrousness of this belief (Stolley, toxic minerals into medicine in very small 1995). The bubonic plague caused by doses. He used arsenic, sulphur, lead, an- Yersinia pestis ravaged Europe for timony and particularly mercury, not just many centuries although at the Interna- for syphilis but also for many other dis- tional Medical Congress in Berlin, 1890, eases. In time, physicians came to view Robert Koch reminded his contemporaries mercury as a panacea and Paracelsus era that so-called vibroids seen in anthrax and marked the beginning of (Cas- relapsing fever are the cause of these dis- tleman, 2002). That notwithstanding, eases (Moxon, 1990). The most vocal about 40,000 people died of ergotism in champion of the Doctrine of signative was 1992 in France and Spain. Some people of Phillippus Aureolus Theophrastus Bom- , Africa and Asia living in jungles bastus Von Hohenheim (1493–1541 have good knowledge of poisonous plants A.D.), the founder of toxicology, the son and animals and so they use them to catch of a Swiss alchemist/physician. A brilliant fish, poisoning arrow heads for hunting as arrogant student of medicine, he lived up warfare and assassination of their foes to his name, Bombastus, but later changed (Garg, 2004). Even in the Europe, cap- it to Paracelsus (i.e. greater than Celsius), tives of war have poisoned their masters convinced that he was more brilliant than using poisonous plants (Castleman, 2002). the Roman Physician Celsius (53 B.C.–7 By 1500 B.C., written recordings indi- A.D.). He was born in the village of Ein- cated that hemlock, opium, arrow poisons siedeln near Zurich, Switzerland on 14 and certain metals were used to poison November, 1493. In 1502, following the enemies or for states executions. Notable death of his mother, the family moved to poisoned victims include Socrates, Cleo- Villach in Carinthia, Southern Austria, patra and Claudius (Radenkova-Saeva, where he further became municipal physi- 2008). cian and also taught chemistry. Paracelsus With roots extending to antiquity, received his baccalaureate in medicine in toxicology is a profession which recog- 1510 and addoclorate in 1516 from the nises that the past is often a prologue to University of Ferrara. He travelled the present (Stirling, 2006). Watson et al. throughout Europe, British Isles, Egypt (2000) provide a concise and informative and the holy land to broaden his know- review of toxicology from its Greek roots ledge (Borzelleca, 1999). He returned to to the ubiquitous use of risk assessment. It Villach in 1524 and remained there until has grown to be a science with change, 1527. Because of his fame, his lectures evolution and even controversy based on were well attended and he burnt medical toxicological method, concept or theory textbooks of Avicenna and Galen. He dividing the science of toxicology to ana- challenged them which led to his leaving lytical toxicology, , ar- the university. Paracelsus taught at the chaeological toxicocology, biochemical University of Basel, Switzerland in the toxicology, clinical toxicology, environ- 1520s and discovered the dose-response mental toxicology, experimental toxicolo- relationship of a poison. This insight came gy, , genetic toxicolo- too late to save thousands of alleged gy, immunotoxicology, industrial toxico- witches executed in part of Europe for logy, inhalation toxicology, medical toxi- possession of poisons but it allowed cology, neurotoxicology, ocular toxico-

BJVM, ××, No × 3 Toxicity studies of drugs and chemicals in animals: An overview logy, ophthalmic toxicology, pathological sis was laid by regulatory agencies on toxicology, pesticide toxicology, photo- determining toxicity profiles of all phar- toxicology, radiation toxicology and vete- maceutical substances available for animal rinary toxicology. The knowledge is based and human patients. In the late 1980s, the on Haber’s law hormesis and Pare’s law OECD and ICH brought out the guide- (Stirling, 2006). Those who developed lines for toxicity testing of pharmaceutical specific techniques, theories or cannons of substances (Parasuraman, 2011). The the field were Albert Roy, Barnes John, Ebers papyrus (corca 1500 B.C.) contains Bernard Claude, Bucheri Wolfgang, many poisons including aconite, opium, Casarett Louis, Christian Mildred, Clark- lead, copper and antimony. Sumerian texts son Thomas, Cook Warren, Doull John, (1400 B.C.) had referred to Gula (a fe- Dover Thomas, Ealon David, Feldman male deity) who was associated with Robert, Fintana Abbe, Furst Arthur, Gol- charms and poisons. Hippocrates also berg Leon, Guengerich F. Peter, Haber wrote on poison in 400 B.C., as Homer Fritz, Hamilton Alice, Hodge Harold Car- (850 B.C.) wrote on the use of arrow poi- penter, Hunt Virlma, Kaye Sidney, Ken- son and in the epic tale of The neway Ernest, Klaassen Curtis, Legge Odyssey and lliad. Cleopatra (60–30 Thomas, Lehman Arnold, Lewin Louis, B.C.), queen of Egypt, experimented with Mably Thomas, Maimonides Moses, Nel- strychnine and other poisons on prisoners son Norton, Saunders Paul, Sterner James, and poor people. She committed suicide Taylor Alfred, Trasko Victoria, Warkany with Egyptian cobra (asp) sometimes used Josef, Watanabe Philip, Watin Jean, in executions. Socrates (470–399 B.C.) Widmark Erik, Wiley Harvey, Wilson was poisoned with coniine and Agrippina James G., Zbinden Gerhard and Orfila killed Claudius to make Nero the emperor Mateo, a Spanish physician who estab- of Rome, who also poisoned Britannicus, lished toxicology as a distinct scientific Claudius’ son using arsenic, under the discipline (De Sosa, 1992). Mathieu Or- directive of Locusta, a professional poi- fila (1787–1853), a Spanish physician soner attached to the family. Herculaneum determined the relationship between poi- was destroyed by mountain Vesuvius sons and their biological properties and eruption in 79 L.E. and Pliny was suffo- demonstrated specific organ damage cated by volcani gases. An Italian lady, caused by . Orfila is referred to as Toffana prepared arsenic-containing cos- the father of modern toxicology. The use metic. Moses Maimonides (1135–1204), a of animals in toxicity studies began in famous Jewish philosopher, born in Spain 1920, when J. W. Trevan introduced the and was educated at the University of Fes use of median lethal dose (LD50). Thereaf- wrote Treatise on poisons and their anti- ter John Draize, an FDA scientist devel- dotes. In the quest for political stability in oped a method for testing eye and skin the Republic of Venice, the Council of ten irritation using rabbits. Later, US National (1310–1797) used poison to kill contro- Cancer Institute (NCI) developed a test to versial figures. The Borgias, Alexander identify carcinogenic chemicals through VI, his son Cesare and Lucrezia were well the daily dosing of rats and mice for 2 known Italian assassins during the 15th years. William MacBride discovered that and 16th century. The Borgias used arsenic thalidomide was the cause of phocomelia to poison for political and monetary gain. in about 1,000 babies. After this, empha- Pope Clement VII (1478–1534) died after

4 BJVM, ××, No × S. A. Saganuwan eating Amanita phalloides. Catherine toxicity studies are carried out to know the Medici, a queen of France used poisoning long term effect of a toxicant (1–1½ as a political tool during her reign. Hiero- years). Subacute is sometimes called sub- nyma Spara (1659) formed Spara’s secret chronic, sub means under i.e. considered organisation in Rome and helped women under acute or below chronic (Saganuwan, plot to kill their wealthy husbands to be- 2012). Other forms of toxicity studies are come wealthy widows. Catherine Mon- explained below. voisin (1640–1680) was accused sorcerer Single dose toxicity study and convicted poisoner in France and un- fortunately she was burnt at the stake Feb- Classical hazard and risk assessment is ruary 22, 1860. Guilia Tophania (1635– largely based on adverse health effects 1719) was a supplier of poison to wives findings in experimental animals accor- who wanted to be widows, she was later ding to globally agreed test guidelines identified and executed by strangulation. (Piersma, 2014). Zhang et al. (2015) de- In respect of that, King Louis XIV passed termined maximum tolerated dose and a decree forbidding the sale of poisons toxicity effects of Inauhzin a novel non- from apothecaries to unknown persons. genotoxic p53-activating compound in Mattieu Joseph Bonaventure Orfila mice. The toxicity effects exhibited by the (1787–1853), a Spanish physician to animals were acute pain, anorexia, immo- Louis XVII of France who occupied a bility and coma. The affected animals chair at the University of Paris was the were euthanised. Animals sacrificed at an first to attempt a systemic correlation be- unscheduled interval were anesthetised tween the chemical and biological effects and necropsied (Zhang et al., 2015). of the known poisons which is applied on Small animals such as rats, mice, forensic toxicology. Bernadino Ramazzini guinea pigs and rabbits are most suitable (1700) is credited with knowledge of sili- for determining the mechanism of drug cosis and plumbism. Percival Potts (1700) absorption and bioavailability values from associated cancer of scrotum to Chimney powder or solution formulation, whereas sweeps (Radenkova-Saeva, 2008). larger animals (dogs, pigs and monkeys) are used to assess absorption from formu- lations. Therefore, the understanding of TOXICITY STUDIES physiological, anatomical and biochemical There are many different types of toxicity differences between gastrointestinal tracts studies carried out for evaluation of toxic of different animal species can lead to the effects of therapeutic agents or potential selection of the correct animal models to toxicants that could pose threat to the mimic the bioavailability of compounds in lives of humans and animals. The tradi- the human species. However, current data tional methods of determining toxic ef- indicate that no single animal can mimic fects of chemicals and drugs include acute gastrointestinal characteristics of humans, toxicity study which is carried out to de- so human studies can never be substituted termine the short time toxicity effect of a by animals. For a given purpose, selection toxicant (1 s to 2 weeks), whereas of the right animal model is possible. For subacute toxicity study is carried out to example, dogs and humans have similar know the relative long term effect of a stomach morphology and emptying char- toxicant (4 weeks – 6 months). Chronic acteristics. Pig and human colon mor-

BJVM, ××, No × 5 Toxicity studies of drugs and chemicals in animals: An overview phology appears similar. The microbial & Whitehead, 1995). Animals of same content of the rabbit intestinal microflora species are used at each level using four is similar to that of humans. In reality, our pre-identified starting doses which can be understanding of the species differences in selected based on the globally harmonized gastrointestinal anatomy, physiology and classification system (Saganuwan, 2012). biochemistry is incomplete. From in vivo Up-and-down procedure drug absorption studies, it is difficult to assign the observed differences in Up-and-down procedure (UDP) was de- bioavailability to any factor. Overall, we veloped by OECD in 1981 and revised need more mechanistic studies to deline- many times. For an agent suspected to be ate the effects of anatomical, physiologi- relatively safe, either 2000 mg/kg or 5000 cal and biochemical differences between mg/kg can be administered orally to the species on overall drug absorption first experimental animal and observed for (Kararli, 1995). The canine neck may be 48 h. if the animal dies, a second and third used as a modelling tool for biomechani- animal can be dosed concurrently and cal investigations of the human cervical observed for 48 h. If these two die and the region (Shairr et al., 2006). test dose is 2000 mg/kg, the LD50 may be There are currently several factors assumed to be below 2000 mg/kg (OECD, driving a move away from the reliance on 1987; 2000a,b; 2001a,b,c). Although 5– in vivo toxicity testing for the purposes of 15 animals can be used, Saganuwan et al. chemical safety assessment. Progress has (2011) has reduced the number of animals started in the development and validation to 6 using 3 reversals. However, 2 animals of non-animal methods. However, recent can be used with one reversal, i.e. 1 dead advances in the biosciences provide excit- and 1 survivor (Saganuwan, 2014). The ing opportunities to accelerate this process prepared solution of a toxicant should be and to ensure that the alternative para- uniform and given by gavage. In rodents, digms of hazard identification and risk the volume should not exceed 1 mL/100 g assessment deliver lasting 3Rs benefits, body weight (NIEHS, 2000). whilst improving the quality and relevance Toxicity study of snake venom of safety assessment. The NC3Rs, a UK- based scientific organisation that supports The calculation of median lethal dose the development and application of novel (LD50) and effective dose fifty (ED50) 3Rs techniques and approaches held a have been developed and modified. LD50 workshop recently, which brought to- value of snake venom can be calculated gether over 20 international experts in the using 1–4 animals, whereas Spearman field of clinical safety assessment (Burden Karber’s method (Carter, 1980) and ideal et al., 2015). median lethal dose formula developed by Saganuwan (2011) can be used for estima- Fixed dose procedure tion of ED50 for snake antivenom. Since Fixed dose procedure (FDP) is used to ED50 is equal to 2 LD50 or 3 LD50, the LD50 assess the non-lethal toxicity rather than of snake venom can be calculated from lethal dose. The investigational product is the ED50 formula most especially for Mi- administered at fixed dose levels of 5, 50, crarus fulvius venom and anti-venom us- 500 and 2000 mg/kg and the animals are ing mice of about 20 g and safety margin observed for a short time period (Stallard of 10,000 (Saganuwan, 2016).

6 BJVM, ××, No × S. A. Saganuwan

Repeated dose acute toxicity testing week repeated dose toxicity studies and a comprehensive assessment of cardiovas- Repeated dose acute toxicity testing cular safety, a potential 50% reduction in (RDATT) lasts for a minimum of 28 days attrition in the GLP phase can be (1 month). The test agent is administered achieved. This will reduce animal use daily using uniform concentration at de- overall, save significant R&D costs and termined dose orally or parenterally. Ro- improve drug pipeline quality (Kavanagh dents 5–7 weeks of age are preferred with et al., 2014). Micro sampling techniques average weight of 20% the standard de- and enhanced analytical sensitivity pro- viation. Animals are observed for toxicity vide scope for reducing numbers of satel- signs (Saganuwan, 2012). Since gestation lite animals on rodent toxicology studies. period of rodents especially mice and rats It is possible to obtain individual serial is about 3 weeks, mutagenicity testing can toxicokinetic profiles from reduced num- be done to assess the effect of drug on bers of satellites attached to each group especially deoxyribonucleic acid (Lawrence et al., 2014). Metabolome (DNA). Expected toxicity signs include analysis of two different mixtures and but are not limited to chromosomal aber- their respective enantiomers indicated no ration, DNA duplication, insertion, inver- biological relevant differences regarding sion and translocation that may lead to the qualitative toxicity towards the as- carcinogenesis. In vivo mutagenicity sumed target organs as well as quantita- which is dose dependent is used to deter- tive amount of toxicity, thereby confirm- mine the case-by-case basis risk assess- ing the toxicity equivalence of the enanti- ment of the test substance. Mutagenicity omers (Parra et al., 2014). An analysis of studies with transgenic animals are more target organ in man toxicity appropriate assay techniques to determine studies for 77 Astra Zeneca candidate the toxicity of a test substance (Eastmond drugs was conducted across a range of et al., 2009; Gholami et al., 2010). therapy areas. In the rodents, the most Repeated dose toxicity study frequently affected organ was the liver, followed by adrenal glands, kidney, Repeated dose toxicity (RDT) is one of spleen, bone marrow, and thymus. In non- the key regulatory endpoints in the hazard rodents, liver and thymus were the most assessment of chemical substances (Ya- frequently affected organs, followed mada et al., 2014). In most cases, the closely by the testes and gastrointestinal safety evaluation of new anti-cancer small tract. Therefore, the use of non-rodents as molecule pharmaceuticals includes repea- a second species in toxicity testing to sup- ted dose toxicity studies in both rodents port human safety is important (Lees et and non-rodents. But retrotoxicology data al., 2014). Aged rats are more prone to illustrate that non-rodent studies make an ecstasy neurotoxicity than adolescent rats important contribution to human risk as- suggesting that age is an important deter- sessment (Deavall et al., 2014). Entry into minant for the neurotoxic events promoted the critical pre-clinical “Good Laboratory by ecstasy (Azevedo et al., 2014). Practice” stage of toxicology testing trig- gers significant R&D investment yet Specific organ acute toxicity study >20% Astra Zeneca’s potential new medi- Specific organ acute toxicity study cines were stopped for safety reasons in (SOATS) is very necessary for drugs de- this GLP phase alone. By smart loading 2

BJVM, ××, No × 7 Toxicity studies of drugs and chemicals in animals: An overview veloped for treatment of specific organ based on a weight-of-evidence argument diseases as seen in respiratory diseases in to improve human risk assessment while which rats are used. The animal is ex- reducing, refining and replacing (3R) posed to aerosol-like preparation for 4 h animal testing. In case-by-case situations, and monitored for 14 days (OECD, the alternative mouse models have the 2000a,b). For ophthalmic agent use, 0.5 potential to contribute to this weight-of- mL of a test substance (1% solution) is evidence, and may be the only carcino- administered to an animal eye and ob- genicity study needed to adequately assess served as for respiratory and for skin 0.5 g carcinogenic risk (Bower, 2014). The of a test substance is applied to surface of impact of testing in a second species for animal’s skin (Curren & Harbell, 1998; embryofoetal developmental toxicity was York & Steilling, 1998). Skin sensitisation discussed during a 2010 ICH workshop in test can be carried out using guinea pig as Tallinn, Estonia. It was proposed to re- a model. The tests used are Draize test, view the frequency with which the results open epicutaneous test, optimisation test, from studies in the rat versus rabbit have split adjuncut test, guinea pig maximisa- driven the lowest observed adverse effects tion test, Buelier test and murine local level (LOAEL) and exposure margins in lymph node assay (WHO, 2008). Neuro- risk assessment for pharmaceuticals (Van toxicological study is carried out in adult Der Laan et al., 2014b). Nonhuman pri- rodents. The test substance is adminis- mates (NHP) models play an important tered for 30–90 days. An in vitro model is role in the development and safety as- preferred for safety value (OECD, 2010). sessment of biopharmaceuticals depend- ing on the particular properties of the drug Reproductive toxicity study candidate. In many instances, the experi- With respect to reproductive toxicity, it is mental use of NHPs is subject to special clear that vaccination during pregnancy regulations and animal welfare considera- might be important to protect the mother tions among which the 3R efforts should as well as the child, but an adjuvant might focus on reduction and refinement. For be a risk especially very early in preg- reduction, some guideline changes related nancy (Van Der Laan et al., 2014a). The to biologics – ICH M3 (R2) and ICH S6 P53+/- mouse model may be the preferred (R1) – can provide an option for using model for compounds with direct or lesser number of NHPs. ICH S6 (R1) equivocal evidence of genotoxicity, while makes specific recommendations on how the rasH2 mouse model developed by the many live infants are required per group Central Institute for Experimental Ani- for a NHP pre-and postnatal development mals in Japan, is the only model accept- study. This recommendation in conjunc- able for compounds with positive, equivo- tion with the use of reference data and cal or negative genotoxicity findings. The normogram data can be used to reduce the rasH2 model is becoming the choice of number of NHPs needed for developmen- pharmaceutical companies that have tal toxicity testing. Also, under certain adopted an alternative mouse model into circumstances, the conduct of dedicated their carcinogenicity risk assessment reproductive toxicity evaluation as per paradigm. The proposed modification of ICH S5 (R2) can be incorporated in the ICHSI guideline addresses the elimi- chronic toxicity studies thus further lower- nation or replacement of a 2-year rat study ing the demand for NHPs. Therefore, re-

8 BJVM, ××, No × S. A. Saganuwan finement should be a key, focus of any 3R recorded and live pups are weighed daily efforts in order to optimise the use of until they are weaned. Very few pups can NHP models. Recent work has provided be sacrificed for histopathology (OECD, statistical power estimates for some fertil- 2015a). ity parameters relative to group size that Two generation reproduction toxicity will be used in developing appropriate studies study designs. Also, the potential of com- bining pre-and postnatal testing with ju- They are similar to one-generation repro- venile toxicity testing has been raised. duction toxicity testing. After the admini- Various options are available to optimise stration, the animals are mated after which NHP DART testing in the spirit of 3R female animals are separated. Sperms are (Weinbauer, 2014). In the case of drugs collected for analysis. Thereafter, the test developed for children, juvenile animals substance is continuously administered to are used as models of human children. pregnant females which are observed Dermal drugs are normally developed regularly for toxicity signs including mor- using the minipig as a testing species due tality. After parturition, nursing rats are to similarities between human and minipig administered the test drug and the mortal- skin. For minipigs, dosing of young ani- ity of the pups (1st filial generation) is mals from weaning at 4 weeks of age is observed. From the F1 generation, one straight forward. However, for younger male and one female animal are selected. preweaned animals, there is a high risk of F1 offspring are not allowed to mate until interference with dosing site by the they have attained full sexual maturity, mother or siblings. This potentially pre- and pairs without a pregnancy are evalu- vents studies that would evaluate effects ated for infertility (Oliveira et al., 2010). of drugs in animals of equivalent human Necropsies and histopathological exami- age 0 to 2 years. However, dermal studies nations are carried out. At the end of the on minipigs younger than 4 weeks of age study, the animals are sacrificed for gross can be performed (Makin et al., 2014). pathological and histopathological exami- nations (OECD, 2015b; Matsuura et al., One-generation reproduction toxicity 2005; Ganiger et al., 2009). During the testing reproduction toxicity studies, develop- This type of test is conducted in some mental and embryo toxicity can be carried generation of animals comprising male out together or independently. Rodents are and female animals. The agent is adminis- preferred. The compound is administered tered for a duration of one complete between 8th and 14th day of pregnancy and spermatogenic cycle in male animals and embryolethal effects are studied. On the for two complete estrus cycles for fe- 21st day of the study a caesarean section is males. Rodents are preferred. After the performed and felaser with haemorrhagic completion of administration, the animals bullae, limb malformations, exencephally, are allowed to mate. The test compound is cleft palates, open eyelids, detail deformi- administered to the females during the ties, mortality and the number of live and period of pregnancy and nursing. The dead animals are noted. Embryo toxicity sperms are collected for analysis. Number assays such as embryonic stems test, of offspring and their sexes are recorded. micromass embryotoxicity assay and The number of live and dead pups are also whole rat embryotoxicity assays can be

BJVM, ××, No × 9 Toxicity studies of drugs and chemicals in animals: An overview done (Kimm-Brinson & Ramsdell, 2001; etc. (Saganuwan, 2012). A satellite group Hofmann et al., 2002; Booth et al., 2010). may be included in the study protocol. During generation reproduction toxicity This group has both a control group and a study, genetic toxicity testing can be car- high-dose group. The animals are ob- ried out to identify gene mutations, chro- served for normal and abnormal body mosomal changes and alterations on the functions and biochemical parameters DNA sequency. This test is conducted in should be measured. Tissue are collected all species of animals. But in the whole for gross and histopathology (Jaijoy et al., animal model, rodent species is preferred. 2010). Carcinogenicity testing is under Genetic toxicity is assessed using the ro- chronic toxicity testing in which both ro- dent chromosomal assay, dominant lethal dent and non-rodent species of animals assay, mouse-specific locus test, micronu- are used. The test can be terminated after cleus test, heritable translocation assay 1½ years in case of mice and hamsters and and sister chromatid exchange assay after 2 years in case of rats. Haematologi- (Oliveira et al., 2014). cal analysis is performed in healthy ani- mals after 1 and 1½ years in mice and rats Subchronic toxicity testing respectively and the study is terminated. The test is conducted for a period of 90 The animals are sacrificed for gross pa- days (3 months). In this type of study, thology and histopathology (Saganuwan, subchronicity factor gives an indication of 2012). the cumulative effects of poisons. It is the ratio of acute LD to 90 days LD . A 50 50 ADVANTAGES OF TOXICITY compound though may have low acute STUDIES toxicity, but it has the tendency to accu- mulate in the body tissues and can cause Apart from accidental discoveries of toxi- subacute or chronic toxicity. Such toxi- cants, malicious killings with some toxic cants are termed as cumulative poisons, agents have also helped recognition of the e.g. DDT, lead, fluorides etc. (Saganu- importance of toxicology. Kothari et al. wan, 2012). A satellite group may be in- (2015) prepared and tested a conjugate cluded in the study protocol, and this vaccine using pneumococcal surface pro- group has both a control group and a high tein A (PSPA) from Streptococcus pneu- dose group (Muralidhara et al., 2010). monia as the carrier and Piao et al. Tissues are collected for gross pathology (2014) generated and tested three and histopathology. comprising PSPA families 1 and 2 in mice Chronic toxicity testing using the method of Saganuwan (2011). Ahmed (2008) isolated and characterised It is a long term toxicity study that last as Bdellovibrionacceae – unique, predatory, long as the life-span of the test animals endoparasitic Gram-negative bacteria. usually 1–2 years. Rodents such as mice They prey in other Gram-negative bacteria and rats are usually used. Chronicity fac- giving them potential as biological control tor gives an indication of the cumulative agents and so its LD50 was determined in effects of poisons. It is the ratio of acute the host using the method of Saganuwan LD50 to 1–2 years LD50. This type of test (2011). In this regard quick chick embryo can be conducted on drugs developed for chorioallantoic membrane (CAM) assay terminal diseases such as cancers, AIDs was used as an alternative predictive

10 BJVM, ××, No × S. A. Saganuwan model in acute toxicological studies for reduce variability and as a means of cyclophosphamide, cisplatin, vincristine, minimising the number of animals used carmustine, campothecin, aloin, mitomy- (OECD, 2000a, b). In a situation where cin C, actinomycin D, melphalan and pa- sensitivity is noticed between the sexes, clitaxel (Kue et al., 2015) using the me- females could be more sensitive and can thod of Saganuwan (2011). Any drug has be used (ASTM, 1987). However, experi- the potential to cause an adverse reaction ence has shown that male mice are more which is unwanted, unpleasant, noxious or sensitive to hydrocortisone and piroxicam harmful consequence usually associated in comparison with female mice. There- with the use of a medication that has been fore, knowledge that metabolic activation administered at a standard dose by the is required for a chemical’s toxicity can proper route, for the purpose of prophy- also indicate that males may be a more laxis, diagnosis or treatment (Wen et al., sensitive sex (NIEHS, 2000). Since de- 2014). Cao et al. (2014) identified a Bac- termination of acute oral toxicity is usu- teriovorax sp. isolate as a potential bacte- ally an initial screening step in the as- rium against snake head fish pathogen sessment and evaluation of the toxic cha- Aeromonas veronii using the method of racteristics of all compounds, the methods Saganuwan (2011). Aeromonas veronii so far utilised for the determination of affects the quality of fish leading to severe LD50 went through entire process of vali- losses to marketing (Cao et al., 2014). dation with different categories of sub- However, a glycoprotein-based native- stances before its final acceptance by subunit vaccine for fish against Aeromo- regulatory bodies (Akhila et al., 2007). nas hydrophila known to be causative The rule of thumb is that all animals agent of bacterial haemorrhagic septicae- should be handled according to the inter- mia or red pest in freshwater fish was de- national guiding principles for biomedical veloped and validated (Ciftci et al., 2016). research involving animals (CIOMS, Hepatitis A virus and its indicator were 1985). Ameni et al. (2014) reported that concentrated and detected from artificial the LD50 of Casearia sylvestris Sw (Sali- seawater using zeolite (Cormier & Janes, caceae) was greater than 2100 mg/kg. 2016) using the method of Saganuwan Saganuwan et al. (2008) estimated LD50 (2011). Death is the ultimate adverse drug of potassium permanganate at dose level event (Litt, 2015). Ibiam et al. (2014) of 1499.7 mg/kg body weight and the arti- determined median lethal concentration cle has been indexed by PubMed. Sa- (LC50=3.259–6.06 mg/L) of Psychotria ganuwan et al. (2011) determined LD50 of microphylla in Clarias gariepinus. Sa- aqueous extract of precatorius ganuwan & Onyeyili (2016) extrapolated at dose level of 2823.7 mg/kg body LD50 of mice to rat, hamster, guinea-pig, weight in mice. The article was published rabbit, monkey, cat, dog, baboon, children by Herba Polonica based in Europe. The and adult humans using human equivalent of Abrus has antidiabetic effect but dose formula in recognition with princi- should be boiled to reduce strength of the ples of reducing, refining and replacing toxic principle abrin present in the (3R). The preferred rodent species for (Saganuwan, 2009). After Paracelsus toxicity studies is the rat, although other (1493–1541) recognised that all sub- rodent species may be used. The test is stances are poisons, the right dose diffe- conducted using a single sex in order to rentiates a poison from a remedy and puts

BJVM, ××, No × 11 Toxicity studies of drugs and chemicals in animals: An overview forth the concept of dose-dependency of tion world-wide would be about 150,000 the toxic-effects of substances. This prin- rats annually (Oberg et al., 2014a, b). ciple holds true even today (Garg, 2004). Suggesting that median lethal doses are During the and develop- still used, compounds that have negligible ment research, to prioritise a compound tendency to accumulate and are rapidly with less toxicity is important especially metabolised have the same chronic LD50 in the early drug discovery stage. Since values as the acute LD50 values (Ham- only a limited amount of samples can be burger, 1989). However, with toxicants to available at this stage, mammalian in vivo which the subjects develop tolerance, the test systems are not adequate for that pur- dose required to produce lethality on re- pose. A variety of cell-based assay sys- peated exposure increases. For such tems for toxicity screening has been estab- agents, a chronicity factor is determined. lished, but these assays do not necessarily Chronicity factor gives an indication of reflect the actual phenotype of mammalian the cumulative effects of poisons. It is the toxicity, which makes it difficult to predict ratio of acute LD50 to chronic LD50 values the precise safety profile of compounds (Saganuwan, 2012). Dogs have histori- (Yamashita, 2015). Generation of animal cally been useful, informative models in data using ever greater dose groups and the development and discovery of many numbers of animals is probably not the novel cancer strategies (Gordon et al., appropriate solution not only on ethical or 2009). The pharmacological manipulation cost reasons, but also in relation to ad- of CB2 receptor in Sprague-Dawley rats dressing the issue. The solution is more may represent a potential therapeutic tool likely to be found by exploring the for the treatment of migraine (Greco et al., mechanistic basis of how such substances 2014), suggesting that animal models of act at the molecular and cellular level and migraine reflect distinct facets of this these interactions may lead to toxicity at clinically heterogeneous disorder and con- the level according to dose, du- tribute to a better understanding of its ration and life stage of exposure (Munn, pathophysiology and pharmacology (Gi- 2014). The principles of the 3R, Re- kermann-Haerter & Moskowitz, 2008). placement, Reduction and Refinement, are Acute poisoning and snake bite kill up being increasingly incorporated into legis- to half a million people in the world each lations, guidelines and practice of animal year (Buckley, 2014). Snakes and other experiments in order to safe-guard animal venomous animals bite humans and other welfare. Data from 36 reductions projects animals for protection or food. Of the were collected retrospectively from work 2800 species of snakes, only about 200 between 2006 and 2010. Substantial re- species have caused death or permanent duction in animal use was achieved by disability by biting humans. In Asia, the different strategies, including improved highest incidence of snake bite to death study design, method development and was 162 per 100,000, in Africa 497 per project coordination. Major animal sav- 100,000, Australia 4 per 1000–2000, in ings were shown in both regulatory and Europe 14 per 200 in the last hundred investigative safety studies. If a similar years, in Latin America (Brazil) 25% of (i.e. 53%) reduction had been achieved death per 19,200 each year and in the simultaneously within the 12 largest USA 12–15 deaths per 7,000 bites (Sawai pharmaceutical companies, the equivalua- et al., 1976; Warrell & Amett, 1976; Lal-

12 BJVM, ××, No × S. A. Saganuwan loo et al., 1995; Warrell, 2004; 2005; ciated with hypothyroidism, polyneuropa- Cook & Zumila, 2009). One million peo- thy, impairment of cranial nerves and car- ple throughout the world are bitten each diomyopathy (Deschamps, 2014). Low- year by poisonous snakes. Of this number level metal and insecticide exposure con- some 100,000 die and another 300,000 tributes much more towards causation of will suffer some form of disabilities for chronic disease and the impaired function- the remainder of their lives. In view of ing than previously thought. It will go a this Odessa (2014) invented and patented long way in developing new approaches alkaline sodium silicate composition to for determining the safety of pesticides inhibit the toxic effects of venom and treat and the need for innovative regulatory venomous bites and stings based on the policy to protect human and environ- method of Saganuwan (2011) whereas the mental health (Hundal, 2014). Octa- effective dose 50 of the antivenom was decanoic acid was reported to induce lung determined by Sanchez et al. (2003). Asi- damage in fish (Ibiam et al., 2014). Ufot edu-Gyekye (2014) determined LD50 of et al. (2014) reported that crude oil polyhexamethylene guanidine and polyex- caused decreased litter size, follicular amethylene guanide used in the treatment stimulating, lutenizing hormone and pro- of raw water at dose levels of 600 and 16 gesterone in rats which may decrease rat mg/kg body weight respectively. Exposure population. They concluded that repro- to a wide range of toxic products with ductive toxicity is associated with oral spectrum being broadened due to intro- exposure to crude oil. Tityus serrulatus duction of a diverse range of pesticides, venom induces a severe systemic inflam- household cleaners and drugs from time to matory condition caused by the produc- time could cause implementation of pre- tion of eicosanoids (Faccioli, 2014). Inha- caution programmes of prevention and lation of airborne nanoparticles has been identification of high risk circumstances, reported during manual activities using which can help in reducing morbidity and typical fume hoods and manufacturing mortality (Peshin et al., 2014). processes. The current occupational expo- sure limits to nanoparticles were under development and there are dose metric DISADVANTAGES OF TOXICITY needs to be harmonised for exposure as- STUDIES sessment and toxicological studies. The The indiscriminate use of pollutants, such challenge of current efforts is to further as heavy metals, surfactants and drugs, investigate occupational exposure and the generated by anthropogenic activities is associated biological responses (Tsai, causing a tremendous hazard to both 2014). Camberos (2014) fed mice with aquatic and terrestrial habitats and to hu- Jatropha curcas fermented with Rhizomu- man health. Therefore, in recent years the cor sp. for a period of 2 weeks. The other interest towards the toxic effects of these group of mice was fed unfermented J. molecules on living has in- curcas. Unfermented J. curcas killed all creased (Forni, 2014). Cobalt intoxication the mice in that group. He concluded that has become more frequent due to the wide Jatropha seed cakes treated by fermenta- use of metal cobalt hip implants. Only a tion were adequately detoxified and few patients have systemic symptoms of hence, can be fed to cattle. Jatropha con- poisoning. Cobalt toxicosis may be asso- tains antinutritional products such as tryp-

BJVM, ××, No × 13 Toxicity studies of drugs and chemicals in animals: An overview sin inhibitors, lectins, phytates and pher- objective evaluation of the available data, bol esters that limit its use as a comple- and regulatory decisions that defy com- mentary food for livestock (Camberos, mon sense (Vale, 2014). Dose-response 2014). Before use, plants that have toxic curves are often drawn and considered in or anti-nutritional elements such as oxa- the context of the “dose makes the poi- lates, tannins, phytates, nitrates/nitrites son”. This is described simplistically as and others should be subjected to soaking increasing dose levels result in increasing and or boiling in water, toasting or fer- adverse effects. This can be result of a mentation to remove the toxic elements monotonic increase or decrease in a re- (Mahajan & Dua, 1994). Therefore, plants sponse that is associated with an adverse should not be abused, as the lack of con- effect. However, non-monotonic dose trolled experiments means toxic levels responses (NMDRs) are biological effects have not been defined and the plant con- with dose-response curves deviating from stituents may affect more than one body the usual shape of a continuous response system. Use of more than therapeutic va- reaching saturation at high doses and lues may lead to overdoses with serious showing no measurable response at low consequences (Saganuwan, 2009). Styrax doses. Some of these effects may be ad- camporum extract showed genotoxicity to verse on highly sensitive signalling path- Chinese hamster lung fibroblasts (V79 ways triggered by steroid hormones or cells) at 60 μg/mL. The genotoxic effect thyroid hormones (Tsatsakis et al., 2014). was attributed to the presence of benzofu- ran lignans, egonol I and homoegonol POSSIBLE SOURCES OF (Tavares et al., 2014) which were also UNCERTAINTY responsible for antitumour activity at 9.1 μg/mL against the Hela cancer cell lines Toxicogenomics is a mature field, which (Oliveira et al., 2014). Lee et al. (2014) provides invaluable information on the conducted 90-day repeated dose toxicity molecular events preceding or accompa- and genotoxicity of Evodia officinalis and nying toxicity. Most traditional uses of concluded that the plant did not show transcriptional profiling and other- mutagenic potential, chromosomal aberra- data in toxicology are: mode-of-action tion and micronucleus formation. Every analysis, classification/prediction, and year, more than hundred new drugs are biomarker discovery. While these applica- approved in Japan. All of them have some tions are very informative for the regula- scientific uncertainty in terms of efficacy tory decisions, they do not contribute to and safety at the time of drug approval. A quantitative evaluation of the margin of better understanding of regulatory science safety and/or characterisation of the un- in the field will contribute to increasing certainties in species-to-species extrapola- efficiency of drug development and pro- tion and population variability. Studies viding a better drug to patients (Uyama, that explore the shape of dose-response 2015). The challenge for toxicologists in relationships at the molecular pathway future will be for them to take an even level, and the impact that inter-individual more active role in opposing more effec- differences in given expressions patterns tively poor science, including inadequate may have on both efficacy and safety out- interpretation of data such as those about comes, represent some of the novel fron- supposed risk that are not based on an tiers in toxicogenomics. Dose-response

14 BJVM, ××, No × S. A. Saganuwan toxicogenomic data may be used for cal- is generally assumed that no threshold culating the point-of-departure at which dose level exists. In this case determina- either adverse or adaptive “molecular pa- tion of dose level for regulation relies on thology” phenotypes are observed after mathematical model to relate predicted exposure to chemicals. Aggregation of the risks to dosage (Barnes & Dourson, dose-response into pathways further 1988). Originally, tolerance distribution improves the linkages between mechanis- like the logit and probit models used for tic understanding and estimating the mar- non-carcinogenic responses were used gin of safety. In addition, the challenge of (Krewski & Brown, 1981). A model elucidating the genetic determinants of called the multistage model developed by inter-individual’s differences in toxicity Armitage & Doll (1961) has become very may be met through a combined analysis popular for cancer risk estimation. The of the toxicity phenotypes, genetic poly- statistical model involves up to about morphisms, and data seven stages and appears to fit observed from the population-base experimental in cancer incidence curves reasonably well. vivo and in vitro model systems. The in- A modified form, referred to as the linear- tricate interplay between genetic poly- ised multistage model is described by morphisms and gene expression requires Howe et al. (1986) and dates risk to low careful consideration of the genetic back- doses. Perhaps the most difficult problems ground dependent and independent toxic- in toxicologic risk assessment are the con- ity pathways. Therefore, as the number of version of animal data to humans. Ideally, toxicogenomic studies which incorporate quantitative interspecies extrapolation dose-response and/or population-based should take into account known species study designs is in the rise, the applicabili- differences. Dose equivalency between ty of such data to next-generation health species has traditionally been based on risk assessments will also increase body weight and body surface area. Travis (Ruzyn, 2014). For example, there is un- & White (1988) have recommended an certainty concerning the safety implica- intermediate scale based on body weight tions of toxic xenobiotics as they are also to the ¾ power for dose conversion be- associated with a variety of diseases such tween species. The application of trans- as inborn errors of metabolism. Phospho- genic animal models to elucidate human lipidosis is also a common finding in non- pathways of drug metabolism and chemi- clinical toxicology studies designed to cal toxicity has been proposed (Wolf et enable clinical trials and marketing appli- al., 2014). The omic technologies have cations for new drugs. It is usually associ- not been, and never will be the panacea ated with cationic amphiphibic drugs for all the challenges in toxicology for the (CADs) with distinct structures; they con- fact that they have yet to find their ways tain both hydrophobic and hydrophilic into regulatory acceptance (Gant, 2015). domains, which enables transit through Both animal tests, as well as their various cellular membranes and intracellular con- in/ex vivo, in vitro and in silico replace- centrations. This chemical property may ments are often not sufficiently predictive be desirable for drugs that act on intracel- of their intended toxic endpoint (Bender, lular targets and could be related to phar- 2014). For example, human oligodendro- macodynamics and drug efficacy (Hast- genesis is the most sensitive endpoint be- ings, 2014). For carcinogenic endpoint, it ing 10 times more sensitive than human

BJVM, ××, No × 15 Toxicity studies of drugs and chemicals in animals: An overview neurogenesis which has the least sensitive city and sensitisation are actual topics endpoint (IC50: μM and 23 μM). Sensitivi- with promising perspective to reduce ani- ty of murine neurogenesis and oligoden- mal use (Van Der Laan, 2014). ICH M3 drogenesis differs only slightly, IC50: 10 (R2) guidance has helped clarify the tim- μM and 14 μM (Dach et al., 2014). Also, ing of reproductive toxicology studies in cleft palate is a craniofacial birth defect drug development. Toxins may be evalu- that affects one in every 700 newborns ated qualitatively using nature of toxins, worldwide. This defect results from a dis- quantitatively using chemistry of toxin ruption in the development of the secon- and their concentration. Colorimetry and dary palate (palatogenesis) which can be spectrophotometry can be used for quali- caused by several genetic and environ- tative analysis of toxins whereas infrared mental factors. Morphologically, palato- spectroscopy, gas chromatography, high genesis in mice, rats and human consists pressure liquid chromatography and im- of palatal shelves, development, growth, munoassay techniques may be used to elevation, content and fusion. The mouse quantify toxins (Parasuraman, 2011). is the mostly commonly used species for However, the guidance remains general the study of palatogenesis and the rat is and may not apply to all drug candidates the species used in human risk assessment for all circumstances. Therefore, in addi- studies; although it is assumed that the tion to the guidance document, other fac- molecular pathways underlying palato- tors such as pharmacological trial design genesis in mice, rats and humans are simi- should be considered when planning the lar, similar expression patterns between timing of these studies. In embryo-foetal mice and rats suggest that the principal development studies, exposure to drug pathways underlying pathogenesis are could be indicated by the presence of ma- conserved in both species (Vicario et al., ternal toxicity, and ICH did not require 2014). collection of toxicokinetic data in these studies. This results in the absence of toxicokinetic data in some studies, and ANIMAL WELFARE ISSUES assessment of risk of pregnant mothers is Animal welfare comprises the state of the done using the conversion of animal dose animal body and mind, and the extent to to human-equivalent dose based on body which its nature (genetic traits manifest in surface area. This approach is less precise breed and temperament) is satisfied (Dun- and occasionally not accepted by regula- can & Fraser, 1997). The discussion about tory agencies. In rabbit embryo-foetal the protection of animals for scientific development study, the absence of purposes is always important in the Euro- PK/ADME data, coupled with the occa- pean Union. A recent directive (2010/63) sional absence of TK data, has led many renewed the previous one after 25 years, to question the relevance of the absence of further emphasising that animals should this species in risk assessment. A survey only be used when clearly needed. In cos- has been conducted to address this ques- metics legislation, animal data are no tion. Under the EMA regulation, planning longer allowed for regulatory purposes. for the juvenile animal study should be Stimulation of in vitro approaches is submitted early in development. Two ap- therefore important in the research activi- proaches are generally considered when ties. Reproduction toxicity, carcinogeni- designing the juvenile animal study. In the

16 BJVM, ××, No × S. A. Saganuwan targeted design approach, the study will holds true for more complex toxicological evaluate large organs of concern in the endpoints related to repeated dose toxici- target population. In the absence of target ty. Using omics data provides a better organs, the modified general toxicity prediction of repeated dose toxicity (Jen- screening approach may be used to iden- nen et al., 2014). In vitro testing is carried tify hazards in the young and developing out in two or three different bacteria and animals (Hew, 2014). Before conducting mammalian cells to cover the end points any toxicological testing in animals or of genemutations clastogenicity and ane- collecting tissue/cell lines from animals, uploidy. The test generally includes a bac- the study should be approved by the Insti- terial reverse mutation assay. The chance tute of Animal Ethics (IAEC) or the pro- of an additional test depends on the tocol should satisfy the guidelines of the chemical structure/class of the substance local governing body. The guidelines dif- (Gholami et al., 2010). A major goal of fer from region to region, country to coun- human in vitro toxicogenomics is to im- try and institution to institution (Schedule, prove the assessment of the toxic hazard 1995). Low, medium and high doses se- and ultimately the risk due to the exposure lected should be sufficient to identify the to chemicals with assays based on human target organ toxicity (Robinson & Chap- cells lines rather than in vivo in rodents. man, 2009). Using high-throughput gene expression read-outs, this approach requires solid and robust computational pipelines and tools ALTERNATIVE METHODS OF in order to identify responsive genes and TOXICOLOGICAL RESEARCH biological pathways capable of predicting According to Assuring Safety Without toxicity of chemicals (Van Delft et al., Animal Testing (ASAT) principle, risk 2004). Classification of untested chemi- assessment may ultimately become possi- cals, which make up more than 95% of all ble without the use of animals (Fenton et chemicals on the market, is not possible al., 2014). The ASAT concept takes hu- without some form of prediction based on man disease mechanisms as starting point a simplified alternative biological model and tries to define if activation of these or a simplified chemical structure-activity mechanisms by chemical exposure in in relationship. The OECD QSAR Toolbox vitro models can be used for toxicological is a freely available software that imple- risk assessment. Chemical analogues iden- ments structure based approaches to the tified by chemo-informatic tools are pre- prediction of toxicological data: it exploits sumed to induce similar biological re- the possibility of combining chemical and sponses, such as similar toxicities. There- biological information to assess the toxici- fore, toxicity may be predicted by read- ty of a query chemical starting from the across from members of the same chemi- toxicity and structural data of analogue cal class. However, compounds with chemicals. A priority of OECD for the seemingly subtle changes in chemical near future is to implement Adverse Out- structure, e.g. the classical example of 2- come Pathways (AOPs) into the Toolbox, AAF and 4-AAF, show relevant changes and provide the investigators with addi- in toxicological properties, thus making tional tools to predict complex toxicologi- prediction solely based on chemo- cal endpoints (Benigni & de Knecht, informatics insufficient. This especially 2014). The use of engineered nanomate-

BJVM, ××, No × 17 Toxicity studies of drugs and chemicals in animals: An overview rial has grown rapidly in a number of in- on the inclusion of recovery animals to dustrial and consumer products. At the toxicology studies to best assess human same time, concerns of possible harmful- safety, while reducing animal use. The health effects of engineered nanomaterial initiative is led by the NC3R in collabora- have increased, and this development may tion with the Medicines and Healthcare seriously jeopardise the successful use of Products Regulatory Agency (MHRA). these valuable materials (Savolainen et Recovery animals are used in pharmaceu- al., 2013). A key element of using in vitro tical development to provide information toxicity data in chemical risk assessments on the potential for a toxic effect observed is linking the effect of concentrations from in animals to translate into long-term hu- in vitro assays to those in the target tissue man risk. They are included in toxicology in vivo. This extrapolation requires an studies to assess whether effects observed estimate of in vivo uptake and clearance. during dosing persist or reverse once However, currently available in vitro ki- treatment ends. An analysis of individual netic assays have significant limitations in study and programme design shows that the in vitro system (Yoon et al., 2014). there are opportunities to reduce the use Therefore, there is need for functional of recovery animals in certain circum- association of the identified biomarker stances, which would not impact drug activities with each cell system that can be development (Sewell & Chapman, 2014). used to support the elucidation of adverse In human risk assessment any relevant outcome pathways connecting molecular exposure route has to be addressed. Con- in initiating event to specific human ad- sequently, for numerous chemicals no verse effects (Berg et al., 2014). The use levels have to be deter- of recreational drugs has changed in re- mined for all three routes (dermal, oral, cent years due to the rapid emergence of inhalation), leading to testing that de- new psychoactive substances known as mands a large number of animals. On the “smart drugs” or “legal highs” which have other hand, the 3R principles (reduction, the biased reputation of being safe, the refinement and replacement of animal vast majority, has hitherto not been tested testing) shall be implemented in risk as- and several fatal cases have been reported, sessment according to several regulations, namely for synthetic cathinones, with e.g. REACH. A reduction of animal num- pathological patterns comparable to am- bers could be achieved by using route-to- phetamines (Araujo et al., 2014). There- route (R2R) extrapolation factor (EF) to fore, there is need to assess toxicity ef- derive the relevant route-specific no- fects of cathinones in experimental ani- adverse-effect-level. One prerequisite for mals with a view to exploring their toxic- this approach is the use of scientifically ity effects. An international expert group sound extrapolation factors, which are which includes 32 organisations (pharma- based on a high quality and broad data ceutical and biotechnology companies, basis (Schroeder et al., 2014). It is contract research organisations and regu- meritable to accumulate and manage suf- latory bodies) has shared data on the use ficient chemical information and toxicity of recovery animals in the assessment of from experiment and, if not possible, pre- pharmaceutical safety for early develop- dicted-evaluation results. The prediction ment. The group has used these data as an of toxicity and harmfulness are known as evidence base to make recommendations very important, for its cost-effective bene-

18 BJVM, ××, No × S. A. Saganuwan fit and handling the newly designed which may or may not lead to death there- chemicals (Cha et al., 2014). In the field fore, animal welfare is a public good that of human exposure sciences, chemical- should be protected. Consensus on how specific exposure information is needed to best to protect welfare and to balance it assess the extent and risk of exposure and with legitimate human interests requires to identify important exposure sources. informed public debate. Veterinarians are However, due to the large amount of uniquely placed to inform the public about available data, finding relevant exposure the issues, and most particularly about studies and identifying knowledge gaps is animal’s interest (Hewson, 2003). extremely time-consuming. To overcome these challenges and make scientific lite- CHALLENGES TO TOXICOLOGICAL rature more assessable for researchers, RESEARCH “text mining techniques” have shown great potential. The applicability of text Benchmark dose modelling is increasingly mining approaches to support the risk used as the preferred approach to define assessment process requires validation the point-of-departure for health risk as- (Larsson et al., 2014). Vitamins and mine- sessment of chemicals. As data are inhe- rals are micronutrients, essential compo- rently variable, there is always a risk to nents of the human diet, and may have select a model that defines a lower confi- adverse effects if consumed in excessive dence bound contrary to expected. Non- amounts. There is also a lower level of sigmoidal model should be used with cau- intake below which risk of deficiency can tion as it may underestimate the risk occur, and an exposure assessment is re- (Oberg et al., 2014a). Therefore, there is quired to identify the correct level of sup- need for revision of the current dose- plementation to meet the needs of each response model selection paradigm, e. g. age group. The daily exposure for some as expressed in the expert opinion from micronutrients shows that total intake European Agency (EFSA) could potentially exceed the tolerable up- and in the current modelling software in per intake level for various age groups. which dose-response/effect models are However, the intake data used in the pub- selected based on principle of Ocham’s lished calculations is often old, limited to razor (Oberg et al., 2014b). In the Euro- adults or by country and can be skewed to pean Union, under REACH regulation, high-end-exposure. The crème global die- companies are faced with the need to tary exposure models are based on Monte phase out chemicals of very high concern Carlo simulations and look at the full dis- and are challenged to identify and demon- tribution of intake for the population or strate the safety of chemical alternatives. subpopulation of interest. This realistic These substances are mainly identified by approach is common in risk analysis for their harmonised classification and label- food safety and accepted by regulators, ling according to the CLP regulation but is seldom used in the field of nutri- based on their intrinsic hazard properties. tional regulation (O’Keefe & Pigat, 2014). Green Screen® for safer chemicals is pro- Suggesting that some experiments may posed as a tool that can help companies have to be performed on some animals to assess and avoid chemicals of high con- explore, discover and evaluate toxicity cern under REACH. In the , effects of some drugs and chemicals, leading companies, driven by the desire to

BJVM, ××, No × 19 Toxicity studies of drugs and chemicals in animals: An overview

be competitive and stay ahead of future Armitage, P. & R. Doll, 1961. Stochastic regulations, are using Green Screen®. In models for carcinogenesis. In: Proceedings the EU, the method is included in aspect of the Fourth Berkeley Symposium on of the training programme offered by Mathematical Statistics and Probability, University of California Press, Berkeley SUBSUPPORT Project. Although Green CA, 4, 1938. Screen® is freely available, technical ex- pertise is required to complete its assess- Asiedu-Gyekye, I. J., 2014. Toxicological study of new molecules in treating raw wa- ment (Zachary et al., 2014). In Europe, ter to make it possible. Journal of Clinical scientific and public demand has driven Toxicology, 4, 49. development of regulatory in vitro assays ASTM, 1987. Standard Test Method for Esti- across a range of disciplines (Akhurst et mating Acute Oral Toxicity in Rats. al., 2014). American Society for Testing and Materi- In conclusion, since the results from in als, Philadelphia P.A, USA. vitro assay cannot give true reflection of Azevedo, R., V. M. Costa, D. Barbosa, A. in vivo parameters, the rule of thumb is Gomes, I. Pita, A. Sequeira, F. Pereira, M. the use of animals for toxicity studies. J. A. Duarte-Araujo Duarte, E. Fernandes, This can be complemented by other meth- M. L. Bastos, F. Carvalho & J. Capela, ods aimed at encouraging replacement, 2014. Aged rats are more prone to “ectasy” refinement and reduction. neurotoxicity than adolescent rats. Toxi- cology Letters, 299S, S241. Barnes, D. G. & M. Dourson, 1988. Reference REFERENCES dose (R & D): Description and use in health risk assessment. Regulatory Toxi- Ahmed, M., 2008. Isolation, characterization cology & Pharmacology, 8, 471–486. and possible biocontrol application of Bdellovibrionaceae (BD) isolated from Bender, A., 2014. Toxicity prediction using New Zealand Sources. PhD Thesis, heterogenous biochemical and biological Massey University, New Zealand. data sources. Toxicology Letters, 299S, S4. Akhila, J. S., S. Deepa & M. C. Alwar, 2007. Benigni, R. & J. de Knecht, 2014. The future Acute toxicity studies and determination of of the QSAR Toobox: Moving to less un- median lethal dose. Current Science, 93, certainty in predictive toxicology. Toxico- 917–920. logy Letters, 229S, S7. Akhurst, L., J. Carter, B. Burlonson & M. Berg, E., M. Polokoff, A. O’Mahony & J. Chapman, 2014. Development of regula- Yang, 2014. Elucidating adverse outcome tory in vitro models. Toxicology Letters, pathways with phenotypic data from pri- 299S, S40–S252. mary human cell system. Toxicology Let- ters, 299S, S55. Ameni, A., A. Latorre, L. Torres & S. Gorniak, 2014. Risk assessment of medicinal plants Booth, A., R. J. Ameh, M. Scott & F L. Caseria Sylvestris Sw (Salicaceae). Toxi- Greenway, 2010. Oral dose-ranging devel- cology Letters, 299S, S40–S252. opmental toxicity study of an herbal sup- plement (NT) and garlic acid in rats. Ad- Araujo, A. M., M. Valente, D. D. da Silva, M. vanced Therapy, 27, 250–255. Carvalho, F. Carvalho, M. D. L. Bastos & P. G. de Pisho, 2014. Smart but not safe: Borzelleca, J. F., 1999. Profiles in toxicology, The potential hepatoloxicity of synthetic Paracelsus: Herald of modern toxicology. cathimones. Toxicology Letters, 299S, Toxicological Sciences, 53, 2–4. S64.

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Science and Technology, Nagoya Con- toxicity of Inauhzin in mice. Toxicology gress, Centre, March 18–20, 2015. Reports, 2, 546–554. Yoon, M., J. Pedersen, E. LeChyse, M. Andersen & H. Clewell, 2014. Liver bio- reactor as an in vitro metabolism system for quantitative in vitro to in vivo extrapo- lation. Toxicology Letters, 299S, S13. Paper received 10.12.2015; accepted for York, M. & W. A. Steilling, 1998. A critical publication 26.02.2016 review of the assessment eye irritation po- tential using the Draize rabbit eye test. Correspondence: Journal of Applied Toxicology, 18, 223– 240. S. A. Saganuwan Zachary, M., A. M. Gechart & M. Whittaker, Department of Veterinary Physiology, 2014. Green Screen® for safer chemicals, Pharmacology and Biochemistry, as a support tool for the EU reach regula- College of Veterinary Medicine, tion. Identifying chemicals of very high University of Agriculture, concerns (SVHC) and safer alternatives. P.M.B. 2373, Makurdi, Toxicology Letters, 299S, S40–S52. Benue State, Nigeria Zhang, Q., S. X. Zeng & H. Lu, 2015. Deter- tel: +2348027444269, +2347039309400 mination of maximum tolerated dose and e-mail: [email protected]

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