Basic toxicology and biomaterials testing

Transcription

1 Biomateriaalitiede 2015 Basic toxicology and biomaterials testing Matti Viluksela University of Eastern Finland Department of Environmental Science and National Institute for Health and Welfare Chemicals and Health Unit Kuopio, Finland

2 Contents Some basics of toxicology Risk and risk assessment Testing for toxicity Biomaterials and biocompatibility testing 2

3 Basic definitions Toxicology Study of adverse effects of chemical substances on living organisms The relationship between dose and its effect is of high significance Toxicokinetics What the body does to the chemical? 1. Absorption 2. Distribution 3. Metabolism 4. Excretion Toxicodynamics What the chemical does to the body? 3

4 Reasons for outcome of toxicity in certain organ, site, species, stage of development 1. High concentration / dose Liver Kidney Sites of entry: skin, lungs, gi-tract 2. Sensitive target (organ, stage of development, species) High oxygen demand Inefficient protection mechanisms Specific actions on receptors, enzymes, ion channels Critical (time) window of sensitivity 4

5 Toxicokinetics and toxicodynamics Toxicokinetics What the body does to the chemical Toxicodynamics What the chemical does to the body 5

6 Toxicokinetics and toxicodynamics A bsorption D istribution M etabolism E xcretion ADME studies 6

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8 Concentration of the chemical in the body C max Concentration AUC area under the curve T 1/2 T max Time 8

9 Test chemical tissue concentration determines toxicity Influence of absorption rate Severe toxic effects Concentration Toxic effects No toxic effects Time 9

10 Influence of dosing regimen Concentration (µg/ml) Oral 1 x 480 mg Oral 4 x 120 mg Oral 16 x 30 mg Continuous dermal exposure Time (h) 10

11 Bioavailability 11

12 Risk Risk = severity x probability Risk = Hazardous property (severity) x Exposure (probability) Environmental hazard, Health hazard Acute toxicity, chronic toxicity, skin irritation, sensitization, reproduction and developmental toxicity, genotixicity, carcinogenicity What dose / exposure level causes the hazard? The amount of chemical entering the body Risk is not only mathematics, but also mental impressions risk perception! 12

13 Hazard Health hazard Toxicity acute toxicity (sub)chronic, repeated dose toxicity, delayed toxicity, 90 days organ toxicity (liver toxicity, kidney toxicity, neurotoxicity ) Local effects, irritation, corrosion skin eyes Sensitization, allergy Reproduction and developmental toxicity Mutagenicity Carcinogenicity If hazardous properties are observed only at high exposure levels, exposure does not necessarily constitute a risk! 13

14 Exposure 14

15 Exposure pathways 15

16 Exposure assessment chemical analyses (working environment. air, water, food ) biomonitoring analysis of tissue levels intake calculations questionnares, interviews If there is no exposure, the hazard does not constitute a risk! 16

17 Risk assessment Hazard identification What is the level of exposure in comparison with the dose level causing toxicity? Exposure assessment ---???--- Determination of doseresponse relationship = Hazard characterization Risk characterization 17

18 Hazard identification Can the chemical cause a hazard? How to study? epidemiological studies clinical trials experimental studies environmental studies QSAR What is found out? toxic effects (ideally also mechanisms) (dose-responses, NOAEL) kinetics sensitivity differences 18

19 Exposure assessment How, when and how much are humans exposed? How to study? chemical analyses (air, water, food ) intake calculations interviews analysis of tissue (blood, urine) levels What is found out? exposed populations identified routes of exposure exposure levels 19

20 Hazard characterization, determination of doseresponse relationships Dependence of the toxic effect on exposure level? What is a safe dose for humans? How to study? What is found out? comparison of effects at different exposure levels extrapolation to humans animal fi human high dose fi low dose Response Control LOAEL NOAEL Dose 20

21 Risk characterization What is the probability for an adverse effect in different populations? How to study? analysis of all data available from the earlier steps of RA outline of needs for risk management What is found out? a comprehensive view on the risk(s) uncertainties needs and extent of risk management 21

22 Toxicity depends on Compound Chemical structure Effects Potency Kinetics Exposure Dose / concentration Time course, frequency Route of exposure Exposed individual Species, gender, age Genome Environment Health status, diseases 22

23 Toxicological information and degree on uncertainty Quantitative structure-activity correlations (QSARs), in silico Experimental studies in vitro short-term in vivo long-term in vivo mechanistic Human studies case reports clinical epidemiological Weight of evidence: a process in which all of the evidence considered relevant to a decision is evaluated and weighted 23

24 Potency and efficacy Potency dose or concentration of a chemical that will cause a certain response, e.g. half of maximal (ED50) Efficacy magnitude of the maximal response efficacy of a full agonist = 1 efficacy of a full antagonist = 0 24

25 Potency and efficacy Response (%) Dose ED50 Response (%) Emax Dose Compounds with different potency, but same efficacy. Compounds with different efficacy. 25

26 Risk extrapolation from animals to man 100 HUMANS sensitive individuals HUMANs population mean ANIMALS population mean Response INTRASPECIES INTERSPECIES ADI NOEL Dose 26

27 Margin of exposure Response Human exposure level Margin of exposure LOAEL NOAEL Tissue concentration / Dose NOAEL = no observable adverse effect level LOAEL = lowest observable adverse effect level 27

28 An example of a toxic effect of chemical: Ethanol induced live injury Normal liver Fatty liver Liver chirrosis Kemikaaliriskit / Matti Viluksela

29 Ethanol induced liver injury: pathophysiological mechanisms Mechanisms of alcoholic fatty liver. (1) Alcohol consumption can directly (via acetaldehyde) or indirectly (via regulation of multiple factors) up-regulate the expression of SREBP-1c and downregulate the expression of PPAR-α, leading to the induction of fatty acid synthesis and inhibition of fatty liver β-oxidation, which results in the development of alcoholic fatty liver. Alcohol exposure also inhibits AMPK and subsequently increases ACC activity but decreases carnitine palmitoyltransferase 1 (CPT-1) activity, leading to an increase in fatty acid synthesis and a decrease in fatty acid β-oxidation. (2) Alcohol consumption can also modify many factors, including HIF-1, C3, C1qa, PKCε, and inos, that subsequently contribute to the development of fatty liver. The mechanisms underlying the effects of these factors remain unclear. Figure 2. Proposed mechanism for alcoholic liver disease. Chronic alcohol abuse increases gut permeability resulting in high circulating endotoxin that reaches the liver via portal circulation. Endotoxin (lipopolysaccharide or LPS) is recognized by the Toll-like receptor (TLR)-4 complex on resident macrophages or Kupffer cells in the liver, leading to production of proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α, and resulting in injury to liver cells (hepatocytes). Kemikaaliriskit / Matti Viluksela

30 Hepatocellular carcinoma Kemikaaliriskit / Matti Viluksela

31 Risk perception Paul Slovic 31

32 Expert vs. layman: different risk assessment Action / exposure Estimated risk ranking Expert Layman Motor vehicles 1 2 Smoking 2 4 Alcoholic beverages 3 6 Hand guns 4 3 Surgical operations 5 10 X-rays 7 22 Production of electricity (not nuclear) 9 18 Nuclear energy

33 Particulate matter in urban air Annually about PM related deaths in Europe, in Finland about 1300 Sensitive individuals: small children, asthmatics, people with cardiovascular diseases, elderly people Mad cow disease (bovine spongiform encephalopathy = BSE) vcjd: altogether about 200 persons died Kemikaaliriskit / Matti Viluksela 33

34 Risk management risk communication, training, classification and labeling monitoring and limiting of exposure restrictions in the use of chemical banning the use of chemical 34

35 Particulate matter in urban air

36 Deaths attributed to 19 leading factors, by country income level, 2004 (World Health Report 2009) 5.1 million deaths 2.0 million deaths

37 Scope of safety evaluation studies Products pharmaceuticals agrochemicals biocides industrial chemicals cosmetics consumer products Safety evaluation studies acute toxicity dermal / ocular irritation sensitization subchronic / chronic toxicity reproduction toxicity developmental toxicity genotoxicity carcinogenicity Test guidelines, Good Laboratory Practice (GLP) OECD Guidelines for Testing of Chemicals ICH (International Congress on Harmonisation) 37

38 Safety evaluation studies Properties to be studied physical and chemical properties toxicity ecotoxicity Types of studies laboratory investigations in vitro experiments animal experiments clinical trials epidemiological studies 38

39 A typical safety evaluation program Chemical Identification and Characterization Literature Review, Structure-Activity Relationships Local Toxicity Acute Toxicity Sensitization Pharmacokinetics Toxicokinetics Subchronic Toxicity Genotoxicity Reproductive and Developmental Toxicity Chronic Toxicity Special Studies Carcinogenicity 39

40 Selection of doses Therapeutic dose range Safety range Toxic dose range Lethal dose range NO(A)EL MTD Control Low dose Medium dose High dose Safety Evaluation Studies 40

41 Subchronic and chronic toxicity testing (1) Subchronic toxicity the adverse effects resulting from repeated exposure over a portion of the average life span (<10%) of an experimental animal duration usually 1-3 months Chronic toxicity the adverse effects resulting from prolonged and repeated exposure duration usually at least 6-12 months 41

42 Subchronic and chronic toxicity testing (2) Objectives define the target organs establish the nature and reversibility of toxic effects establish dose-responses and NO(A)EL observe accumulation, tolerance determine differences in sex / species sensitivity select dose-levels for chronic studies 42

43 Subchronic and chronic toxicity testing (3) Methods and procedures at least 2 animal species (rat and dog / minipig) routes of administration: oral, dermal, inhalation etc. preferably the same route that man is likely to be exposed) number of animals: rodents 10 (5-20)/sex/group large animals 4 (3-8)/sex/group daily administration, 3 dose-levels and a control group 43

44 Subchronic and chronic toxicity testing (4) Observations clinical signs and symptoms, behaviour body weights food consumption water consumption 44

45 Subchronic and chronic toxicity testing (5) Laboratory investigations haematology clinical chemistry, hormones urine analysis ophthalmoscopy electrocardiography, blood pressure etc. toxicokinetics (rodents: separate satellite groups) organ weights macroscopic pathology and histopathology liver, kidney, nervous system, reproductive organ etc. function tests upgrades : endocrine system, pharmacological effects 45

46 Subchronic and chronic toxicity testing (6) Growth rate and food consumption sensitive indicators of general toxicity monitoring the health status of individual animals Animal welfare if an animal shows severe signs of toxicity, e.g. decreased body weight development (>25% below controls), ill health, suffering dosing should be discontinued if needed, the animal should be euthanized 46

47 Subchronic and chronic toxicity testing (7) Organ weights may reveal a specific target organ response body weight related organ weights decrease in body weight decrease in fat deposits increased liver / kidney weight may be an adaptive response to increased functional load, not necessarily a true toxic event associated with morphological chances? associated with functional changes? 47

48 Subchronic and chronic toxicity testing (8) Reversibility of effects additional recovery groups may be included usually controls + high dose, both genders an off-dose period, e.g. 2 weeks similar observations and investigations as for main study animals 48

49 Carcinogenicity testing 2 animal species, usually rat and mouse duration for the major portion of the life span of the animals: rat 24 months mouse 18 months number of animals: at least 50/sex/group routes of administration: usually oral, dermal or inhalation macroscopic pathology and histopathology are extremely important 49

50 Predictive ability of animal experiments predictability for organ toxicity fairly good sensitivity for predicting carcinogenesis fairly good, but not all animal carcinogens are human carcinogens (false positives) concordance in carcinogenicity between rats and mice only 70%; predictive ability for humans is likely to be lower understanding mechanisms of toxicity reduces uncertainty 50

51 ISO : Biological evaluation of medical devices 51

52 ISO :2003(E) 52

53 Biological safety assessment of a medical device 53

54 Biological safety testing: some principles Certain tests (e.g. testing for systemic effects) may not be applicable where the presence of leachable materials has been excluded, or where leachables have a known and acceptable toxicity profile. Protection of humans is the primary goal, a secondary goal is to ensure animal welfare and to minimize the number and exposure of test animals. The test shall be performed on either a) an extract of the material and/or b) the material itself Extraction conditions should attempt to simulate or exaggerate the conditions of clinical use so as to determine the potential toxicological hazard, without causing significant changes in the test material such as fusion, melting or alteration of the chemical structure. 54

55 Biological safety assessment of a medical device 1. Chemical nature of the materials (including additives, contaminants, leachables, degradation products) Existing knowledge Prior use of the materials 2. Intended use: conditions of exposure, nature, degree, frequency and duration of exposure 3. Biological safety test data All potential biological hazards should be considered for every material and final product, but this does not imply that testing for all potential hazards will be necessary or practical certain tests (e.g. testing for systemic effects) may not be applicable where the presence of leachable materials has been excluded, or where leachables have a known and acceptable toxicity profile 55

56 Documentation of a biological safety assessment 1. A general description or drawing of the device 2. Quantitative formulations for all materials in contact with tissues or infusable fluid 3. A review of available toxicity and prior use data for each material and chemical in contact with tissues or infusable fluid 4. Information on previous experience with each material in related applications 5. Reports of biological safety tests 6. An assessment of the data reviewed 56

57 Biological safety assessment of a medical device Testing shall be performed on the final product, or on representative samples taken from the final product or from materials processed in the same manner as the final product. Initial evaluation tests 1. Cytotoxicity 2. Sensitization 3. Irritation / intracutaneous reactivity 4. Systemic toxicity Acute toxicity Subacute and subchronic toxicity 5. Genotoxicity 6. Implantation 7. Haemocompatibility Supplementary evaluation tests 1. Chronic toxicity 2. Carcinogenicity 3. Reproductive and developmental toxicity 4. Biodegradation 57

58 Biocompatibility testing: Irritation / intracutaneous reactivity Objective Irritation: To estimate the irritation potential using appropriate sites for implant tissue such as skin, eye and mucous membrane in a suitable model. Intracutaneous reactivity: To assess the localized reaction of tissue to medical device extracts. Applicable where determination of irritation by dermal or mucosal tests are inappropriate (e.g. medical devices having access to the blood path). May also be useful where extractables are hydrophobic. 58

59 Biocompatibility testing: Sensitization Objective To estimate the potential for contact sensitization. These tests are appropriate because exposure or contact to even minute amounts of potential leachables can result in allergic or sensitization reactions. Methods Currently only in vivo methods available Local lymph node assay (LLNA) Guinea pig maximization test (GPMT) Several in vitro methods under development 59

60 Biocompatibility testing: Implantation Objective To assess local pathological effects on tissues Methods The material or product is surgically implanted in implantation site or in appropriate (clinically relevant) tissue Test specimen is compared with control material (wellknown, widely used, acceptable biocompatibility) Equivalent to subchronic / chronic toxicity tests if systemic effects are also investigated 60

61 ISO :2003(E) Annex B (informative) 61

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63 ISO :2003(E) 63