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  1. 1. Chapter 4 The Dose-Response Relationship
  2. 2. Introduction • The need to establish some criteria to base the relative safety of chemicals is essential. • Several methods are used to obtain data in order to establish safe levels and provide information about the relative toxicity of chemicals.
  3. 3. Acute Toxicological Studies (1) • Information concerning the toxicity of a substance is obtained primarily from either acute or chronic toxicity studies. • Acute toxicity studies are the most commonly performed studies for obtaining information on the effects of chemical exposure. They are short-term, relatively inexpensive tests with death of the test animal being the useful observed effect. • Information obtained from acute toxicity tests can be used to – determine the relative toxicity of different chemicals comparing the respective LD50 values, which are the doses that prove to be lethal for 50 percent of the test animals; – identify the target organs (heart, liver, kidneys, etc.) that are affected as a result of exposure; and – determine the appropriate doses for long term, chronic studies.
  4. 4. Acute Toxicological Studies (2) • To perform acute toxicity studies the test animal population is divided into several groups, with an equal number of individuals (usually 10 to 50) in each group. One group of test animals –referred to as the control group – is exposed to all the same experimental conditions except exposure to the toxic substance. • The groups are observed for 14 days, and the number of deaths in each group is recorded.
  5. 5. Acute Toxicological Studies (3) • LC50 is a measure of chemical toxicity resulting from inhalation. LC means “lethal concentration” and the subscript has the same meaning as described previous for LD. The unit of measure for the LC50 is usually expressed as part per million (ppm).
  6. 6. Chronic Toxicological Studies (1) • Chronic toxicity tests may be performed over a period of months, years, or the lifetime of the test animal. Doses of the toxic substance are selected to assure that most of the animals will survive the entire time the study is performed. • Different species of test animals may be more or less sensitive to the same toxic chemical. • Males and females of the same species may respond differently to the same substance. • Although acute and chronic studies provide useful information in evaluating chemical toxicity, it is important to understand that they are not truly representative of the environment to which people are exposed in everyday life.
  7. 7. Chronic Toxicological Studies (2) • Characteristics of animal studies – A single dose – A single route of exposure – The number of animals exposed is small. – The genetic make-up of the population is not very diverse. – Only individuals in good health and/or of the same sex are selected. • In reality – Several different routes – Exposure to more than one chemical at a time – Greater genetic variability in a larger population – the range of responses to a given chemicals more diverse. – The collective interactive effect of all these factors on toxicity makes it difficult to establish a cause-effect relationship.
  8. 8. Dose-Response Curves (1) • The major purpose for performing acute and chronic toxicity studies is to establish a cause-effect relationship between exposure to a toxic substance and an observed effect in order to determine a safe exposure level. • A curve can be drawn that illustrates the relationship between the dose administered and the observed response. This curve is referred to as the dose-response curve. • A dose-response curve can be developed form most chemicals. From these curves the threshold level and the relative toxicity of chemicals can be obtained to help establish safe levels of chemical exposure.
  9. 9. Dose-Response Curves (2) • The threshold is the dose below which no effect is detected or above which an effect is first observed. • The threshold information is useful information in extrapolating animal data to humans and calculating what may be considered a safe human dose for a given toxic substance. • The threshold dose (ThD0.0) is measured as mg/kg/day. It is assumed that humans are as sensitive as the test animal used. To determine the equivalent dose in man the ThD0.0 is multiplied by the average weight of a man, which is considered to be 70 kg.
  10. 10. Dose-Response Curves (3) • The calculation used to determine the safe human dose (SHD) is as follows: substance toxic of mg/day Amount SF (kg) 70 ThD SHD 0.0    Where SHD = Safe Human Dose ThD0.0 = Threshold dose at which no effect is observed. 70 Kg = Average weight of a man SF = Safety factor (ranges from 10 to 1000, which varies according to the type of test and data used to obtain the ThD0.0.
  11. 11. Terminology Associated with Dose-Response Curves (1) • No observed effect level (NOEL) – The highest tested dose of a substance that has been reported to have no harmful (adverse) health effects on people or animals. • No observed adverse effect level (NOAEL) – It denotes the level of exposure of an organism, found by experiment or observation, at which there is no biologically or statistically significant (e.g. alteration of morphology, functional capacity, growth, development or life span) increase in the frequency or severity of any adverse effects in the exposed population when compared to its appropriate control.
  12. 12. Terminology Associated with Dose-Response Curves (2) • lowest-observed-effect-level (LOEL) – Lowest concentration or amount of a substance, found by experiment or observation, that causes any alteration in morphology, functional capacity, growth, development, or life span of target organisms distinguishable from normal (control) organisms of the same species and strain under the same defined conditions of exposure • lowest-observed-adverse-effect-level (LOAEL) – Lowest concentration or amount of a substance, found by experiment or observation, which causes an adverse alteration of morphology, functional capacity, growth, development, or life span of a target organism distinguishable from normal (control) organisms of the same species and strain under defined conditions of exposure.
  13. 13. Terminology Associated with Dose-Response Curves (3) • Frank-effect level (FEL) – Level of exposure that produces unmistakable and irreversible effects (such as impairment or mortality) at statistically significant increased severity or frequency. – The level where overt effects are observed is referred to as the Frank-effect level.
  14. 14. Threshold Dose • Dose below which no adverse effects are observable in a population of exposed individuals. • Threshold dose approximated by a NOAEL (No Observed Adverse Effect Level)
  15. 15. Non-threshold Effects • Thresholds are assumed not to exist for genotoxic carcinogens
  16. 16. Safe Dose for Threshold Effect • NOAEL approach – Identify the adverse effect that occurs at the lowest dose – Determine the NOAEL or LOAEL for that endpoint – Divide NOAEL/LOAEL by uncertainty factors
  17. 17. Acceptable Daily Intake • ADI estimated (maximum) amount of an agent, expressed on a body mass basis, to which a subject may be exposed over his lifetime without appreciable health risk (also TDI, tolerable daily intake) • ADI derived from NOAEL by the use of uncertainty factors UFs (or safety factors SFs)
  18. 18. Reference Dose (RfD) • RfD is an estimate of the daily exposure that is likely to be without appreciable health effect even if continued exposure occurs over a lifetime.
  19. 19. Uncertainty Factors • Default values: UFinterspecies = 10 , UFhuman variability = 10
  20. 20. Additional Uncertainty Factors • Sometimes applied: – UFLOAEL-NOAEL = 3 or 10 – UFsubchronic-chronic = 3 or 10 – UF database insufficiences = up to 10 • MF, modifying factor for professional judgement: up to 10 • RfD = NOAEL (or LOAEL) / UF1 UF2 UF3 MF
  21. 21. Benchmark Dose Approach (1) • NOAEL approach uses only single points, shape of dose-response is ignored. • Bench mark dose (BMD) is calculated from the curve fitted to the dose-reponse data, so all information is used. • BMD can only be used when available data are suitable for modelling. • Not replacement for NOAEL, but additional tool
  22. 22. Benchmark Dose Approach (2) 1. A mathematical model is applied to the experimental data to produce a dose-response curve of best fit. 2. By statistical calculation an upper 95% confidence limit of the curve is determined 3. The Benchmark Response is defined as 10% (or 5%, or 1%). 4. The Benchmark Dose corresponds to the bench mark response on the upper confidence limit curve.
  23. 23. Benchmark Dose Approach (3)
  24. 24. Non-threshold? • Absence of a detectable effect at low doses is either because the dose is below a threshold or because the response is below the level that can be detected by the test sytem.
  25. 25. Threshold or Non-threshold • Presence of a threshold cannot be proven from experimental data. • Conclusions about existence of a threshold are based on biological plausibility and expert judgement. • Genotoxic effects (DNA dammage leading to cancer) are thought to be possible at any level of exposure, so no threshold.
  26. 26. Low-dose Extrapolation • Dose-response curve is use to extrapolate to doses lower than then the experimental region.
  27. 27. Calculated Risk or Safe Dose • Extrapolation may be used to calculate: – the risk associated with a known intake. – intake associated with a de minimis risk, e.g. an increased life time risk of developing cancer of 1 in 106 (‘virtually safe dose’, VSD).
  28. 28. Linear Low-dose Extrapolation • Assumption: linear relationship between dose and biological response
  29. 29. Linear Extrapolation from LED10 • LED10 – low confidence limit of dose with response of 10%
  30. 30. From LED10 to VSD • If LED10 = 57 mg/kg/day (response 10% or 0,1) • then VSD (virtual safe dose, response 1 in 106 or 10-6) = 57 x 10-6 / 0,1 = 5,7 x 10-4 mg/kg/day
  31. 31. ALARA Approach • For genotoxic carcinogens (non-threshold effects) also the ALARA-approach may be followed: exposure to be reduced to as low as reasonably achievable (ALARA) or as low as reasonably practical (ALARP)
  32. 32. Relative Toxicity (1) • A comparison of the dose-response curves for two or more different chemicals – administered to the same type of test animal – can be used to determine the relative toxicity of the chemicals. • The LD50 for chemical A is less than the LD50 for chemical B. Therefore, chemical A is considered more toxic than chemical B.
  33. 33. Relative Toxicity (2) • Chemical A is more toxic than chemical B at the higher doses, but at the lower doses chemical B is more toxic than chemical A.
  34. 34. Relative Toxicity (3) • Relative toxicity also be evaluated by comparing the slope of each dose-response curve. A steep dose-response curve is indicative of a chemical that is rapidly and extensively absorbed and slowly detoxified: therefore, the chemical is more toxic.
  35. 35. Variables Influencing Toxicity • These factors include: – the dose-time relationship, – the route of exposure, – the physical and chemical properties of the toxic substance, – the chemical interactions, – the interspecies and intraspecies variability, – genetics, – age, – sex, and – health of the individual collectively. • The toxicity of a given substance is the net result of the complex interaction between all of these various factors.
  36. 36. Dose-Time Relationship • Not only is the dose of a toxic substance important in determining toxicity, but so is the frequency and length of exposure. • The rapid elimination of the substance from the blood stream decreases the probability of an adverse effect. • Gradual accumulation increases the probability that an adverse effect will occur. – Symptoms of acute exposure to sublethal quantities of chlorinated solvents, such as chloroform or carbon tetrachloride, are primarily associated with the nervous system, such as excitability, dizziness and narcosis. – Chronic exposure to these solvents is primarily associated with liver damage.
  37. 37. Routes of Exposure • LD50 values for various organochlorine and organophosphate pesticides resulting from ingestion and skin exposure. • The different absorption rates for various regions of the skin in the human male
  38. 38. Physical and Chemical Factors • Chemical toxicity can be affected by the arrangement of the atoms in a molecule. • The structural differences can result in different toxic effects being observed as a result of exposure. – The LD50 for 1,1-dichloroethylene is 5,750 mg/kg and the LD50 for the 1,2-dichloroethylene isomers is 770 mg/kg. – Based on animal studies, 1,1-dichloroethylene is a suspected human carcinogen. Its structure is similar to a known human carcinogen, vinyl chloride. – The 1,2-dichloroethylene isomers are not carcinogenic, but they do have different effects. The cis isomer is an irritant and acts as a narcotic (causes drowsiness). The trans isomer affects the central nervous system (brain and spinal cord) and the gastrointestinal system causing weakness, tremors, cramps, nausea; it may also cause liver and kidney damage.
  39. 39. Interspecies and Intraspecies Variability • Interspecies variability – The variability in response between species is primarily the result of the evolution of different biological mechanisms involved in the metabolism of toxic substances. – The differences in toxicity may be due to the rate at which metabolism occurs, or whether metabolism produces chemical intermediates more toxic than the parent compound. • For example, the pesticide malathion is metabolized at a different rate in mammals than in insects. In mammals, the chemical intermediates are rapidly metabolized to deliver end products that are excreted from the body. In insects metabolize malathion more slowly and produce a chemical intermediate --- malaoxon --- that is toxic to the insect. • Methanol intoxication results in ocular damage and blindness. However, in other species methanol is less toxic and the symptoms associated with human intoxication are not observed. • Rats do not have a vomiting reflex. Rodenticides, such as squill, are more effective if they are ingested because the rat can not discard the substance by vomiting.
  40. 40. Intraspecies (individual) variability • Individuals within a population ---exposed to a single dose --- will demonstrate a wide array of responses. • This type of variability can be caused by differences associated with individual genetics and sex of the individuals within the population.
  41. 41. Genetics • The differences in genetics makeup both within species and between species markedly influence the disposition and the metabolism of toxic substances. – For example, some individuals have an enzyme (glucose-6- phosphate dehydrogenase) deficiency resulting in their red blood cells being more fragile than normal. Consequently, red blood cells of these individuals are more susceptible to chemicals such as phenylhydrazine and primaquine, which cause hemolysis. – The enzyme aryl hydrocarbon hydroxylase (AHH) transforms the chemical benzo[a]pyrene to its carcinogenic metabolite. Some individuals produce lower amounts of AHH; benzo[a]pyrene is therefore less toxic to these individuals. Similarly, low AHH individuals may be less likely to develop cancer from cigarette smoke.
  42. 42. Age • In general, younger and developing individuals are more sensitive to toxic substances than older individuals. The difference can be primarily attributed to differences in metabolisms; specially to differences in the functioning of the liver. • In younger individuals the mechanisms for detoxifying substances are less developed than in older individuals. However, some substances are less toxic to the young than to adults. • The lead absorption rate is four to five times greater in young individuals than in adults; the cadmium absorption rate is 20 times greater than in adults.
  43. 43. Sex • The difference in toxicity between genders seems to be primarily influence by the sex hormones (estrogen and testosterone), which can affect metabolism. • Some organophosphate pesticides are more toxic to females than males. – Parathion is metabolized more rapidly in females resulting in a higher concentration of the more toxic intermediate, paraoxon. – Male rats are 10 times more susceptible to liver damage than female rats as a result of chronic oral exposure to DDT.
  44. 44. State of Health • The liver and the kidney are important organs for detoxifying and removing toxic substances. Therefore, conditions that lead to liver or kidney disease enhance the toxic effects of substances normally detoxified by these organs.
  45. 45. Chemical Interactions (1) • The interaction between two or more chemicals and their subsequent effects have not been widely studied; not because these interactions are not important, but rather because the results are difficult to interpret and extrapolate, and also because the studies are longer and costlier. • The presence of other chemicals at the time of exposure or as a result of previous exposure can affect the response to the specific toxicant being studied. These effects can be additive, synergistic, antagonistic, or they may demonstrate the characteristics associated with sensitization or potentiation
  46. 46. Chemical Interactions (2) • Additive effects – An additive effect occurs when the combined effect of the two chemicals is equal to the sum of the effects of each individual chemical. • Synergistic effect – A synergistic effect occurs when the observed effects are greater than what would be predicted by simply summing the effects of each individual chemical.
  47. 47. Chemical Interactions (3) • Antagonistic effects – Antagonistic effects are the basis on which antidotes are developed to counteract the effects of toxic substances that enter the body. – Antagonistic effects occur when the sum of the effects of each individual chemical is less than the sum of the effects for both chemicals.
  48. 48. Chemical Interactions (4) • Four major types of antagonism – Functional antagonism • It refers to a type of reaction two chemicals have on a specific physiological function in the test organism, usually producing counterbalancing or opposite effects. – Chemical antagonism • It may be referred to as chemical inactivation. This type of interaction is the result of a reaction between two chemicals to produces a less toxic product. – Dispositional antagonism • The toxic substance is either transformed into a less toxic substance or it is eliminated from the body more rapidly, then the observed effects will be less than the expected ones.
  49. 49. Chemical Interactions (5) – Receptor antagonism • It occurs when two chemicals require binding to the same receptor in order to exert their effect. The result is that when the two chemicals are administered together the observed effect is less than the sum of the effects for each chemical. – Sensitization • Initial exposure to toxic chemicals does not always result in an observable or measurable effect. However, subsequent exposure to the same chemical may result in an observable or measurable effect. • Sensitization occurs as a result of the interaction of the body’s immune system with the toxic chemical during the initial exposure.
  50. 50. Chemical Interactions (6) – Potentiation • At first glance it would appear that synergism and potentiation are the same. In A potentiation, only one of the chemicals has an observable effect when administered alone. • Chemical A has no effect on mortality (0%). Chemical B causes 25% mortality. When the two chemicals are administered together the effect is greater than the sum of their individual effects (0% + 25% = 100 %).
  51. 51. LD50 of Chemicals Substance Animal, Route LD50 Reference Sucrose (table sugar) rat, oral 29,700,000,000 ng/kg [7] Vitamin C (ascorbic acid) rat, oral 11,900,000,000 ng/kg [8] Grain alcohol (ethanol) rat, oral 7,060,000,000 ng/kg [10] Melamine rat, oral 6,000,000,000 ng/kg Table Salt rat, oral 3,000,000,000 ng/kg [12] Paracetamol (acetaminophen) rat, oral 1,944,000,000 ng/kg [13] Metallic Arsenic rat, oral 763,000,000 ng/kg [15] Aspirin (acetylsalicylic acid) rat, oral 200,000,000 ng/kg [17] Caffeine rat, oral 192,000,000 ng/kg [18] Cadmium oxide rat, oral 72,000,000 ng/kg [22] Nicotine rat, oral 50,000,000 ng/kg [23] Strychnine rat, oral 16,000,000 ng/kg [24] Arsenic trioxide rat, oral 14,000,000 ng/kg [25] Metallic Arsenic rat, intraperitoneal 13,000,000 ng/kg [26] Sodium cyanide rat, oral 6,400,000 ng/kg [27] Beryllium oxide rat, oral 500,000 ng/kg [30] Aflatoxin B1 (from Aspergillus flavus) rat, oral 480,000 ng/kg [31] Dioxin (TCDD) rat, oral 20,000 ng/kg [33] Polonium-210 human, inhalation 10 ng/kg (estimated) [37] Botulinum toxin (Botox) human, oral, injection, inhalation 1 ng/kg (estimated) [38] Ionizing radiation human, irradiation 6 Gy
  52. 52. Typical Costs of Various Types of Toxicity Tests
  53. 53. Typical Testing protocols for Acute and Chronic Toxicity Studies
  54. 54. Dose-Response Curve
  55. 55. LD50 Values of Organochlorine Pesticides
  56. 56. LD50 Values of Organophosphate Pesticides
  57. 57. Regional Differences of Dermal Absorption in Human Male
  58. 58. Toxicity of various Compounds to Different Test Species
  59. 59. No-effect Levels on a Dose-Response Curve
  60. 60. Acute Toxicity of N-hydroxy acetylaminofluorene in Various Strains of Rats
  61. 61. Five Threshold Doses • 美國環境保護署(EPA)對動物毒性實驗的反應程度確立了五種界限值(threshold), 用以評估毒性強弱而發展出各類毒性物質的每日允許攝取量。這五個界限值為: • 一 ﹑沒有可觀察到的影響值(no-observed-effect level,NOEL)。 • 二﹑沒有可觀察到的不良影響值(no-observed-adverse-effect level,NOAEL)。 • 三﹑可觀察到有影響的最低值(lowest-observed-adverse-drrect level,LOEL)。 • 四﹑可觀察到有不良影響的最低值(lowest-observed-adverse-effect level,LOAEL)。 • 五﹑有直接顯著影響值(frank effect level,FEL)。 • 美國是以NOAEL值經過體重權衡後,除以安全因素而得到每日允許攝取量。而這一限 值就是表示,對設定毒性物質可允許它每天進入體內之總和量。 • • 圖片連結-各種劑量反應關係 各種劑量反應關係 圖片連結-各種劑量反應關係
  62. 62. LOEL • 最低作用值(LOEL,Lowest-observed effect level) • 是指在研究人體或動物接觸某種物質時產生任何反應的頻率或嚴重性在生物學上顯著 增加的最低劑量。 相關名詞 • 最低作用值:不良效應級(有害作用劑量):最低濃度或數量的物質,通過實驗或觀察 發現,這會導致不利的改變形態,機能,生長,發育,壽命或生物體的目標分辨正常 (對照)的生物物種和同一菌株在規定條件下的暴露。 逆轉錄不利影響 , 最低的觀察 效果級別 , 不遵守,不利的效應級別 , 無觀測效應水平 • 在劑量反應曲線中,我們常常會應用到幾個相關名詞,包括: • 無反應劑量(no-effect level, NEL)或無觀察反應劑量(no-observed effect level, NOEL ):無顯著反應的最低劑量。 • 最低觀察反應劑量 (最低作用值)lowest observed effect level,LOEL):可以觀察到反應 之最低劑量,不過此反應為次要之反應而非引起主要想要測量之反應。 • 無觀察危害反應劑量(no observed adverse effectlevel, NOAEL):一些非欲觀察之毒性 反應(危害性低)發生之劑量,但在高劑量下觀察得到的反應,在此劑量並不會出現 。 (無顯著危害的最高劑量,並非無反應而是與對照組無統計差異) • 最低觀察危害反應劑量(lowest observed adverseeffect level, LOAEL):第一個可以觀 察得到危害反應的最低劑量濃度。 • 法蘭克反應劑量(frank effect level, FEL):引起嚴重危害反應發生之劑量稱之。 訂定