poisons and drugs

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Poisons and DrugsPoisons and Drugs

Prof. Monzir S. Abdel-LatifProf. Monzir S. Abdel-Latif

Chemistry DepartmentChemistry Department

Islamic University of GazaIslamic University of Gaza

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SyllabusSyllabus

In this course, it is anticipated to cover the In this course, it is anticipated to cover the following topics:following topics:

Introduction to toxicologyIntroduction to toxicology Toxicodynamics and ToxicokineticsToxicodynamics and Toxicokinetics Data interpretationData interpretation Risk assessment and managementRisk assessment and management Exposure and monitoringExposure and monitoring

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Methods of AnalysisMethods of Analysis1.1. Gas ChromatographyGas Chromatography2.2. Liquid chromatographyLiquid chromatography3.3. Mass SpectrometryMass Spectrometry4.4. Hyphenated TechniquesHyphenated Techniques5.5. AAS and AESAAS and AES6.6. Spectrophotometry and FluorometrySpectrophotometry and Fluorometry Sample PreparationSample Preparation Classes of drugs and poisonsClasses of drugs and poisons1.1. According to site of actionAccording to site of action2.2. Grouping of drugsGrouping of drugs

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AmphetaminesAmphetamines Halaucination drugsHalaucination drugs Cannabis SativaCannabis Sativa Diamorphine and heroinDiamorphine and heroin CocaineCocaine QatQat PsilocybinPsilocybin Marijuana and its metaboliteMarijuana and its metabolite BenzodiazepinesBenzodiazepines OpiatesOpiates Phenyl cyclidinePhenyl cyclidine PesticidesPesticides Prediction of HazardPrediction of Hazard AntidotesAntidotes Diagnosis and treatmentDiagnosis and treatment

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We will follow presentation chapters from several We will follow presentation chapters from several textbooks including “Fundamantal Toxicology” by textbooks including “Fundamantal Toxicology” by John Duffus and Howard Worth, published by RSC John Duffus and Howard Worth, published by RSC in 2006, Poisoning and Toxicology handbook, 4in 2006, Poisoning and Toxicology handbook, 4 thth Ed., Ed., A Guide to Practical Toxicology, 2A Guide to Practical Toxicology, 2ndnd Ed, Woolley, Ed, Woolley, Toxicological Chemistry and Biochemistry, 3Toxicological Chemistry and Biochemistry, 3rdrd Ed., Ed., as well as others .as well as others .

However, other books in Instrumental Analysis and However, other books in Instrumental Analysis and related research papers will be found very helpful.related research papers will be found very helpful.

I’ll try to maintain a web page for the course and I’ll try to maintain a web page for the course and regularly post reading material for you to look at. regularly post reading material for you to look at.

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Introduction to ToxicologyIntroduction to Toxicology

Toxicology is the fundamental science of poisons.Toxicology is the fundamental science of poisons. A poison is a substance that can cause severe injury A poison is a substance that can cause severe injury

or death as a result of interaction with living tissue.or death as a result of interaction with living tissue. Therefore, in principle, all chemicals can be Therefore, in principle, all chemicals can be

considered as potential poisons causing injury or considered as potential poisons causing injury or death upon excessive exposure. At the same time all death upon excessive exposure. At the same time all chemicals can be regarded as safe if exposure to chemicals can be regarded as safe if exposure to chemicals was kept below a tolerable limit. chemicals was kept below a tolerable limit.

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Exposure to toxinsExposure to toxins

Exposure is a function of the following factors:Exposure is a function of the following factors: Amount or concentration of the target Amount or concentration of the target

chemicalchemical Time of interaction of the chemical with the Time of interaction of the chemical with the

target organtarget organ Frequency of interaction of the chemical with Frequency of interaction of the chemical with

the target organthe target organ

For highly toxic chemicals, the tolerable For highly toxic chemicals, the tolerable exposure is close to zeroexposure is close to zero

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Determination of tolerable exposureDetermination of tolerable exposure

In fact, this constitutes a problem since we do In fact, this constitutes a problem since we do need reliable data relating exposure to injury need reliable data relating exposure to injury or adverse effect.or adverse effect.

Unfortunately, what can be considered as an Unfortunately, what can be considered as an injury or an adverse effect is not well defined injury or an adverse effect is not well defined and debatable. We will look at this problem and debatable. We will look at this problem laterlater

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Adverse effectsAdverse effects

An adverse effect can be defined as an An adverse effect can be defined as an abnormal, undesirable, or harmful change of abnormal, undesirable, or harmful change of people or organs following exposure to the people or organs following exposure to the potentially toxic substancepotentially toxic substance

Although the ultimate adverse effect is death, Although the ultimate adverse effect is death, the following are definite adverse effects:the following are definite adverse effects:

Altered food consumptionAltered food consumptionAltered body or organ weightAltered body or organ weightAltered enzyme or hormone levels, ..etcAltered enzyme or hormone levels, ..etc

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Harmful effectsHarmful effects

An effect is considered harmful if it causes a An effect is considered harmful if it causes a functional damage to an organ, irreversible change in functional damage to an organ, irreversible change in homeostasis or increased susceptibility to chemical or homeostasis or increased susceptibility to chemical or biological stress including infectious diseases.biological stress including infectious diseases.

One should consider the degree of alteration from One should consider the degree of alteration from normality and the relation of the altered property to normality and the relation of the altered property to the total well-being of the personthe total well-being of the person

In some cases, a person can adapt to the irreversible In some cases, a person can adapt to the irreversible alteration and practice normal lifealteration and practice normal life

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In some cases of immune reactions In some cases of immune reactions leading to allergy:leading to allergy:

The first exposure may not cause an adverse The first exposure may not cause an adverse effect of allergy, however, it may sensitize the effect of allergy, however, it may sensitize the organism to respond adversely to future organism to respond adversely to future exposures even at low levelsexposures even at low levels

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Amount of exposureAmount of exposure

The amount of exposure to a chemical that The amount of exposure to a chemical that causes injury varies over a very wide range causes injury varies over a very wide range depending on the type of chemical and its form depending on the type of chemical and its form (liquid, solid, or gas)(liquid, solid, or gas)

For example:For example:Ethanol>>NaCl>>DDT>Nicotine>>dioxinEthanol>>NaCl>>DDT>Nicotine>>dioxin

This can be quantified using the median lethal This can be quantified using the median lethal dose (LDdose (LD5050) concept ) concept

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Median Lethal Dose (LDMedian Lethal Dose (LD5050), mg ), mg

toxin/kg body weighttoxin/kg body weight LDLD50 50 is a statistically derived single dose of a is a statistically derived single dose of a

chemical that can be expected to cause the chemical that can be expected to cause the death of 50% of organisms of a given death of 50% of organisms of a given population, under a defined set of population, under a defined set of experimental conditions.experimental conditions.

LDLD5050 when reported for human beings are when reported for human beings are obtained by extrapolation from studies on obtained by extrapolation from studies on mammals, or observations following mammals, or observations following accidental or suicidal exposures. accidental or suicidal exposures.

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The LDThe LD5050 is used to classify and compare toxicity of is used to classify and compare toxicity of

chemicals, although it is of limited merits. For chemicals, although it is of limited merits. For example, the LDexample, the LD5050 classification orally to rats are: classification orally to rats are:

Very toxicVery toxic less than 25 mg/kgless than 25 mg/kg ToxicToxic from 25 -199 mg/kgfrom 25 -199 mg/kg HarmfulHarmful from 200 - 2000 mg/kgfrom 200 - 2000 mg/kg

However, it is not convincing to label a substance as However, it is not convincing to label a substance as toxic because its LDtoxic because its LD5050 is 199, while labeling another is 199, while labeling another

as harmful since its LDas harmful since its LD5050 is 200. That is why the LD is 200. That is why the LD5050

values need more refinements.values need more refinements.

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In addition, when using LDIn addition, when using LD5050 values, there is values, there is

no simple relationship between lethality and no simple relationship between lethality and sub lethal toxic effects.sub lethal toxic effects.

In other words also, it is not informative to In other words also, it is not informative to what is the minimum dose that can be lethal, what is the minimum dose that can be lethal, and thus no indication of what can be and thus no indication of what can be considered a safe exposure level.considered a safe exposure level.

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Toxicity versus RiskToxicity versus Risk

With regards to chemical safety, risk With regards to chemical safety, risk assessment can be more important than actual assessment can be more important than actual toxicity of chemicals. toxicity of chemicals.

Risk can be regarded as the probability that a Risk can be regarded as the probability that a substance would impart unacceptable harm or substance would impart unacceptable harm or unacceptable effects to an organ or to unacceptable effects to an organ or to ecosystems upon exposure.ecosystems upon exposure.

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SafetySafety

It is possible to define safety as the practical certainty It is possible to define safety as the practical certainty that injury will not (high probability) result from that injury will not (high probability) result from exposure to a hazard under defined conditions.exposure to a hazard under defined conditions.

Practical certainty is a numerically specified low risk Practical certainty is a numerically specified low risk (or socially acceptable risk). (or socially acceptable risk).

Assessment of risk depends on scientific data, but Assessment of risk depends on scientific data, but acceptability is influenced by social, economic, acceptability is influenced by social, economic, political and benefits arising from a chemical or a political and benefits arising from a chemical or a process.process.

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Uncertainty (safety) factorsUncertainty (safety) factors

A threshold of exposure above which an adverse A threshold of exposure above which an adverse effect can occur and below which no such effect is effect can occur and below which no such effect is observed, is obtained from available data. observed, is obtained from available data.

The threshold of exposure is then divided by a factor The threshold of exposure is then divided by a factor (uncertainty or safety factor) to lower it to a new (uncertainty or safety factor) to lower it to a new value that toxicologists can regard as safe beyond value that toxicologists can regard as safe beyond doubt.doubt.

US National Academy of Sciences safe drinking US National Academy of Sciences safe drinking water committee proposed the following guidelines water committee proposed the following guidelines for selecting the safety factors, to be used with no for selecting the safety factors, to be used with no observed effect level (NOEL) data.observed effect level (NOEL) data.

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Safety Factor Selection:Safety Factor Selection:1.1. An uncertainty (safety) factor of 10 is used An uncertainty (safety) factor of 10 is used

when valid human data based on chronic when valid human data based on chronic exposure is availableexposure is available

2.2. An uncertainty (safety) factor of 100 is used An uncertainty (safety) factor of 100 is used when human data is inconclusive or limited when human data is inconclusive or limited to acute exposure, but reliable data on to acute exposure, but reliable data on animals is availableanimals is available

3.3. An uncertainty (safety) factor of 1000 is used An uncertainty (safety) factor of 1000 is used when no human data is available and when no human data is available and experimental animal data is limited experimental animal data is limited

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Exposure to potentially toxic Exposure to potentially toxic substancessubstances

Toxins can cause injury when they reach Toxins can cause injury when they reach sensitive parts of an organism at a sensitive parts of an organism at a sufficiently high concentration.sufficiently high concentration.

Exposure can occur through:Exposure can occur through:

1.1. Skin (dermal or percutaneous) AbsorptionSkin (dermal or percutaneous) Absorption

2.2. InhalationInhalation

3.3. IngestionIngestion

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Skin absorptionSkin absorption Among the chemicals that are absorbed through the Among the chemicals that are absorbed through the

skin are aniline, hydrogen cyanide, some steroid skin are aniline, hydrogen cyanide, some steroid hormones, organic mercury compounds, hormones, organic mercury compounds, nitrobenzene, organophosphate compounds and nitrobenzene, organophosphate compounds and phenol. Some chemicals, such as phenol or phenol. Some chemicals, such as phenol or methylmercury chloride, can be lethal if absorbed methylmercury chloride, can be lethal if absorbed from a fairly small area (a few square centimeters) of from a fairly small area (a few square centimeters) of skin. If protective clothing is being worn, it must be skin. If protective clothing is being worn, it must be remembered that absorption through the skin of any remembered that absorption through the skin of any chemical that gets inside the clothing will be even chemical that gets inside the clothing will be even faster than through unprotected skin because the faster than through unprotected skin because the chemical cannot escape and contact is maintained chemical cannot escape and contact is maintained over a longer time.over a longer time.

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InhalationInhalation

Gases and vapors are easily inhaled but inhalation of Gases and vapors are easily inhaled but inhalation of particles depends upon their size and shape. The particles depends upon their size and shape. The smaller the particle, the further into the respiratory smaller the particle, the further into the respiratory tract it can go. Dusts with an effective aerodynamic tract it can go. Dusts with an effective aerodynamic diameter of between 0.5 and 7 µm can persist in the diameter of between 0.5 and 7 µm can persist in the alveoli and respiratory bronchioles after deposition. alveoli and respiratory bronchioles after deposition. Peak retention depends upon the aerodynamic shape Peak retention depends upon the aerodynamic shape but is mainly of those particles with an effective but is mainly of those particles with an effective aerodynamic diameter of between 1 and 2 µm.aerodynamic diameter of between 1 and 2 µm.

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Physical irritation by dust particles or fibers can cause Physical irritation by dust particles or fibers can cause very serious adverse health effects but most effects very serious adverse health effects but most effects depend upon the solids being dissolved. Special depend upon the solids being dissolved. Special consideration should be given to asbestos fibers consideration should be given to asbestos fibers which may lodge in the lung and cause fibrosis and which may lodge in the lung and cause fibrosis and cancer even though they are mostly insoluble and cancer even though they are mostly insoluble and therefore do not act like classical toxicants: care therefore do not act like classical toxicants: care should also be taken in assessing possible harm from should also be taken in assessing possible harm from manmade mineral fibers that have similar properties.manmade mineral fibers that have similar properties.

Some insoluble particles such as asbestos, coal dust and Some insoluble particles such as asbestos, coal dust and silica dust will readily cause fibrosis of the lungsilica dust will readily cause fibrosis of the lung

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IngestionIngestion

A chemical may accumulate if absorption A chemical may accumulate if absorption exceeds excretion; this is particularly likely exceeds excretion; this is particularly likely with substances that combine a fairly high with substances that combine a fairly high degree of lipid solubility with chemical degree of lipid solubility with chemical stability. Such chemicals are found in the stability. Such chemicals are found in the group of persistent organic pollutants (POPS), group of persistent organic pollutants (POPS), including several organochlorine pesticides, including several organochlorine pesticides, which are now largely, but not entirely, which are now largely, but not entirely, banned from use banned from use

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Divalent lead ions accumulate in bone where Divalent lead ions accumulate in bone where they replace the chemically similar calcium they replace the chemically similar calcium ions. While in the bone, they cause little harm ions. While in the bone, they cause little harm but when bone breaks down, or during but when bone breaks down, or during pregnancy or illness, the lead ions enter the pregnancy or illness, the lead ions enter the blood and may poison the person who has blood and may poison the person who has accumulated them or, in the case of pregnancy, accumulated them or, in the case of pregnancy, the unborn child.the unborn child.

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Local versus systemic effectsLocal versus systemic effects

Adverse effects may be local or systemic. Local Adverse effects may be local or systemic. Local effects occur at the site of exposure of the effects occur at the site of exposure of the organism to the potentially toxic substance. organism to the potentially toxic substance. Corrosives always act locally. Irritants Corrosives always act locally. Irritants frequently act locally. Most substances that are frequently act locally. Most substances that are not highly reactive are absorbed and not highly reactive are absorbed and distributed around the affected organism distributed around the affected organism causing general injury at a target organ or causing general injury at a target organ or tissue distinct from the absorption site.tissue distinct from the absorption site.

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The target organ is not necessarily the organ of The target organ is not necessarily the organ of greatest accumulation. For example, adipose greatest accumulation. For example, adipose (fatty) tissue accumulates organochlorine (fatty) tissue accumulates organochlorine pesticides to very high levels but does not pesticides to very high levels but does not appear to be affected by them. Some appear to be affected by them. Some substances produce both local and systemic substances produce both local and systemic effects. For example, tetraethyl lead damages effects. For example, tetraethyl lead damages the skin on contact, and is then absorbed and the skin on contact, and is then absorbed and transported to the central nervous system transported to the central nervous system where it causes further damage.where it causes further damage.

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Effects of a chemical can accumulate even if the Effects of a chemical can accumulate even if the chemical itself does not. There is some chemical itself does not. There is some evidence that this is true of the effects of evidence that this is true of the effects of organophosphate pesticides and other organophosphate pesticides and other neurotoxins on the nervous system. This may neurotoxins on the nervous system. This may lead to poor functioning of the nervous system lead to poor functioning of the nervous system in humans in old age. Because of the time in humans in old age. Because of the time difference between exposure and effect, difference between exposure and effect, establishing the relationship between such establishing the relationship between such delayed effects and the possible cause, no delayed effects and the possible cause, no longer present in the body, is often difficult.longer present in the body, is often difficult.

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Adverse effects related to allergies appear to be Adverse effects related to allergies appear to be increasing. Allergy (hypersensitivity) is the increasing. Allergy (hypersensitivity) is the name given to disease symptoms following name given to disease symptoms following exposure to a previously encountered exposure to a previously encountered substance (allergen) that would otherwise be substance (allergen) that would otherwise be classified as harmless. Essentially, an allergy classified as harmless. Essentially, an allergy is an adverse reaction of the altered immune is an adverse reaction of the altered immune system. The process, which leads to the system. The process, which leads to the disease response on subsequent exposure to disease response on subsequent exposure to the allergen, is called sensitization.the allergen, is called sensitization.

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Another important aspect of adverse effects to be Another important aspect of adverse effects to be considered is whether they are reversible or considered is whether they are reversible or irreversible. For the liver, which has a great capacity irreversible. For the liver, which has a great capacity for regeneration, many adverse effects are reversible, for regeneration, many adverse effects are reversible, and complete recovery can occur. For the central and complete recovery can occur. For the central nervous system, in which regeneration of tissue is nervous system, in which regeneration of tissue is severely limited, most adverse effects leading to severely limited, most adverse effects leading to morphological changes are irreversible and recovery morphological changes are irreversible and recovery is, at best, limited. Carcinogenic effects are also is, at best, limited. Carcinogenic effects are also irreversible, but suitable treatment may reduce the irreversible, but suitable treatment may reduce the severity of such effectsseverity of such effects

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Chemical InteractionsChemical Interactions

1.1. Additive effectAdditive effectThe simplest interaction is an additive effect: this The simplest interaction is an additive effect: this

is an effect, which is the result of two or is an effect, which is the result of two or more chemicals acting together and is the more chemicals acting together and is the simple sum of their effects when acting simple sum of their effects when acting independently. In mathematical terms,independently. In mathematical terms,

1 + 1 = 2, 1 + 5 = 6, 1 + 1 = 2, 1 + 5 = 6, etcetc..The effects of organochlorine pesticides are The effects of organochlorine pesticides are

usually additive.usually additive.

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2. 2. Synergistic (multiplicative) effectSynergistic (multiplicative) effect

this is an effect of two chemicals acting together, which this is an effect of two chemicals acting together, which is greater than the simple sum of their effects when is greater than the simple sum of their effects when acting alone; it is called synergism. In mathematical acting alone; it is called synergism. In mathematical terms,terms,

1 + 1 = 4, 1 + 5 = 10, 1 + 1 = 4, 1 + 5 = 10, etcetc..

Asbestos fibers and cigarette smoking act together to Asbestos fibers and cigarette smoking act together to increase the risk of lung cancer by a factor of 40, increase the risk of lung cancer by a factor of 40, taking it well beyond the risk associated with taking it well beyond the risk associated with independent exposure to either of these agents.independent exposure to either of these agents.

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3. 3. PotentiationPotentiation

In potentiation, a substance that on its own causes no In potentiation, a substance that on its own causes no harm makes the effects of another chemical much harm makes the effects of another chemical much worse. This may be considered to be a form of worse. This may be considered to be a form of synergism. In mathematical terms,synergism. In mathematical terms,

0 + 1 = 5, 0 + 5 = 20, 0 + 1 = 5, 0 + 5 = 20, etcetc..

For example, isopropanol, at concentrations that are not For example, isopropanol, at concentrations that are not harmful to the liver, increases (potentiates) the liver harmful to the liver, increases (potentiates) the liver damage caused by a given concentration of carbon damage caused by a given concentration of carbon tetrachloride.tetrachloride.

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4. 4. AntagonismAntagonism

The opposite of synergism is antagonism: an The opposite of synergism is antagonism: an antagonistic effect is the result of a chemical antagonistic effect is the result of a chemical counteracting the adverse effect of another; in other counteracting the adverse effect of another; in other words, the situation where exposure to two chemicals words, the situation where exposure to two chemicals together has less effect than the simple sum of their together has less effect than the simple sum of their independent effects. Such chemicals are said to show independent effects. Such chemicals are said to show antagonism. In mathematical terms: antagonism. In mathematical terms:

1 + 1 = 0, 1 + 5 = 2, 1 + 1 = 0, 1 + 5 = 2, etcetc..

For example, For example, histaminehistamine lowers arterial pressure, while lowers arterial pressure, while adrenalineadrenaline raises arterial pressure raises arterial pressure

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Toxicity testingToxicity testing

Dose response and Concentration responseDose response and Concentration response

A dose–response (concentration–response) A dose–response (concentration–response) relationship is defined as the association relationship is defined as the association between dose (concentration) and the between dose (concentration) and the incidence of a defined biological effect in an incidence of a defined biological effect in an exposed population, usually expressed as exposed population, usually expressed as percentage. Historically the defined effect was percentage. Historically the defined effect was death.death.

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The classic dose–response or concentration–response The classic dose–response or concentration–response relationship is shown in the figure below. This is a relationship is shown in the figure below. This is a theoretical curve and in practice such a curve is rarely theoretical curve and in practice such a curve is rarely found. Curves of this kind form the basis for the found. Curves of this kind form the basis for the determination of the LDdetermination of the LD5050 or the LC or the LC5050 (the median (the median

lethal concentration). The LDlethal concentration). The LD5050 and LC and LC5050 are specific are specific

cases of the generalized values LDcases of the generalized values LDnn and LC and LCnn. The LD. The LDnn

is the dose of a toxicant lethal to is the dose of a toxicant lethal to nn% of a test % of a test population. The LCpopulation. The LCnn is the exposure concentration of is the exposure concentration of

a toxicant lethal to a toxicant lethal to nn% of a test population.% of a test population.

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the LDthe LD5050 is the statistically derived single dose of is the statistically derived single dose of

a chemical that can be expected to cause death a chemical that can be expected to cause death in 50% of a given population of organisms in 50% of a given population of organisms under a defined set of experimental conditions. under a defined set of experimental conditions. Similarly, the LCSimilarly, the LC5050 is the statistically derived is the statistically derived

exposure concentration of a chemical that can exposure concentration of a chemical that can be expected to cause death in 50% of a given be expected to cause death in 50% of a given population of organisms under a defined set of population of organisms under a defined set of experimental conditions.experimental conditions.

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Another important value that may be derived Another important value that may be derived from the relationship shown is the threshold from the relationship shown is the threshold dose or concentration, the minimum dose or dose or concentration, the minimum dose or concentration required to produce a detectable concentration required to produce a detectable response in the test population. The threshold response in the test population. The threshold value can never be derived with absolute value can never be derived with absolute certainty and therefore the lowest observed certainty and therefore the lowest observed effect level (LOEL) or the NOEL have effect level (LOEL) or the NOEL have normally been used instead of the threshold normally been used instead of the threshold value in deriving regulatory standards.value in deriving regulatory standards.

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There is a move to replace these values by the There is a move to replace these values by the benchmark dose (BMD). This is defined as the benchmark dose (BMD). This is defined as the statistical lower confidence limit of the dose that statistical lower confidence limit of the dose that produces a defined response (called the benchmark produces a defined response (called the benchmark response or BMR, usually 5 or 10%) in a given response or BMR, usually 5 or 10%) in a given population under defined conditions compared to population under defined conditions compared to background, defined as 0%. background, defined as 0%. It involves fitting a mathematical model to the entire dose-response dataset, and allowing the model to estimate the threshold dose corresponding to a level of benchmark response (BMR).

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The use of the LDThe use of the LD5050 in the classification of in the classification of

potentially toxic chemicals has been described; potentially toxic chemicals has been described; it must be emphasized that such a it must be emphasized that such a classification is only a very rough guide to classification is only a very rough guide to relative toxicity. The LDrelative toxicity. The LD5050 tells us nothing tells us nothing

about sub lethal toxicity. Any classification about sub lethal toxicity. Any classification based on the LDbased on the LD5050 is strictly valid only for the is strictly valid only for the

test population and conditions on which it is test population and conditions on which it is based and on the related route of exposure.based and on the related route of exposure.

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The LDThe LD5050 tells us nothing about the shape of the tells us nothing about the shape of the

dose–response curve on which it is based. dose–response curve on which it is based. Thus, two chemicals may appear to be equally Thus, two chemicals may appear to be equally toxic since they have the same LDtoxic since they have the same LD5050, but one , but one

may have a much lower lethal threshold and may have a much lower lethal threshold and kills members of an exposed population at kills members of an exposed population at concentrations where the other has no effectconcentrations where the other has no effect

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The determination and use of the LDThe determination and use of the LD5050 are likely are likely to decline in future as fixed dose testing to decline in future as fixed dose testing becomes more widely used. In fixed dose becomes more widely used. In fixed dose testing, the test substance may be administered testing, the test substance may be administered to rats or other test species at no more than to rats or other test species at no more than three dose levels: the possible dose levels are three dose levels: the possible dose levels are preset legally to equate with a regulatory preset legally to equate with a regulatory classification or ranking system. Dosing is classification or ranking system. Dosing is followed by an observation period of 14 days. followed by an observation period of 14 days. The dose at which toxic signs are detected is The dose at which toxic signs are detected is used to rank or classify the test materials.used to rank or classify the test materials.

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Fixed dose testing (no mortality)Fixed dose testing (no mortality)

The initial test dose level is selected with a view The initial test dose level is selected with a view to identifying toxicity without mortality to identifying toxicity without mortality occurring. Thus, if a group of five male and occurring. Thus, if a group of five male and five female rats is tested with an oral dose of five female rats is tested with an oral dose of 500 mg/ kg body weight and no clear signs of 500 mg/ kg body weight and no clear signs of toxicity appear, the substance should not be toxicity appear, the substance should not be classified in any of the defined categories of classified in any of the defined categories of toxicity. If toxicity is seen but no mortality, the toxicity. If toxicity is seen but no mortality, the substance can be classed as ‘harmful’.substance can be classed as ‘harmful’.

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If mortality occurs, retesting with a dose of 50 If mortality occurs, retesting with a dose of 50 mg/ kg body weight is required. If no mg/ kg body weight is required. If no mortality occurs at the lower dose but signs of mortality occurs at the lower dose but signs of toxicity are detected, the substance would be toxicity are detected, the substance would be classified as ‘toxic’. If mortality occurs at the classified as ‘toxic’. If mortality occurs at the lower dose, retesting at 5 mg/ kg body weight lower dose, retesting at 5 mg/ kg body weight would be carried out and if signs of toxicity would be carried out and if signs of toxicity were detected and mortality occurred, the were detected and mortality occurred, the substance would be classified as ‘very toxic’.substance would be classified as ‘very toxic’.

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Body weight versus areaBody weight versus area

On a body weight basis, it is assumed for toxicity data On a body weight basis, it is assumed for toxicity data extrapolation that humans are usually about 10 times extrapolation that humans are usually about 10 times more sensitive than rodents. On a body surface–area more sensitive than rodents. On a body surface–area basis, humans usually show about the same basis, humans usually show about the same sensitivity as test mammals, sensitivity as test mammals, i.e. i.e. the same dose per the same dose per unit of body surface area will give the same given unit of body surface area will give the same given defined effect, in about the same percentage of the defined effect, in about the same percentage of the population. Knowing the above relationships, it is population. Knowing the above relationships, it is possible to estimate the exposure to a chemical that possible to estimate the exposure to a chemical that humans should be able to tolerate.humans should be able to tolerate.

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Epidemiology and human toxicologyEpidemiology and human toxicology

Epidemiology is the analysis of the distribution Epidemiology is the analysis of the distribution and determinants of health-related states or and determinants of health-related states or events in human populations and the events in human populations and the application of this study to the control of application of this study to the control of health problems. It is the only ethical way to health problems. It is the only ethical way to obtain data about the effects of chemicals on obtain data about the effects of chemicals on human beings and hence to establish beyond human beings and hence to establish beyond doubt that toxicity to humans exists. The doubt that toxicity to humans exists. The following are the main approaches that have following are the main approaches that have been used in epidemiology.been used in epidemiology.

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11 . .Cohort studyCohort study

A cohort is a component of the population born during a A cohort is a component of the population born during a particular period and identified by the period of birth particular period and identified by the period of birth so that its characteristics (such as causes of death and so that its characteristics (such as causes of death and numbers still living) can be ascertained as it enters numbers still living) can be ascertained as it enters successive time and age periods. The term ‘cohort’ successive time and age periods. The term ‘cohort’ has broadened to describe any designated group of has broadened to describe any designated group of persons followed or traced over a period of time.persons followed or traced over a period of time.

Therefore, the group is selected before the study is Therefore, the group is selected before the study is done. done.

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In a cohort study, one identifies cohorts of In a cohort study, one identifies cohorts of people who are, have been, or in the future people who are, have been, or in the future may be exposed or not exposed, or exposed in may be exposed or not exposed, or exposed in different degrees, to a factor or factors different degrees, to a factor or factors hypothesized to influence the probability of hypothesized to influence the probability of occurrence of a given disease or other occurrence of a given disease or other outcome. An essential feature of the method is outcome. An essential feature of the method is the observation of a large population for a the observation of a large population for a sufficient (long) period, to generate reliable sufficient (long) period, to generate reliable incidence or mortality rates in the population incidence or mortality rates in the population subsets. subsets.

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Cohort studyCohort study of lung cancer and of lung cancer and smokingsmokingLung cancer Lung cancer

casescasesNo lung cancer No lung cancer

controlcontrol

SmokersSmokers127 (a)127 (a)35 (b)35 (b)

NonsmokersNonsmokers73 (c)73 (c)165 (d)165 (d)

400 persons were selected and followed for a long period of time, with regards 400 persons were selected and followed for a long period of time, with regards to smoking and development of lung cancer.to smoking and development of lung cancer.

These were classified according to being smoking or nonsmoking, developed These were classified according to being smoking or nonsmoking, developed cancer or notcancer or not

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No. of smokers with lung cancerNo. of smokers with lung canceraa127127

No. of smokers without cancerNo. of smokers without cancerbb3535

Total number of smokersTotal number of smokersa + ba + b162162

No of non smokers with cancerNo of non smokers with cancercc7373

No of non smokers without No of non smokers without cancercancer

dd165165

Total No. of non smokers Total No. of non smokers c + dc + d238238

Proportion of smokers who Proportion of smokers who develop cancerdevelop cancer

a/(a+b)a/(a+b)0.7840.784

Proportion of non smokers who Proportion of non smokers who develop cancerdevelop cancer

c/(c+d)c/(c+d)0.3070.307

5555

Relative riskRelative risk

Relative risk (RR) is defined as:Relative risk (RR) is defined as: RR = {(a/a+b)}/{c/(c+d)}RR = {(a/a+b)}/{c/(c+d)} RR = {127/(127+35)}/{73/(73+165)}RR = {127/(127+35)}/{73/(73+165)} RR = 2.6RR = 2.6

This means that the probability of developing This means that the probability of developing cancer is 2.6 times as high in smokers as in cancer is 2.6 times as high in smokers as in nonsmokers. This is an evidence of association nonsmokers. This is an evidence of association of cancer with smoking.of cancer with smoking.

5656

22 . .Case-control studyCase-control study

A case-control study starts with the identification of persons with A case-control study starts with the identification of persons with the disease (or other outcome variable) of interest, and a the disease (or other outcome variable) of interest, and a suitable control (comparison and reference) group of persons suitable control (comparison and reference) group of persons without the disease. The relationship of an attribute to the without the disease. The relationship of an attribute to the disease is examined by comparing the diseased and non-disease is examined by comparing the diseased and non-diseased with regard to how frequently the attribute is present diseased with regard to how frequently the attribute is present or, if quantitative, the levels of the attribute, in the two groups.or, if quantitative, the levels of the attribute, in the two groups.

Example: cancer suspected due to nuclear material exposureExample: cancer suspected due to nuclear material exposureTo examine the effect of exposure of pregnant women to

pesticides on the birth weight of the infants

5757

Case control study of lung cancer Case control study of lung cancer and smokingand smoking

Lung cancer Lung cancer casescases

No lung cancer No lung cancer controlcontrol

SmokersSmokers127 (a)127 (a)35 (b)35 (b)

NonsmokersNonsmokers73 (c)73 (c)165 (d)165 (d)

TotalTotal200200200200

200 persons with lung cancer (cases) and 200 persons without lung cancer 200 persons with lung cancer (cases) and 200 persons without lung cancer (control) were selected and categorized to whether they are smokers or (control) were selected and categorized to whether they are smokers or

nonsmokersnonsmokers

5858

Odds ratioOdds ratio OR is a measure of association between exposure and OR is a measure of association between exposure and

outcome. outcome. OR = (a/c)/(b/d)OR = (a/c)/(b/d) Odds of exposure among cases = a/c =127/35 = 1.7397Odds of exposure among cases = a/c =127/35 = 1.7397 Odds of exposure among control = b/d = 35/165 = 0.2121Odds of exposure among control = b/d = 35/165 = 0.2121 Odds ratio = 1.7397/0.2121 = 8.2Odds ratio = 1.7397/0.2121 = 8.2 This means that the odds of exposure to smoking among cases This means that the odds of exposure to smoking among cases

of lung cancer are 8.2 times as large as the odds of smoking of lung cancer are 8.2 times as large as the odds of smoking among control. This indicates an important association among control. This indicates an important association between lung cancer and smoking. between lung cancer and smoking.

5959

Risk factorsRisk factors

Risk factors are factors that increase the Risk factors are factors that increase the probability of having the diseaseprobability of having the disease

Protective factors are factors that will decrease Protective factors are factors that will decrease the probability of having the disease. This is the probability of having the disease. This is implied by an odd ratio less than 1implied by an odd ratio less than 1

6060

Case control study of obesity and Case control study of obesity and regularly eatig vegetableregularly eatig vegetable

Obese casesObese casesNo obese controlNo obese control

Eat vegetableEat vegetable121 (a)121 (a)171 (b)171 (b)

Do not eat vegetDo not eat veget129 (c)129 (c)79 (d)79 (d)


6161

Odds ratioOdds ratio

OR = (a/c)/(b/d)OR = (a/c)/(b/d) Odds of exposure among cases = a/c =121/129 = 0.938Odds of exposure among cases = a/c =121/129 = 0.938 Odds of exposure among control = b/d = 171/79 = 2.1646Odds of exposure among control = b/d = 171/79 = 2.1646 Odds ratio = 0.938/2.1646 = 0.43Odds ratio = 0.938/2.1646 = 0.43 This means that the odds of exposure to eating veget among This means that the odds of exposure to eating veget among

obese persons were 0.43 times as large as the odds among non obese persons were 0.43 times as large as the odds among non obese control. This indicates that eating vegets could be a obese control. This indicates that eating vegets could be a protective factor decreasing the probability of obesityprotective factor decreasing the probability of obesity

6262

Case-control study of depression and Case-control study of depression and regularly eatig vegetableregularly eatig vegetable

Depressed casesDepressed casesundepressed undepressed controlcontrol

Eat vegetableEat vegetable90 (a)90 (a)90 (b)90 (b)

Do not eat vegetDo not eat veget130 (c)130 (c)130 (d)130 (d)


6363

Odds ratioOdds ratio OR = (a/c)/(b/d)OR = (a/c)/(b/d) Odds of exposure among cases = a/c = 90/130 = 0.6923Odds of exposure among cases = a/c = 90/130 = 0.6923 Odds of exposure among control = b/d = 90/130 = 0.6923Odds of exposure among control = b/d = 90/130 = 0.6923 Odds ratio = 0.6923/6923 = 1.0Odds ratio = 0.6923/6923 = 1.0 This means that the odds of exposure to eating veget among This means that the odds of exposure to eating veget among

depressed persons were the same as the odds among depressed persons were the same as the odds among undepressed control. This indicates that eating vegets has no undepressed control. This indicates that eating vegets has no association with depression.association with depression.

Conclusion: OR>1 suggests a possible risk factor, an OR<1 Conclusion: OR>1 suggests a possible risk factor, an OR<1 suggests a possible protective factor, while an OR=1 suggests suggests a possible protective factor, while an OR=1 suggests no association between exposure and outcomeno association between exposure and outcome

6464

Advantages of a case-control studyAdvantages of a case-control study

Can be easily used for studying infrequent Can be easily used for studying infrequent diseasesdiseases

Relatively inexpensive as no follow-up is Relatively inexpensive as no follow-up is necessarynecessary

Fast process, as no need to wait for the Fast process, as no need to wait for the accumlation of enough cases as in cohort accumlation of enough cases as in cohort studiesstudies

Cheaper to do than cohort studiesCheaper to do than cohort studies

6565

Limitations of case-control studiesLimitations of case-control studies

Unlike cohort, Cannot be used to calculate the incidence rate, Unlike cohort, Cannot be used to calculate the incidence rate, which is necessary to calculate the relative risk.which is necessary to calculate the relative risk.

It is not possible to elucidate the chronologic order of It is not possible to elucidate the chronologic order of exposure and disease, which one occurred firstexposure and disease, which one occurred first

Results of a case control study is affected by selection bias if Results of a case control study is affected by selection bias if the control group does not come from the same population as the control group does not come from the same population as the casesthe cases

Recall bias between cases and control may be a real limitation Recall bias between cases and control may be a real limitation as cases remembrance are usually betteras cases remembrance are usually better

Although good for rare diseases, the study is not suitable for Although good for rare diseases, the study is not suitable for rare exposures, like studing the risk of asthma among workers rare exposures, like studing the risk of asthma among workers in a nuclear submarine.in a nuclear submarine.

6666

33 . .Confounding studiesConfounding studies

6767

A confounding variable is a variable (say, A confounding variable is a variable (say, pollution) that can cause the disease under pollution) that can cause the disease under study (cancer) and is also associated with the study (cancer) and is also associated with the exposure of interest (smoking). The existence exposure of interest (smoking). The existence of confounding variables in smoking studies of confounding variables in smoking studies made it difficult to establish a clear causal link made it difficult to establish a clear causal link between smoking and cancer unless between smoking and cancer unless appropriate methods were used to adjust for appropriate methods were used to adjust for the effect of the confoundersthe effect of the confounders

6969

An example of a confounding studyAn example of a confounding study

We are studying categories which developed a disease and We are studying categories which developed a disease and did not develop a disease as a consequence of exposure. did not develop a disease as a consequence of exposure.

First a table like the one below is constructed:First a table like the one below is constructed:

diseaseddiseasedNo diseaseNo diseaseTotalTotal

ExposedExposedaabba+ba+b

Not exposedNot exposedccddc+dc+d

7070

No. of people with diseaseNo. of people with diseaseaa

No. of people without diseaseNo. of people without diseasebb

Total number of exposedTotal number of exposeda + ba + b

No of unexposed but diseasedNo of unexposed but diseasedcc

No of unexposed without diseaseNo of unexposed without diseasedd

Total No. of unexposed Total No. of unexposed c + dc + d

Proportion of exposed who developed Proportion of exposed who developed diseasedisease

a/(a+b)a/(a+b)

Proportion of unexposed who developed Proportion of unexposed who developed diseasedisease

c/(c+d)c/(c+d)

7171

Three cases:Three cases:

1.1. a/(a+b) > c/(c+d) a/(a+b) > c/(c+d)

This means that exposure and disease are positively This means that exposure and disease are positively associatedassociated

2. a/(a+b) < c/(c+d) 2. a/(a+b) < c/(c+d)

This means that exposure and disease are negatively This means that exposure and disease are negatively associated. Exposure is thus a protective factorassociated. Exposure is thus a protective factor

3. a/(a+b) = c/(c+d) 3. a/(a+b) = c/(c+d)

This means that there is no association between This means that there is no association between exposure and diseaseexposure and disease

7272

The relative risk (RR) is a measure of the The relative risk (RR) is a measure of the degree of association between the exposure degree of association between the exposure and development of the diseaseand development of the disease

RR = {(a/a+b)}/{c/(c+d)}RR = {(a/a+b)}/{c/(c+d)} RR>1 means that exposed individuals have RR>1 means that exposed individuals have

higher probability of developing the diseasehigher probability of developing the disease RR<1 means that exposure leads to less risk of RR<1 means that exposure leads to less risk of

the disease, i.e. exposure is a protective factorthe disease, i.e. exposure is a protective factor RR=1 suggests no association between RR=1 suggests no association between

exposure and diseaseexposure and disease

7373

An Example: Bedsores and mortalityAn Example: Bedsores and mortality

dieddiedDid not Did not diedie

TotalTotal

BedsoreBedsore7979745745824824

No bedsoreNo bedsore2862868290829085768576

TotalTotal3653659035903594009400

7474

No. of people with bedsore who diedNo. of people with bedsore who diedaa7979

No. of smokers with bedsore who did No. of smokers with bedsore who did not dienot die

bb745745

Total number of people with bedsoreTotal number of people with bedsorea + ba + b824824

No of people without a bedsore who No of people without a bedsore who dieddied

cc286286

No of people without bedsore who did No of people without bedsore who did not dienot die

dd82908290

Total No. of people without a bedsore Total No. of people without a bedsore c + dc + d85768576

Proportion of people with a bedsore Proportion of people with a bedsore who diedwho died

a/(a+b)a/(a+b)9.6%9.6%

Proportion of people without a Proportion of people without a bedsore who diedbedsore who died

(c/(c+d)(c/(c+d)3.3%3.3%

7575

RR = (79/824)/(286/8576) = 2.9RR = (79/824)/(286/8576) = 2.9 This suggests that the probability of death was 2.9 This suggests that the probability of death was 2.9

times as high in people with bedsores as in people times as high in people with bedsores as in people without bedsores. This may imply a strong without bedsores. This may imply a strong association between bedsores and death.association between bedsores and death.

Do not rush with conclusions as there is a possibility Do not rush with conclusions as there is a possibility of a confounding factor; where when patients where of a confounding factor; where when patients where admitted to hospital the medical severity of their admitted to hospital the medical severity of their cases was recorded, leading to two categories: high cases was recorded, leading to two categories: high severity and low severity groups. Our task now is to severity and low severity groups. Our task now is to check whether medical severity is a confounding check whether medical severity is a confounding factor or not. Two tables will be necessary, one for factor or not. Two tables will be necessary, one for each category:each category:

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Collected infoCollected info::

Of the 79 people who had bedsores and died 55 had high Of the 79 people who had bedsores and died 55 had high medical severity and 24 had low medical severitymedical severity and 24 had low medical severity

Of the 745 people who had bedsores and did not die, 51 had Of the 745 people who had bedsores and did not die, 51 had high medical severity and 694 had low medical severityhigh medical severity and 694 had low medical severity

Of the 286 people who had no bedsores and died, 5 had high Of the 286 people who had no bedsores and died, 5 had high medical severity and 281 had low medical severitymedical severity and 281 had low medical severity

Of the 8290 people who had no bedsores and did not die, 5 Of the 8290 people who had no bedsores and did not die, 5 had high medical severity and 8285 had low medical severity.had high medical severity and 8285 had low medical severity.

Now construct a table for each category:Now construct a table for each category:

7777

High medical severity groupHigh medical severity group


TotalTotal




7878

The relative risk in the high medical severity The relative risk in the high medical severity group group

RR = (55/106)/(5/10) = 1.04RR = (55/106)/(5/10) = 1.04

7979

Low medical severity groupLow medical severity group


TotalTotal



TotalTotal3053058979897992849284

8080

The relative risk in the low medical severity The relative risk in the low medical severity group group

RR = (24/718)/(281/8566) = 1.02RR = (24/718)/(281/8566) = 1.02

8181

This process is called stratification where two strata This process is called stratification where two strata where created. In each stratum, the association where created. In each stratum, the association between bedsores and death cannot be explained by between bedsores and death cannot be explained by medical severity because in each stratum medical medical severity because in each stratum medical severity was kept constant. severity was kept constant.

Stratification is used to adjust for a confounding Stratification is used to adjust for a confounding factor.factor.

Both relative risks of both strata are close to 1. This Both relative risks of both strata are close to 1. This means that the risk of death has no association to means that the risk of death has no association to bedsores, provided that adjustment is made for bedsores, provided that adjustment is made for medical severity. medical severity.

8282

Bedsores Death

Medical severity

associationassociation

Association of interest

8383

The unadjusted relative risk of 2.9 is thus The unadjusted relative risk of 2.9 is thus misleading. Therefore, trying to eliminate bedsores misleading. Therefore, trying to eliminate bedsores will not affect probability of death. If A does not will not affect probability of death. If A does not cause B, then eliminating A will not affect the cause B, then eliminating A will not affect the occurrence of B. occurrence of B. Bedsores are said to be guilty by Bedsores are said to be guilty by association.association.

If the adjusted and unadjusted relative risk have If the adjusted and unadjusted relative risk have been similar, this means that medical severity did been similar, this means that medical severity did not confound the association between bedsores and not confound the association between bedsores and death. death.

For confounding to occur:For confounding to occur:1.1. There must be an association between cofounder There must be an association between cofounder

and the diseaseand the disease2.2. There must be an association between cofounder There must be an association between cofounder

and exposure and exposure

8484

44 . .Cross-sectional studyCross-sectional study

A cross-sectional study examines the relationship between A cross-sectional study examines the relationship between diseases (or other health-related characteristics) and other diseases (or other health-related characteristics) and other variables of interest as they exist in a defined population at one variables of interest as they exist in a defined population at one particular time. Disease prevalence rather than incidence is particular time. Disease prevalence rather than incidence is normally recorded in a cross-sectional study and the temporal normally recorded in a cross-sectional study and the temporal sequence of cause and effect cannot necessarily be determined. sequence of cause and effect cannot necessarily be determined.

A type of study that is referenced about a single point in time. In A type of study that is referenced about a single point in time. In other words, information about the exposure factor and other words, information about the exposure factor and outcome variables are collected at the same point of time. outcome variables are collected at the same point of time. Example: conducting a survey in which information about Example: conducting a survey in which information about exposure and information about disease outcome are collected exposure and information about disease outcome are collected at the same time. at the same time.

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adverse effect of allergy

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