In this article we will discuss about the toxicant dose-response relationship observed in animals.
The term ‘dose’ is defined as the quantity of toxicant administered to, or received by, the animal at one time or in given period of time. ‘Dosage’ refers to the administration of toxicant in doses.
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The amount is readily ascertained when the toxicant/chemical/substance is given by a route such as oral, subcutaneous, or intraperitoneal. This definition is not applicable to exposure by inhalation, because the amount that is retained in the animal depends on the concentration of the toxicant in the inhaled air as well as the duration of the exposure (contact reaction between a chemical and biological system).
Selection of Doses:
To observe the responses, in acute lethality studies, one concentration should be aimed causing the death of about 50% of the animals. At least two other concentrations are selected, one killing more than 50% but less than 90% and the other killing 10-50% of the animals.
In longer-term studies, at least three concentrations should be selected. The highest concentration is intended to cause some toxic effects and the lowest concentration should have no apparent effects. At present, for long term toxicity studies of any toxicant, 8 or 10% concentration (dose) of LC50 for 96 hours is selected.
Duration of Exposure to Toxicants:
Overall, based on the duration, the exposure of animals to toxicants may be divided into four categories:
1. Acute Exposure or Short-Term Exposure:
It is exposure of animals to a chemical up to 96 hours.
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The repeated exposure of animals to sublethal doses of toxicants for one month or less duration is termed as subacute exposure.
The repeated exposure of animals to sublethal doses of toxicants for one to three months’ time is known as subchronic exposure.
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4. Chronic or Long-Term Exposure:
The repeated exposure of animals to sublethal doses of toxicants for more than 3 months’ time is known as chronic exposure.
Types of Human Exposure:
The humans are exposed to toxicants by any of the following means:
(a) Intentional Exposure:
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People consume various drugs and food-additives in their daily life. Sometimes, these substances are taken over for long span of time. As an example, many people use alcohol and cigarettes on a long-term basis, which may lead to chronic toxic effects.
(b) Occupational Exposure:
Humans, on account of their occupations, are exposed to a wide variety of xenobiotics. Occupational exposure to toxic substances is mainly chronic and continuous. The routes of exposure are either inhalation or skin contact. Lung diseases and dermatitis are very common diseases among industrial workers. Sometimes, acute exposure may occur in the event of an accident such as an explosion, spillage, and leakage or because of bad working practices.
(c) Environmental Exposure:
A wide variety of chemical substances in liquid and gaseous state reach the environment on account of various anthropogenic activities and affect the health of humans. Such exposure is recognized as environmental exposure i.e., exposure through the environment.
This form of exposure is usually chronic but sometimes accidents at factories may lead to acute exposure such as, the leakage of Methyl Isocyanate (MIC) by Union Carbide factory at Bhopal. Chronic exposure to gases such as sulphur dioxide, nitrogen oxide, ammonia and carbon mono oxide occur in industrial areas and in the regions of heavy traffic.
(d) Accidental Poisoning:
Mistaken ingestion of poisonous/toxic substances, cleaning fluids and other household products, drugs and excessive doses of drugs cause accidental poisoning. Such type of exposure is normally acute rather than chronic.
(e) Intentional Poisoning:
Intentional poisoning and intentional consumption of poisonous substances for suicidal purposes come under this category of exposure.
Biological Effect Monitoring in Exposed Animals:
Biological effect monitoring (BEM) may be defined as the continuous or repeated measurement and assessment of early biological effects in exposed humans, to evaluate ambient exposure and health risk by comparison with appropriate reference values based on knowledge of the probable relationship between ambient exposure and biological effects.
BEM is used in environmental and occupational toxicology — and also sometimes in forensic toxicology to monitor possible problems at an early stage. Generally, BEM is carried out using readily available specimens such as urine, blood, and feces.
When a BEM parameter is exceeded, the agent non-specific signs and symptoms are not necessarily related to exposure to specific agents, nor to conditions of work environment. BEM parameters are sensitive, but non-selective, and thus may serve as a safety net when the exposure is actually not known (e.g., exposure to various different solvents or a mixture of solvents).
Categories of Toxic Effects:
The exposure of animals to certain doses/ concentrations of toxicants brings about certain alterations in the animals. Such alterations are called responses or effects of toxicants. Toxicant-induced alterations may be at the level of whole animal (death of organisms), at cellular levels (histopathological changes) or at molecular levels (biochemical changes) leading to some physiological responses. When the criterion of effect is lethality or death of organisms, the obtained response is ‘all or none’ type.
Following exposure to a toxicant, the animal either dies or survives i.e., either the animal responds fully to toxicants and the response is death of the animal or it does not respond at all. Hence, this type of effect is termed as ‘all or none’ type.
Responses Produced by the Different Toxicant Doses on the Animals:
Actually responses are the incidences produced by the different doses of different toxicants on the animals. Responses may also be defined as the signals of toxicity in the animals. The ultimate consequences of the toxicity of various toxicants may also be referred to as responses.
The ultimate response, as a result of acute exposure, depends on the amount of toxicant at the critical target sites. In other form, the amount of toxicant at the target site is partly dependent on the dose.
Between exposure to the dose and the response there are four major occurrences. The first three occurrences are absorption, metabolism, and excretion and in this context, metabolism includes both detoxification and toxification processes. The fourth occurrence is distribution within the body, which is critical to the toxicology of xenobiotics.
The nature of toxic effect often varies with the dose. For instance, as the dose of barbiturate increases, the effect progresses through no observable effect to mid-sedation, to loss of righting reflex (sleep), and, finally, death.
Furthermore, because of differences of more individual susceptibility, not all animals exposed to the same dose will respond in the same way. Based on the doses, the effects i.e., responses (incidences) may be assessed by simple statistical analyses. It may yield information on the dose-effect and dose-response relationship.
Dose-Response Relationship:
It may be defined as the graphical relationship between different doses of a toxicant and the corresponding responses produced by them in a test animal.
In simple words, the relationship between the dose and the magnitude of the effect produced by the toxicant may be termed as the dose- response relationship or dose-effect relationship of toxicants.
The exact relationship between the dose of the xenobiotic (toxicant) and the response (effect) depends upon the biological object under observation i.e., the age, size, health, sex and species of the animal and on the nature of chemical (toxicant) used.
Such studies assist medical practitioners to assess the therapeutically effective dose of any drug and the toxicologists to predict the adverse effects of various xenobiotics on biological system.
In toxicology the important concept is the dose. It is also important to recognize the difference between dose and concentration. Dose is actually the concentration multiplied by time.
In the case of essential compounds there is a gradual transition from deficiency to effectiveness depending on the dose given. At low dose, when deficiency occurs, the metabolised or excreted amount of a substance is greater than the uptake, leading to a shortage in the cell or tissue or organ.
A well-known example is vitamin deficiency, leading, in the case of vitamin C, to scurvy. An intake of vitamin C will rectify the balance, and the scurvy will disappear. Thus, a certain minimum amount is necessary to prevent deficiency. This is illustrated in Fig. 17.1, where the effect of an essential compound is shown as a function of dose. There is a gradual change of effects from deficiency to an essential level through to fatal toxicity.
Another known example is Zinc. There is a homeostatic control of zinc in blood plasma, but deficiency may lead to skin healing problems which are overcome by zinc treatment at the wound site. In excess, zinc may cause gastrointestinal problems if the dose is high enough (1 g or more).
Actually the dose-effect relationship was originally developed by pharmacologists to study the relation between a drug and the effects or side- effects on the body. These kinds of relationship can also be used in toxicology. They may be applied to compounds as illustrated in Fig. 17.2, which reveals the relationship between the dose and different effects for a non-essential compound.
The basic difference between Figs. 17.1 and 17.2 is that, in the case of a non-essential compound, no benefit can be expected and, thus, sometimes such compounds are termed toxic. Figs. 17.1 and 17.2 refer to no effects, early effects, and clinical effects.
The no-effect area is clearly concerned with levels that have no effect. The maximum dose that produces no detectable alteration under defined conditions of exposure is referred to as the no-effect level. It is not easy to determine actually whether an effect is detectable, and what the significance of the effect is. Thus a more practical measure is the no observed adverse effect level (NOAEL).
An adverse effect is a change in morphology, physiology, growth, development, or life span of an organism that results in impairment of functional capacity or impairment of capacity to compensate for additional stress or increase in susceptibility to the harmful effects of other environmental influences. Early effects are not necessarily adverse effects and a NOAEL, may be above the level causing an early effect but below the level causing a clinical effect.
Clinical effects are usually mentioned as adverse effects, e.g., occupational asthma (which may be caused by many compounds). Other examples of clinical effects are disturbances in the peripheral and central nervous system (e.g., tremor, polyneuropathy), and disturbances of liver and renal function. As the body has only a few organs and the number of industrially used chemicals is about 70,000, very few early effects and clinical effects are specific for one compound. Thus, one effect may be caused by different compounds.
The branch of science dealing with the production of effects from the structure of a chemical compound is called quantitative structure-activity relationship (QSAR).
Dose-effect relationships are generally S-shaped (Fig. 17.3). In this example there is an exponential rise above the baseline in urinary β2 microglobulin, a parameter for assessing renal tubular damage, as a function of the dose of cadmium in urine. Beyond this, enhancing the dose gives only a moderate rise in the effect. The steepness of the dose-effect relationship determines the value at which the NOAEL must be set. The NOAEL has to be set at a lower dose when the curve rises steeply compared with a low rise.
Early effects are those that can be observed before adverse change has occurred. Examples are compounds that smell offensive at a no-effect level. Mercaptans, for example, are used as a warning odour in natural gas. At higher levels these are toxic.
Thus, in low concentrations, a certain effect that is not adverse is used for a different purpose. Other examples of early effects are the excretion of certain enzymes and specific proteins into urine as a consequence of low exposure to certain compounds such as metals, solvents, pesticides, and drugs.
A dose-effect relationship exists for nearly all organ systems. But there are two exceptions — cancer and the immune system.
If in Fig. 17.3 the effect is replaced by the response, a dose-response curve is obtained. A response may be the reaction of an animal or part of an animal (such as a muscle) to a stimulus, and, as such, is similar to a dose-effect curve. An alternative definition of response is the proportion of a group of individuals that demonstrate a defined effect in a given time at a given dose rate.
These kinds of dose- response relationships are quite different from the dose-effect relationships. Dose- response (D-R) relationships in this sense can be used to differentiate between groups. If a D-R relationship for one group has a steeper slope than for another, the first group will be more sensitive to the dose than the latter. D-R relationships consequently can be used to detect sensitive groups.
Types of Dose-Response Relationships:
These are two basic types of dose-response relationship:
1. Quantitative
2. Quantum.
1. Quantitative Dose-Response Relationship:
It is also known as graded-dose-response- relationship. Occasionally, such quantitative information can be derived from human data. For example, with increasing intakes of methyl mercury, there were paresthesia, ataxia, dysarthria, deafness, and death, and a greater proportions of individuals manifest these effects. Fig. 17.4 shows graphically both the dose-response and dose- effect relationships among individuals exposed to various doses of methyl mercury.
Quantitative-Dose- Response Relationship is also termed as ‘Graded Dose-Response-Relationship’. It is because the degree of response eventually reaches a maximum value corresponding to the subsequent increase in the dose. At this point, further increase in the dose level does not elicit any response at all on behalf of the living subject. This act of cessation with the further increase in the degree of response is termed “ceiling effect”. The dose of the toxicant at which the ceiling effect is produced is called the ‘ceiling-dose’.
The study of the dose-response relationship proves useful in the evaluation of toxicological data. Because of the variation in individual’s sensitivity of any group of organisms, they will not die at the same dose of a chemical. Therefore, the frequency of response, e.g., death, will increase along with the dose. When the mortality or frequency of other effects is plotted against the dose on a logarithmic scale, an S-shaped curve is obtained. The central portion of the curve (between 16% and 84% response) is sufficiently straight for estimating the LD50 or ED50 dose.
2. Quantum-Dose-Response-Relationship:
Such a dose response relationship exhibits quantized alterations in the degree of response elicited by increasing subsequent enhancements of toxicant dose. Hence such a dose-response- relationship is basically ‘all or none’ type relationship.
Alive or dead outcome = Quantal response, but
Alive = Moribund = Dead = Polychotomous Quantal Response.
Quantum-dose-response relationship is observed in case of population studies. In such studies, the percentage of the affected population increases as the dose of the toxicant is raised. The relationship is quantal because a response is stated to be either present (all) or absent (none) in a given individual’s population.
Cumulative Response:
Soon after the toxicant is absorbed and distributed in the animal, it is excreted slowly or rapidly. They may be excreted as the parent chemicals or their metabolites and/or as their conjugates. When the excretion of a toxicant/drug occurs at a slow rate, the complete-response is a result of the sum of the responses, produced by more than one dose of that toxicant/drug. Such a phenomenon is referred to as cumulation of the toxicant and the response produced is referred to as cumulative response.
Saccharin is rapidly excreted; hence its blood level drops rapidly, even after repeated administration. On the other hand, methyl mercury is excreted very slowly; its gradual accumulation culminates in a near plateau only after 270 days. Due to slow rate of excretion of this toxicant its blood level rises resulting in poisoning.
Certain heavy metals, e.g. lead, may accumulate inside the bones without generating deleterious effects although they may produce certain adverse effects on release into the blood circulation. This incidence is referred to as passive cumulation.
Another example of cumulative response is intake of alcohol by human. A single dose of alcohol per day may cause no ill-effect. But if taken repeatedly, the alcohol goes on accumulating slowly in the body because of its slow excretion. The accumulated alcohol damages liver, causes degeneration of intestinal mucosa and, finally, produces cirrhosis of liver, and death. Likewise small doses of X-rays produce slight burns which heal up in a few days. But exposure to these small doses of X-rays over a long period of time causes cumulation which produce deleterious effects, sometimes cancer.
Threshold Limit:
Threshold limit is that concentration of the toxicant/drug beyond which any further increase in the toxicant/drug dose causes progressive changes in the degree of response. Below the threshold limit, there is no apparent change in the degree of responses elicited by an increase in the dose-level. In other words, a dose below which no effect or response is measurable. Such a dose of toxicant is termed as threshold dose. In terms of 50% lethal concentration, it is known as threshold lethal concentration.
The ‘threshold lethal concentration’ is also known as incipient LC50. Threshold has more theoretical significance than the LC50 at an arbitrary time period and is more useful.
A system of classifying different grades of toxicity has been given in a report by an international group (Joint IMCO/FAO/UNESCO, 1969).
The categories given below seem useful for general description of toxicants/pollutants:
Very toxic – Threshold below 1 mg/l
Toxic – Threshold 1-100 mg/l
Moderately toxic – Threshold 100-1,000 mg/l
Slight toxic – Threshold 1,000-10,000 mg/l
Practically non-toxic – Threshold above 10,000 mg/l
Loomis (1978) used the concept of complex (chemical-receptor) to explain the existence of threshold that “there must be necessarily be a quantity of chemical agent below which no biological effect would be achieved”. The existence of threshold doses for chemicals is important in evaluating safety.
Actually safety is the practical certainty that injury will not result from the chemical in question when used in the quantity and in the manner proposed for it. On the other hand, risk denotes the probability (expected frequency) that a chemical will produce undesirable effect under specified conditions.
For workers in industrial environments, standards have been published by the American Conference of Governmental Industrial Hygienists (1974) and their recommendations are given in Table 17.1 for a selected list of pollutants. These data can be used in the design of industrial ventilation system.
Application of Threshold Dose:
The concept of threshold dose is of basic importance because it implies that there is no observed adverse effect level (NOAEL). The NOAEL is important for setting exposure limits. Based on NOAEL, the acceptable daily intake (ADI) may be determined. This factor is used to determine the safe intake of food-additives and contaminants such as pesticides and residues of veterinary drugs, hence to establish safe levels in food.
The ADI is determined by applying the suitable safety factor which is generally 100 –
ADI = NOAEL/100
The NOAEL is determined in mg/kg/day and can be used in setting of occupational exposure limits –
Cppm = Concentration in parts per million
Cmv = Particulate loading of the air – may be defined as total mass of suspended particulate material/ unit volume of mixture
mg/m3 = mg/cubic meter
Therapeutic Index (Margin of Safety):
The concept of the “therapeutic index” was introduced by Paul Ehrlich in 1913.
Therapeutic index indicates the closeness of a lethal-dose to the therapeutically effective dose or therapeutic index is the ratio of LDS0 and ED50 –
T.I. (Therapeutic Index) = LD50/ED50
Therapeutic Index is of great importance to a physician and biochemist, as it clearly indicates the ‘margin of safety’ of a particular drug, by showing that how much extra concentration of a therapeutically effective dose will turn it into a lethal dose.
Thus, the margin of safety is the range of doses of a drug or chemical between a non-effective dose and a lethal dose.
The greater the value (T.I.), the higher the margin of safety.
For example, the therapeutic index of Penicillin is 100 –
i.e., T.I. = 100
i.e. (LD50/ED50) = 100
i.e. 100 × ED50 = LD50
It can be concluded that a hundred-fold increase in the ED50 dose will exert lethal effects. In simple form; this explains the fact that even large variations from the prescribed dose will not prove deleterious in the animals. In this way penicillin accounts for a very high margin of safety.
To sum up, the concept of therapeutic index is very useful in considering the margin of safety for the practical use of pharmaceuticals. Margin of safety is calculated by a safety factor: 100
i.e., Margin of Safety of any chemical = (Threshold concentration/100)
Potency Versus Efficacy:
To compare the toxic effects of two or more chemicals, the dose response to the toxic effects of each chemical must be established. One can then compare the potency and maximal efficacy of the two chemicals to produce a toxic effect. These two important terms can be explained by reference to Fig 17 5. which depicts dose-response curves to four different chemicals for the frequency of a particular toxic effect, such as the production of tumours.
Chemical A is said to be more potent than chemical B because of their relative positions along the dosage axis Potency thus refers to the range of doses over which a chemical produces increasing responses. Thus, A is more potent than B and C is more potent than D. Maximal efficacy reflects the limit of the dose-response relationship on the response axis to a certain chemical. Chemicals A and B have equal maximal efficacy, whereas the maximal efficacy of C is less than that of D.
