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Concepts in Toxicology
By John H. Duffus, Douglas M. Templeton, Monica Nordberg The Royal Society of Chemistry
Copyright © 2009 IUPAC, John H Duffus, Douglas M Templeton, Monica Nordberg
All rights reserved.
ISBN: 978-0-85404-157-2
CHAPTER 1
Concept Group 1. Concepts Applying to Fundamental Principles of Toxicology
Subgroup A – Exposure and Toxicity
1.1 Acute and Chronic 14
1.2 Interaction 18
1.3 Dose 21
1.4 Adverse Effect and Toxicity 22
1.5 Toxicity Classification, Labelling and Material Safety Data Sheets 26
1.6 Terms Applied to Toxic Substances 35
1.1 Acute and Chronic
A. Acute
acute
1 Of short duration, in relation to exposure or effect. In experimental toxicology, acute refers to studies where dosing is either single or limited to one day, although the total study duration may extend to two weeks.
2 In clinical medicine, sudden and severe, having a rapid onset. Antonym: chronic.
acute effect
Effect of finite duration occurring rapidly (usually in the first 24 h or up to 14 d) following a single dose or short exposure to a substance or radiation.
acute exposure
Exposure of short duration.
Antonym: chronic exposure.
acute toxicity
1 Adverse effects of finite duration occurring within a short time (up to 14 d) after administration of a single dose (or exposure to a given concentration) of a test substance or after multiple doses (exposures), usually within 24 h of a starting point (which may be exposure to the toxicant, or loss of reserve capacity, or developmental change, etc.).
2 Ability of a substance to cause adverse effects within a short time of dosing or exposure.
Antonym: chronic toxicity.
In toxicology, 'acute' is a word that is used in combination with exposure, toxicity and effect. Acute exposure is a single or very short-lasting dosing by any route. Talking about acute toxicity addresses adverse effects (further discussed below), i.e. harmful effects, unwanted negative effects that occur immediately after or within a short time after administration of a single dose of a substance, or following short exposure or concurrently with continuous exposure, or recurrently following shortly after multiple doses. 'Short' implies a time of 24 h or less. Some effects considered to be acute can occur up to as long as 96 h after exposure. Uraemia can be an acute effect, but it takes almost 96 h to see such an outcome. In toxicity testing, it is most important to be aware of this in order not to draw any false conclusions from animal studies with agents that cause such an acute effect. Acute effects usually occur or develop rapidly after a single exposure. However, acute effects can also appear immediately after, or during, repeated or prolonged exposure.
Acute Toxicity. Historically, an important aspect of acute toxicity has been the identification of the lethal dose or exposure that kills an organism after a short exposure or a single dose. This has been established by a test in which selected organisms are exposed to a series of increasing dose levels until a dose is reached at which all the organisms die. For regulatory purposes, to permit extrapolation to humans, it is usually performed with at least two mammalian species. From such tests, the LD50 for the test species has been derived and used for the classification of the toxicity of chemicals to humans. Such tests involved killing large numbers of animals to obtain a toxicity classification based on lethality. However, this classification tells us nothing about sublethal effects such as immunotoxicity or teratogenicity. This situation was clearly unsatisfactory and so acute toxicity testing is now designed in such a way as to obtain maximum information about all aspects of acute toxicity using the minimum number of animals.
In Europe, classification of new chemicals for toxicity is no longer based on the LD50. The tests used for this purpose are based on survival rather than on lethality. For example, the method of fixed-dose testing is usually limited to a maximum dose of 500 mg per kg body weight. If five males and five females exposed to a dose of this magnitude survive with no evidence of toxicity, the chemical tested need not be classified as toxic. Toxicity classifications based on this approach can provide a similar classification of toxicity to the old LD50 system but with a huge reduction both in the number of animals used and in animal suffering compared to the traditional LD50 tests. Another approach to reducing the numbers of animals used is the up-and-down procedure, which also produces a value approximating to the LD50. This procedure uses sequential dosing together with sophisticated computational methods. It provides a point estimate of the LD50 while achieving significant reductions in animal use.
Since chronic toxicity testing (see below) is expensive and labour-intensive, there is a great need to replace it where possible with shorter-term predictive acute tests and early identification of biomarkers of toxicity. This has been possible to some extent with carcinogenicity. In the past, a cancer study was designed to expose animals to the toxicant and to follow the animals during their lifetime. Each animal upon death was examined for occurrence and localization of tumours in the body. Since it is expensive to maintain animals over long periods, the need for new tools to identify carcinogens is clear. Many cancers start with mutations or chromosome damage, and this can be assessed with short-term tests such as the Ames test or the host-mediated (Legator) test. The Ames test is based on reversal of a point mutation in a Salmonella strain, which makes it unable to synthesize the amino acid histidine. Back-mutation can be detected by growth of the bacteria in a histamine-depleted medium. Rat liver microsomes are included in the test medium to simulate the metabolic activation of organic compounds that may take place in the intact animal. The host- mediated test looks for chromosome changes in vitro and (or) in vivo, including chromosome breaks and sister chromatid exchanges, in microbial cells introduced (e.g. by intravenous injection) into a host animal. The host animal receives the test compound orally and therefore acts as a source of chemical metabolism, distribution and excretion. Another whole animal test involves looking for the production of micronuclei in animals exposed to possible carcinogens. The micronucleus test is less sensitive than bacterial tests but is a more realistic measure of likely chromosomal damage in mammals at risk.
It is also possible to test quickly for mutagenicity and the possibility of associated carcinogenicity by adding suspect substances to cell cultures and looking for chromosome damage and cell transformation. Another approach to carcinogenicity testing is to apply the substances to tissues in culture and/or to genetically compatible transplants and similarly assess the changes that occur.
While the acute tests for mutagenicity give a quick indication of the mutagenic potential of substances tested, it must be emphasized that the effects observed may not necessarily extrapolate to the intact organism. The bacterial strains used in the Ames test have been selected for the absence of DNA (deoxyribonucleic acid) repair mechanisms so that they are much more sensitive to mutagenicity than any normal organism. Cultured cells and tissues lose differentiated properties and are abnormal in this way. Dedifferentiated cells tend to divide more rapidly than normal, and this may facilitate chromosomal damage. In assessing carcinogenicity, it must be remembered that not all mutations lead to cancer nor are all cancers the result of mutations. Thus, while these acute tests may indicate the possibility of carcinogenicity, they are not sufficient to prove it and can be regarded only as screening tests to select substances for further study in this regard.
B. Chronic
chronic
Long-term (in relation to exposure or effect).
1 In experimental toxicology, chronic refers to mammalian studies lasting considerably more than 90 days or to studies occupying a large part of the lifetime of an organism.
2 In clinical medicine, long-established or long-lasting. Antonym: acute.
chronic effect
Consequence that develops slowly and/or has a long-lasting course: may be applied to an effect that develops rapidly and is long-lasting.
Antonym: acute effect.
Synonym: long-term effect.
chronic exposure
Continued exposures occurring over an extended period of time, or a significant fraction of the test species' or of the group of individuals', or of the population's lifetime.
Antonym: acute exposure.
Synonym: long-term exposure.
chronic toxicity
Adverse effects following chronic exposure. Effects which persist over a long period of time whether or not they occur immediately upon exposure or are delayed.
Antonym: acute toxicity.
Chronic effects usually occur after repeated or prolonged exposures. However, chronic effects can also occur after single exposure if they develop slowly or are long-lasting. They are often irreversible. Chronic effects may follow accumulation of a toxic substance or of metabolites formed by biotransformation of the administered substance. They may also be the result of cumulative irreversible effects of toxicants.
Chronic Toxicity. Chronic toxicity usually results in a progressive loss of organ function, e.g. increasing liver damage following regular ingestion of ethanol. For humans, a particularly serious example of chronic toxicity may be the gradual loss of brain cells due, for example, to excessive exposure to ethanol or other neurotoxic agents. Brain cells do not divide and cannot be replaced once they are lost. Because we have a large reserve capacity of such cells their gradual loss may not be apparent, but this added to the normal loss associated with aging may result in premature dementia and related adverse effects.
For the toxicologist, a particular problem arises when the dose or exposure is low or the effect develops a very long time after exposure as may happen with cancer. In these circumstances, it is very difficult to attribute a cause to the delayed effect. It is also difficult to test substances for such effects. Cancer in humans may take up to 40 years to develop after exposure to a carcinogen. Our normal test animals, rats and mice, have life spans of about 2 years and 18 months, respectively. To cause malignant tumours within such a short time, very large doses of suspect carcinogens must be applied. Thus, test doses are much higher than those to which humans may ever be exposed and may therefore overwhelm metabolic defence mechanisms that work well within normal human exposure ranges.
Subchronic (sometimes referred to confusingly as subacute) toxicity refers to the adverse effects observed when animals are administered a toxicant over time, as a result of repeated daily dosing of a chemical, or exposure to the chemical, for a significant part of an organism's lifespan (usually not exceeding 10%). Observations of acute and subchronic toxicity indicate what the critical (target) organ and the critical effect are. With experimental animals, the subchronic period of exposure may range from a few days to 6 months. The terms 'subchronic' and 'subacute' suffer from many variations in their usage and are best avoided. It is better to replace them by giving precise definition of the times of administration and observation. Subchronic testing has usually been limited to 90 days. Chronic toxicity testing should be over the lifetime of the organism, which means 1.5–2 years in the case of the mouse or the rat.
Chronic toxicity testing in rodent and non-rodent species identifies not only general toxicity but also aspects of mutagenicity, carcinogenicity and reproductive toxicity (in rats and rabbits), including specific effects on the reproductive organs, teratogenicity and reproductive toxicity. Some strains of mice, for example, have different frequencies of naturally occurring effects. Most chronic studies are carried out with at least two animal species, usually rats and a non-rodent species such as dogs or primates. For cancer testing, it is important to choose a species known to have an intrinsically low frequency of tumours. For example, the Syrian golden hamster has a low background frequency of tumours in the trachea and lung and thus may be chosen to test for carcinogens suspected to target these organs.
Currently there is great activity in developing alternatives to chronic animal testing, e.g. the use of stem cells, tissue culture and in silico methods.
1.2 Interaction
additive effect
Consequence that follows exposure to two or more physicochemical agents that act jointly but do not interact: the total effect is the simple sum of the effects of separate exposures to the agents under the same conditions.
potentiation
Dependent action in which a substance or physical agent at a concentration or dose that does not itself have an adverse effect enhances the harm done by another substance or physical agent.
synergism (in toxicology)
Pharmacological or toxicological interaction in which the combined biological effect of two or more substances is greater than expected on the basis of the simple summation of the toxicity of each of the individual substances.
antagonism
Combined effect of two or more factors which is smaller than the solitary effect of any one of those factors. In bioassays, the term may be used when a specified effect is produced by exposure to either of two factors but not by exposure to both together.
When an organism is exposed to two or more substances that produce a particular physiological effect these substances may or may not interact. If there is no interaction the effects would be strictly additive; this is intuitively obvious, and the IUPAC Glossary of Terms Used in Toxicology, 2nd Edition, cited in the Introduction, defines additive effect accordingly. The substances would in general each show a dose response–effect individually, and the effects would be strictly additive at any combination of concentrations. This shifts our focus from the substances to the effect: additivity or other descriptors of interaction do not describe the substances themselves, but rather the effects that they elicit. A corollary is that to assert that two substances behave in an additive fashion requires that no statistically significant difference can be demonstrated between measurements made upon exposure to the substances together compared to the sum of the individual exposures.
Additivity. From the above considerations, additivity is strictly the result of substances acting together but independently in the production of a toxic effect. The following are therefore characteristics of additivity:
1. Additivity (or lack thereof) refers only to what can be measured. Therefore, it refers only to specific effects. Two substances that may be additive with respect to a certain effect may be non-additive with respect to other effects. For example, two drugs might be strictly additive with respect to an effect on blood pressure, but have non-additive effects on liver function. So-called drug–drug interactions often refer to non-additivity with respect to side effects.
2. Additivity may occur at some concentration ratios and not others. Because any substance may be non-toxic at some levels and toxic at others (remember Paracelsus, it is only a question of the dose), non-additive effects may be observed only when one component reaches a critical, threshold concentration. For instance, two anticancer drugs might have additive effectiveness until one reached a threshold concentration for suppressing angiogenesis, at which point the effectiveness of the other might increase based on an ability to target hypoxic tissue.
3. Additivity, in the strictest sense, is in general probably not the norm. The complexity of biological systems is such that multiple effects will probably occur with any bioactive agent, and any overlap with the effects of a second agent will produce candidate effects for non-additivity.
Non-additive Interactions. When two or more substances are related through a common toxic, therapeutic or other biological effect, and yet their effect is non-additive with respect to a measured parameter, this interaction may be described by one of several different terms. When the effect of one substance is diminished by the presence of a second, the situation is fairly straightforward. Antagonism is defined above as the 'combined effect of two or more factors which is smaller than the solitary effect of any one of those factors', with the added comment that, in bioassays, the term antagonism 'may be used when a specified effect is produced by exposure to either of two factors but not by exposure to both together'. In this case, it is not necessary to specify which of the two factors is decreasing the activity of the other; both are assumed to have a certain activity, which when they are present together is less than additive. This distinguishes antagonism from inhibition, where one substance may or may not elicit an effect common with another, but is nevertheless capable of antagonizing that effect. When exposure is to more than two substances, i.e. to mixtures, outcomes are often difficult to predict.
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Excerpted from Concepts in Toxicology by John H. Duffus, Douglas M. Templeton, Monica Nordberg. Copyright © 2009 IUPAC, John H Duffus, Douglas M Templeton, Monica Nordberg. Excerpted by permission of The Royal Society of Chemistry.
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