- Email: [email protected]
Distinguishing between adverse and non-adverse effects – Session summary
Exp Toxic Pathol 2002; 54: 51–55 URBAN & FISCHER http://www.urbanfischer.de/journals/exptoxpath
Session Report from the Joint STP/IFSTP International Symposium “Toxicologic Pathology in the New Millenium” June 24–28, 2001, in Orlando, Florida 1
Co-chairman, Consultant, Langendorfer Str. 17, Wülfrath, Germany Co-chairman and speaker, NYMC, Dept. Pathology, BSB, Valhalla, NY, USA 3 Speaker, Syngenta Central Toxicology Laboratory, Cheshire, England 4 Speaker, CIIT, Centers for Health Research, Research Triangle Park, NC, USA 2
Distinguishing between adverse and non-adverse effects Session summary E. KARBE1, G. M. WILLIAMS2, R. W. LEWIS3, I. KIMBER3, and P. M. D. FOSTER4 Received: October 8, 2001; Revised: December 11, 2001; Accepted: February 18, 2002 Address for correspondence: Prof. Dr. med. vet. EBERHARD KARBE, Langendorfer Str. 17, 42489 Wülfrath; Fax: ++49-(0)2058-80392, e-mail: [email protected] Key words: Adverse effect; non-adverse effect; NOEL; NOAEL; enzyme induction; liver; immune system; endocrine system.
Introduction to the session We in toxicologic pathology have to make decisions on various levels: Firstly, we decide whether there is a lesion, secondly we have to diagnose it and give it the appropriate name, thirdly we have to find out whether it is induced, fourthly to judge whether the lesion is adverse or non-adverse, and lastly decide whether the induced lesion has relevance to man. In this session we are concerned with the characterization of adverse and especially of non-adverse effects. We have to be able to distinguish between the two in toxicity studies to determine the non-adverse effect level (NOAEL). The NOAEL is needed to set the initial dose in phase I studies with regard to drugs and to calculate the admissible daily intake of environmental chemicals. Thus, the topic of our session is important for hazard identification and risk assessment. There is, however, a paucity of references in the literature specifically dealing with this issue. Therefore, the chairmen of this session were pleased that our session topic was accepted in the overall program, since there appears to be an urgent need to present and discuss this issue in a large international forum. The objective of this session is to give some guidance to toxicologic pathologists, study directors, and their colleagues in governmental agencies, on how to define non-adverse effects and to establish the NOAEL in toxicity studies.
The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) also saw the need to deal with this issue on an international basis and estab-
lished a taskforce three years ago. The taskforce’s objective was to draft a paper to define adverse and non-adverse effects, to be published. However, that draft forms the basis of our first presentation in this session, concentrating on definitions and a multi-step approach. RICHARD LEWIS, chairman of the ECETOC taskforce, presented this paper:
Recognition of adverse and non-adverse effects in toxicity studies “One of the most important quantitative outputs from toxicity studies is identification of the highest exposure level (dose or concentration) that does not cause treatment related effects that could be considered relevant to human health risk assessment. A review of regulatory and other scientific literature and of current practices has revealed a lack of consistency in definition and application of frequently used terms such as ‘No Observed Effect Level’ (NOEL), ‘No Observed Adverse Effect Level’ (NOAEL), ‘adverse effect’, ‘biologically significant effect’ or ‘toxicologically significant effect’. Moreover, no coherent criteria were found that could be used to guide consistent interpretation of toxicity studies, including the recognition and differentiation between adverse and non-adverse effects. 0940-2993/02/54/01-051 $ 15.00/0
51
This presentation addresses the issues identified above, first by proposing a standard set of definitions for key terms such as NOEL and NOAEL that are frequently used to describe the overall outcome of a toxicity study. Secondly, a coherent framework is outlined that can assist the toxicologist in arriving at consistent study interpretation. This structured process involves two main steps. In the first the toxicologist must decide whether differences from control values are treatment related or if they are chance deviations. In the second step, only those differences judged to be effects are further evaluated in order to discriminate between those that are adverse and those that are not. For each step criteria will be described that can be used to make consistent judgements. In differentiating an effect from a chance finding, consideration is given inter alia to dose response, spurious measurements in individual parameters, the precision of the measurement under evaluation, ranges of natural variation and the overall biological plausibility of the observation. In discriminating between the adverse and the non-adverse effect consideration is given to: whether the effect is an adaptive response, whether it is transient, the magnitude of the effect, its association with effects in other related endpoints, whether it is a precursor to a more significant effect, whether it has an effect on the overall function of the organism, whether it is a specific effect on an organ or organ system or secondary to general toxicity or whether the effect is a predictable consequence of the experimental model. In interpreting complex studies it is recognized that a weight of the evidence approach, combining the criteria outlined above to reach an overall judgement, is the optimal way of applying the process. It is believed that the use of such a scheme will help to improve the consistency of study interpretation that is the foundation of hazard and risk assessment.” In addition to the above summary, a few detailed aspects from the presentation should be mentioned. Firstly a recommendation for the general definition on non-adverse effects: Non-adverse effects can be defined as those biological effects that do not cause biochemical, morphological or physiological changes that affect the general well-being, growth, development or life span of an animal. Secondly, he cited examples of non-adverse effects, including: • • • •
inhibition of plasma butyrylcholinesterase, inhibition of erythrocyte cholinesterase up to 20%, decrease of plasma alanine and aspartate transaminases, delayed preputial separation associated with retarded body weight gains, • minor increases in liver weight not associated with changes in morphology or function. The last example relates to the most common and important non-adverse effects and leads us to the next presentation by GARY WILLIAMS: 52
Exp Toxic Pathol 54 (2002) 1
Alteration of liver cell function and proliferation: Differentiation between adaptation and toxicity “Exposure of experimental animals to biologically effective levels of chemicals, either endogenous or exogenous, the latter of either synthetic or natural origin, elicits a response(s) which reflects the diverse ways in which the various units of organization of an organism deal with chemical perturbation. For some chemicals, an initial response constitutes an adaptive effect which maintains homeostasis. Disruption of this equilibrium at any level of organization leads to an adverse effect, or toxicity. The livers of laboratory animals and humans, like other organs, undergo programmed phases of growth and development, characterized by proliferation followed by differentiation. With organ maturity, the process of differentiation leads to the commitment of differentiated cells to constitutive functions which maintain homeostasis and to specialized functions which serve organismal needs. In the mature livers of all species, proliferation of all cell types subsides to a low level. Thus, the mature liver consists of 2 types of cells: intermediate cells, the hepatocytes, which replicate infrequently, but can respond to signals for replication, and replicating cells, the stem cells, endothelial, Kupffer, and stellate cells (Ito or pericytes), bile duct epithelium and granular lymphocytes (pit cells). Quantifiable alterations or effects at the molecular level underlie alterations at the organelle level, which in turn lead to alterations at the cellular level, which can ultimately be manifested as a change in the whole organism. Alterations can be quantal (binary), i.e. either all or none, as with cell replication, cell necrosis or apoptosis, and cell differentiation, which take place at the cellular level. They can also be graded or continuous (nonbinary), as with enzyme induction, organelle hypertrophy, and extracellular matrix elaboration, occurring either at the intra- or extra (supra) cellular level. Any quantifiable change induced in the function or structure of a cell or tissue constitutes a response or effect. Each of the several types of cell in the liver responds to a given stimulus according to its localization and function. Generally, renewing cells are more vulnerable to chemical injury than intermediate cells, which are largely quiescent. Hepatic adaptive responses usually involve actions of the chemical on cellular regulatory pathways, often receptor mediated, leading to changes in gene expression and ultimately alteration of the metabolome. The response is directed toward maintaining homeostasis through modulation of various cellular and extracellular functions. At all levels of organization, adaptive responses are beneficial in that they enhance the capacity of all units to respond to chemical-induced stress, are reversible and preserve viability. Such adaptation at subtoxic exposures is also referred to as hormesis. In contrast, adverse or toxic effects in the liver often involve chemical reaction with cellular macromolecules
and produce disruption of homeostasis. Such effects diminish the capacity for response, can be nonreversible at all levels of organization and can compromise viability. An exposure that elicits an adaptive response can produce toxicity with longer or higher exposures (i.e. above a threshold) and the mechanism of action changes with the effective dose. A variety of hepatic adaptive and toxic effects has been identified. Examples of adaptive effects are provided by phenobarbital and ciprofibrate, while pdichlorobenzene and 2-acetylaminofluorene illustrate different toxic effects. The effects of chemicals in the liver are, in general, similar between experimental animals and humans, although exceptions exist. Thus, identification and monitoring of both types of effect are integral in the safety assessment of chemical exposures.” In the above paper, examples of non-adverse effects associated with changes of liver cell function and/or morphology are given, based on recognized mechanisms: Example Effect Proandrogenic compounds Increased hexokinase activity Proestrogenic compounds Increased phosphoenol-pyruvate carboxykinase activity Fibrates, phthalates Induction of cyclooxygenase 2 Phenobarbital Induction of γ-glutamyl transferase Phenobarbital Induction of biotransformation enzymes Erythromycin Increased plasma membrane P-glycoprotein 4-Hydroxynonenal Induction of heat shock proteins Anthracyclines Induction of multidrug resistance membrane pump Ciprofibrate Peroxisome proliferation Di(2-ethylhexyl)phthalate (slight increase) Phenobarbital Cell hypertrophy Rifabutin Multinucleated hepatocytes Liver cells are especially well equipped with cytoprotective agents, antioxidant enzymes, and DNA repair systems, rendering them rather insensitive to potential hazardous effects of xenobiotics, thus maintaining homeostasis at certain dose levels of exposure, visible to the pathologist, if marked, as cytoplasmic change (e.g. ground glass appearance) that can be associated with enlargement of liver cells. However, at high doses for long periods, disruption of the equilibrium may lead to adverse effects, including liver cell degeneration, necrosis, or neoplasia. While an increase of liver weight by induced hypertrophy and hyperplasia, at least up to 20% in the absence of other liver pathology, is often regarded as a nonadverse effect in rodents, comments on such weight increase limits were not available during the session. Obviously, such figures are difficult to establish, since xenobiotics act on liver cells by many different mechanisms,
and a correlation between increased liver weight and enzyme induction, on the one hand, and adverse liver pathology, on the other, is not uniform, but dependent on the compound and its mechanism of action. The aspect of immune effects is of special importance, because of the increase of allergies in the human population. An overview was presented by IAN KIMBER:
Immune responses: Adverse versus non-adverse effects “The adaptive immune system in vertebrates has evolved to provide host resistance to infectious microorganisms and malignant disease. Normal immune function and the induction of specific immune responses require the orchestrated interaction between cells and molecules both within and outside the lymphoid system. Immunotoxicology can be defined as the study of adverse health effects that may result from the interaction of xenobiotics with the immune system. In general terms such effects can take one of two forms. The first of these is immunotoxicity (or immunosuppression) where there is a perturbation of, or damage to, one or more components of the immune system resulting in impaired immune function and reduced host resistance. The design and interpretation of experimental immunotoxicity studies and the investigation of clinical immunosuppression require consideration of the relationship between changes in the structure and/or function of discrete components of the immune system and holistic changes in the susceptibility to infectious and malignant disease. The other main way in which chemicals may cause adverse health effects secondary to interaction with the immune system is through stimulation of specific immune responses that result in allergic disease. Allergy to chemicals and proteins can take many forms, including allergic contact dermatitis, allergic sensitization of the respiratory tract (associated with rhinitis and/or asthma), systemic allergic reactions (associated frequently with drug treatment) and gastrointestinal disease. Here there is a need to distinguish between immunogenic responses per se and those immune responses that are of sufficient vigor and of the quality necessary to provoke allergic sensitization. The purpose of this article is to explore the extent to which distinctions can be drawn between adverse and non-adverse effects in the context of immunotoxicity and allergy.” Parameters relating to immunotoxicity are changes in morphology of lymphoid tissue, the impairment of immunological function of various cell populations, the reduction of the concentration of lymphocyte subpopulations, and the decrease of host resistance realized in in vivo assays. It appears that various changes of above parameters may be induced without affecting host resistance. Obviously, a considerable functional reserve exists in the whole immune system. Thus, small changes in Exp Toxic Pathol 54 (2002) 1
53
immune status can be tolerated without an increase in host susceptibility due to functional redundancy in the immune system and the existence of compensatory and complementary mechanisms. However, such reserves may not be present in infants, the aged or those with congenital or acquired pertubations of immune function; at a population level there may exist a more linear relationship between susceptibility to immunotoxicity and health status, as shown at least for the immunosuppressant cyclophosphamide. Expert panelists at a meeting sponsored by the International Life Sciences Institute came to the conclusion that “any effect on immune reserve could be important to the health of an individual and that immune reserve and redundancy should not be considered when evaluating and interpreting immune function data”. In general, at least for environmental chemicals, risk assessment is based on the highest NOAEL (with functional integrity possibly associated with no adverse effects) and a safety factor, which is raised from 100 to 1000 when considering those most sensitive within the human population. The same approach for potential immunotoxicants would consider effects on immune functions non-adverse, as long as host resistance is not affected in the animal model. The corresponding NOAEL and the same safety factors as above would apply. Considering the above ILSI citation, it appears that the safety factor for the most sensitive people regarding single immune parameters would already be implemented by calling all effects adverse. There are doubts that such a procedure is practical. The conclusion drawn is that the translation of small changes in immunological function to an assessment of health risks for man remains problematic and that there currently exists no clear consensus on what magnitude of change can be defined as being adverse and what non-adverse. Regarding testing for potential allergens, special consideration is given to skin sensitization resulting in allergic contact dermatitis. A suitable animal model to detect such effects is the murine lymph node assay, which measures the increase in cell proliferation of the regional lymph node after challenge with the compound after a single previous sensitization. In practice, the threshold for classification as a skin sensitizer is a 3-fold increase in proliferation relative to vehicle controls and recently a retrospective mathematical analysis has shown that this 3-fold index serves to discriminate accurately between sensitizers (adverse) and non-sensitizers (non-adverse regarding sensitization). Even though this model includes one sensitization only, and multiple sensitizations may increase the effect after the challenge, there is no doubt that chemicals that fail at all concentrations to mount a 3-fold or greater increase in lymph node cell proliferation are not associated with contact allergy in humans. Thus, induced proliferations up to a 3-fold increase can be considered as non-adverse effects regarding sensitization. 54
Exp Toxic Pathol 54 (2002) 1
The last presentation is concerned with endocrine disruption, an issue receiving great attention in recent years. This topic was presented by DR. PAUL FOSTER:
Endocrine active agents: Implications of adverse and non-adverse changes “The USEPA is currently in the process of developing screening and testing methodologies for the assessment of agents that may possess endocrine-like activity – the so-called endocrine disruptors. Moreover, the Agency has signaled its intention of placing information arising from such studies on the worldwide web. This has created significant interest in how such information may be used in risk assessment and by policymakers and the public in the potential regulation or deselection of specific chemical agents. The construction of lists of endocrine disruptors whilst fulfilling the requirements of some parties is really of little use when the nature of the response, the dose level employed and the lifestage of the test species used are not given. Thus we have already seen positive in vitro information available on the interaction with a receptor being used as a key indicator when the results of large, high quality in vivo studies showing no adverse changes have been ignored. Clearly a number of in vitro systems are available to ascertain chemical interaction with specific (mainly steroid) hormone receptors including a number of reporter gene assays. In the main these assays only provide indicators of potential problems and should not be, in isolation, indicators of toxicity. Likewise short-term in vivo screens such as the uterotrophic and Hershberger studies are frequently conducted in castrated animals and thus indicate the potential for a pharmacological response in vivo rather than an adverse effect. A number of new end points have been added to standard rodent testing protocols in the belief of providing more sensitivity to detect endocrine related changes. These include the measurement of anogenital distance (AGD), developmental landmarks (vaginal opening (VO), preputial separation (PPS)) and in some studies the counting of nipples and areolae on males. AGD, VO and PPS are all affected by the size of the pup in which they are measured and should always be compared using bodyweight as a co-variate. The historical control database for such changes is gradually growing, albeit that if pups are not individually identified it becomes problematic to associate any change with a specific malformation or to assess whether a delay or advance in, for example, developmental landmarks is biologically significant. Agents that significantly reduce AGD in males (it is an androgen dependent variable) frequently have other more adverse changes associated with this end point (e.g. reproductive tract malformations), but a 2-3% change in AGD although measurable is unlikely to be biologically of importance and in isolation would not necessarily be considered adverse. Retention of thoracic nipples in male rat pups is also an indicator of impaired androgen status. Recent studies have also shown that this
retention for some endocrine active chemicals is permanent. Thus the presence of a permanent structural change that is rarely found in adult control animals could be considered a malformation and therefore a developmental adverse effect on which risk assessment decisions could be made. The advent of multigeneration reproduction studies as the definitive studies for the assessment of the dose-response relationships and risk assessment for endocrine disruptors has shown that current testing protocols may be inadequate to reliably detect the adverse effects of concern as only 1 adult/sex/litter is examined. A number of the effects on reproductive development whilst due to an in utero exposure will not be manifest until after puberty or at adulthood. The use of only a limited number of animals to examine such changes, particularly for weaker acting materials indicates that some agents may have been examined in well conducted, modern protocols, but have insufficient power to detect low incidence phenomena (e.g. a 5% incidence of malformations).” Almost all effects of endocrine disruptors on hormone-sensitive organs are considered adverse, but even this heterogeneous group of causative compounds can lead to non-adverse effects, such as 1) transient retention of areolae/nipples in males, 2) up to 3% reduction of anogenital distance as isolated finding, 3) effects on anogenital distance, vaginal opening or preputial separation, secondary to reduced pup size/ weight (delayed development). Many in vitro and some manipulated in vivo experiments, such as the uterotrophic or Hershberger studies in
castrated animals, show effects which find no parallel in the relevant non-castrated animals, at least at lower dose levels. While it is important to identify potential endocrine disruptors using sensitive models, the identification of adverse effects to be used for risk assessment should be done in relevant animal models.
Concluding remarks In this session, concentrating on non-adverse effects, we dealt with definitions and general aspects, the liver, the immune system, and hormone-sensitive organs. We could not cover all organ systems, but selected the most important ones. All speakers emphasized the need to consider in each case of hazard identification all other related relevant effects, before deciding that an effect is non-adverse; each case has to be dealt with individually, considering its special circumstances. This session was the first of its kind before such a large international forum, and we realize that the results will need adjustments and additions. We hope we were able to give some guidance to those who have to make decisions on hazard identification for the benefit of producers and users alike.
Reference: Proceedings with complete manuscripts of all presentations, including references, is scheduled to be published in Toxicologic Pathology 2002.
Exp Toxic Pathol 54 (2002) 1
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Session Report from the Joint STP/IFSTP International Symposium “Toxicologic Pathology in the New Millenium” June 24–28, 2001, in Orlando, Florida 1
Co-chairman, Consultant, Langendorfer Str. 17, Wülfrath, Germany Co-chairman and speaker, NYMC, Dept. Pathology, BSB, Valhalla, NY, USA 3 Speaker, Syngenta Central Toxicology Laboratory, Cheshire, England 4 Speaker, CIIT, Centers for Health Research, Research Triangle Park, NC, USA 2
Distinguishing between adverse and non-adverse effects Session summary E. KARBE1, G. M. WILLIAMS2, R. W. LEWIS3, I. KIMBER3, and P. M. D. FOSTER4 Received: October 8, 2001; Revised: December 11, 2001; Accepted: February 18, 2002 Address for correspondence: Prof. Dr. med. vet. EBERHARD KARBE, Langendorfer Str. 17, 42489 Wülfrath; Fax: ++49-(0)2058-80392, e-mail: [email protected] Key words: Adverse effect; non-adverse effect; NOEL; NOAEL; enzyme induction; liver; immune system; endocrine system.
Introduction to the session We in toxicologic pathology have to make decisions on various levels: Firstly, we decide whether there is a lesion, secondly we have to diagnose it and give it the appropriate name, thirdly we have to find out whether it is induced, fourthly to judge whether the lesion is adverse or non-adverse, and lastly decide whether the induced lesion has relevance to man. In this session we are concerned with the characterization of adverse and especially of non-adverse effects. We have to be able to distinguish between the two in toxicity studies to determine the non-adverse effect level (NOAEL). The NOAEL is needed to set the initial dose in phase I studies with regard to drugs and to calculate the admissible daily intake of environmental chemicals. Thus, the topic of our session is important for hazard identification and risk assessment. There is, however, a paucity of references in the literature specifically dealing with this issue. Therefore, the chairmen of this session were pleased that our session topic was accepted in the overall program, since there appears to be an urgent need to present and discuss this issue in a large international forum. The objective of this session is to give some guidance to toxicologic pathologists, study directors, and their colleagues in governmental agencies, on how to define non-adverse effects and to establish the NOAEL in toxicity studies.
The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) also saw the need to deal with this issue on an international basis and estab-
lished a taskforce three years ago. The taskforce’s objective was to draft a paper to define adverse and non-adverse effects, to be published. However, that draft forms the basis of our first presentation in this session, concentrating on definitions and a multi-step approach. RICHARD LEWIS, chairman of the ECETOC taskforce, presented this paper:
Recognition of adverse and non-adverse effects in toxicity studies “One of the most important quantitative outputs from toxicity studies is identification of the highest exposure level (dose or concentration) that does not cause treatment related effects that could be considered relevant to human health risk assessment. A review of regulatory and other scientific literature and of current practices has revealed a lack of consistency in definition and application of frequently used terms such as ‘No Observed Effect Level’ (NOEL), ‘No Observed Adverse Effect Level’ (NOAEL), ‘adverse effect’, ‘biologically significant effect’ or ‘toxicologically significant effect’. Moreover, no coherent criteria were found that could be used to guide consistent interpretation of toxicity studies, including the recognition and differentiation between adverse and non-adverse effects. 0940-2993/02/54/01-051 $ 15.00/0
51
This presentation addresses the issues identified above, first by proposing a standard set of definitions for key terms such as NOEL and NOAEL that are frequently used to describe the overall outcome of a toxicity study. Secondly, a coherent framework is outlined that can assist the toxicologist in arriving at consistent study interpretation. This structured process involves two main steps. In the first the toxicologist must decide whether differences from control values are treatment related or if they are chance deviations. In the second step, only those differences judged to be effects are further evaluated in order to discriminate between those that are adverse and those that are not. For each step criteria will be described that can be used to make consistent judgements. In differentiating an effect from a chance finding, consideration is given inter alia to dose response, spurious measurements in individual parameters, the precision of the measurement under evaluation, ranges of natural variation and the overall biological plausibility of the observation. In discriminating between the adverse and the non-adverse effect consideration is given to: whether the effect is an adaptive response, whether it is transient, the magnitude of the effect, its association with effects in other related endpoints, whether it is a precursor to a more significant effect, whether it has an effect on the overall function of the organism, whether it is a specific effect on an organ or organ system or secondary to general toxicity or whether the effect is a predictable consequence of the experimental model. In interpreting complex studies it is recognized that a weight of the evidence approach, combining the criteria outlined above to reach an overall judgement, is the optimal way of applying the process. It is believed that the use of such a scheme will help to improve the consistency of study interpretation that is the foundation of hazard and risk assessment.” In addition to the above summary, a few detailed aspects from the presentation should be mentioned. Firstly a recommendation for the general definition on non-adverse effects: Non-adverse effects can be defined as those biological effects that do not cause biochemical, morphological or physiological changes that affect the general well-being, growth, development or life span of an animal. Secondly, he cited examples of non-adverse effects, including: • • • •
inhibition of plasma butyrylcholinesterase, inhibition of erythrocyte cholinesterase up to 20%, decrease of plasma alanine and aspartate transaminases, delayed preputial separation associated with retarded body weight gains, • minor increases in liver weight not associated with changes in morphology or function. The last example relates to the most common and important non-adverse effects and leads us to the next presentation by GARY WILLIAMS: 52
Exp Toxic Pathol 54 (2002) 1
Alteration of liver cell function and proliferation: Differentiation between adaptation and toxicity “Exposure of experimental animals to biologically effective levels of chemicals, either endogenous or exogenous, the latter of either synthetic or natural origin, elicits a response(s) which reflects the diverse ways in which the various units of organization of an organism deal with chemical perturbation. For some chemicals, an initial response constitutes an adaptive effect which maintains homeostasis. Disruption of this equilibrium at any level of organization leads to an adverse effect, or toxicity. The livers of laboratory animals and humans, like other organs, undergo programmed phases of growth and development, characterized by proliferation followed by differentiation. With organ maturity, the process of differentiation leads to the commitment of differentiated cells to constitutive functions which maintain homeostasis and to specialized functions which serve organismal needs. In the mature livers of all species, proliferation of all cell types subsides to a low level. Thus, the mature liver consists of 2 types of cells: intermediate cells, the hepatocytes, which replicate infrequently, but can respond to signals for replication, and replicating cells, the stem cells, endothelial, Kupffer, and stellate cells (Ito or pericytes), bile duct epithelium and granular lymphocytes (pit cells). Quantifiable alterations or effects at the molecular level underlie alterations at the organelle level, which in turn lead to alterations at the cellular level, which can ultimately be manifested as a change in the whole organism. Alterations can be quantal (binary), i.e. either all or none, as with cell replication, cell necrosis or apoptosis, and cell differentiation, which take place at the cellular level. They can also be graded or continuous (nonbinary), as with enzyme induction, organelle hypertrophy, and extracellular matrix elaboration, occurring either at the intra- or extra (supra) cellular level. Any quantifiable change induced in the function or structure of a cell or tissue constitutes a response or effect. Each of the several types of cell in the liver responds to a given stimulus according to its localization and function. Generally, renewing cells are more vulnerable to chemical injury than intermediate cells, which are largely quiescent. Hepatic adaptive responses usually involve actions of the chemical on cellular regulatory pathways, often receptor mediated, leading to changes in gene expression and ultimately alteration of the metabolome. The response is directed toward maintaining homeostasis through modulation of various cellular and extracellular functions. At all levels of organization, adaptive responses are beneficial in that they enhance the capacity of all units to respond to chemical-induced stress, are reversible and preserve viability. Such adaptation at subtoxic exposures is also referred to as hormesis. In contrast, adverse or toxic effects in the liver often involve chemical reaction with cellular macromolecules
and produce disruption of homeostasis. Such effects diminish the capacity for response, can be nonreversible at all levels of organization and can compromise viability. An exposure that elicits an adaptive response can produce toxicity with longer or higher exposures (i.e. above a threshold) and the mechanism of action changes with the effective dose. A variety of hepatic adaptive and toxic effects has been identified. Examples of adaptive effects are provided by phenobarbital and ciprofibrate, while pdichlorobenzene and 2-acetylaminofluorene illustrate different toxic effects. The effects of chemicals in the liver are, in general, similar between experimental animals and humans, although exceptions exist. Thus, identification and monitoring of both types of effect are integral in the safety assessment of chemical exposures.” In the above paper, examples of non-adverse effects associated with changes of liver cell function and/or morphology are given, based on recognized mechanisms: Example Effect Proandrogenic compounds Increased hexokinase activity Proestrogenic compounds Increased phosphoenol-pyruvate carboxykinase activity Fibrates, phthalates Induction of cyclooxygenase 2 Phenobarbital Induction of γ-glutamyl transferase Phenobarbital Induction of biotransformation enzymes Erythromycin Increased plasma membrane P-glycoprotein 4-Hydroxynonenal Induction of heat shock proteins Anthracyclines Induction of multidrug resistance membrane pump Ciprofibrate Peroxisome proliferation Di(2-ethylhexyl)phthalate (slight increase) Phenobarbital Cell hypertrophy Rifabutin Multinucleated hepatocytes Liver cells are especially well equipped with cytoprotective agents, antioxidant enzymes, and DNA repair systems, rendering them rather insensitive to potential hazardous effects of xenobiotics, thus maintaining homeostasis at certain dose levels of exposure, visible to the pathologist, if marked, as cytoplasmic change (e.g. ground glass appearance) that can be associated with enlargement of liver cells. However, at high doses for long periods, disruption of the equilibrium may lead to adverse effects, including liver cell degeneration, necrosis, or neoplasia. While an increase of liver weight by induced hypertrophy and hyperplasia, at least up to 20% in the absence of other liver pathology, is often regarded as a nonadverse effect in rodents, comments on such weight increase limits were not available during the session. Obviously, such figures are difficult to establish, since xenobiotics act on liver cells by many different mechanisms,
and a correlation between increased liver weight and enzyme induction, on the one hand, and adverse liver pathology, on the other, is not uniform, but dependent on the compound and its mechanism of action. The aspect of immune effects is of special importance, because of the increase of allergies in the human population. An overview was presented by IAN KIMBER:
Immune responses: Adverse versus non-adverse effects “The adaptive immune system in vertebrates has evolved to provide host resistance to infectious microorganisms and malignant disease. Normal immune function and the induction of specific immune responses require the orchestrated interaction between cells and molecules both within and outside the lymphoid system. Immunotoxicology can be defined as the study of adverse health effects that may result from the interaction of xenobiotics with the immune system. In general terms such effects can take one of two forms. The first of these is immunotoxicity (or immunosuppression) where there is a perturbation of, or damage to, one or more components of the immune system resulting in impaired immune function and reduced host resistance. The design and interpretation of experimental immunotoxicity studies and the investigation of clinical immunosuppression require consideration of the relationship between changes in the structure and/or function of discrete components of the immune system and holistic changes in the susceptibility to infectious and malignant disease. The other main way in which chemicals may cause adverse health effects secondary to interaction with the immune system is through stimulation of specific immune responses that result in allergic disease. Allergy to chemicals and proteins can take many forms, including allergic contact dermatitis, allergic sensitization of the respiratory tract (associated with rhinitis and/or asthma), systemic allergic reactions (associated frequently with drug treatment) and gastrointestinal disease. Here there is a need to distinguish between immunogenic responses per se and those immune responses that are of sufficient vigor and of the quality necessary to provoke allergic sensitization. The purpose of this article is to explore the extent to which distinctions can be drawn between adverse and non-adverse effects in the context of immunotoxicity and allergy.” Parameters relating to immunotoxicity are changes in morphology of lymphoid tissue, the impairment of immunological function of various cell populations, the reduction of the concentration of lymphocyte subpopulations, and the decrease of host resistance realized in in vivo assays. It appears that various changes of above parameters may be induced without affecting host resistance. Obviously, a considerable functional reserve exists in the whole immune system. Thus, small changes in Exp Toxic Pathol 54 (2002) 1
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immune status can be tolerated without an increase in host susceptibility due to functional redundancy in the immune system and the existence of compensatory and complementary mechanisms. However, such reserves may not be present in infants, the aged or those with congenital or acquired pertubations of immune function; at a population level there may exist a more linear relationship between susceptibility to immunotoxicity and health status, as shown at least for the immunosuppressant cyclophosphamide. Expert panelists at a meeting sponsored by the International Life Sciences Institute came to the conclusion that “any effect on immune reserve could be important to the health of an individual and that immune reserve and redundancy should not be considered when evaluating and interpreting immune function data”. In general, at least for environmental chemicals, risk assessment is based on the highest NOAEL (with functional integrity possibly associated with no adverse effects) and a safety factor, which is raised from 100 to 1000 when considering those most sensitive within the human population. The same approach for potential immunotoxicants would consider effects on immune functions non-adverse, as long as host resistance is not affected in the animal model. The corresponding NOAEL and the same safety factors as above would apply. Considering the above ILSI citation, it appears that the safety factor for the most sensitive people regarding single immune parameters would already be implemented by calling all effects adverse. There are doubts that such a procedure is practical. The conclusion drawn is that the translation of small changes in immunological function to an assessment of health risks for man remains problematic and that there currently exists no clear consensus on what magnitude of change can be defined as being adverse and what non-adverse. Regarding testing for potential allergens, special consideration is given to skin sensitization resulting in allergic contact dermatitis. A suitable animal model to detect such effects is the murine lymph node assay, which measures the increase in cell proliferation of the regional lymph node after challenge with the compound after a single previous sensitization. In practice, the threshold for classification as a skin sensitizer is a 3-fold increase in proliferation relative to vehicle controls and recently a retrospective mathematical analysis has shown that this 3-fold index serves to discriminate accurately between sensitizers (adverse) and non-sensitizers (non-adverse regarding sensitization). Even though this model includes one sensitization only, and multiple sensitizations may increase the effect after the challenge, there is no doubt that chemicals that fail at all concentrations to mount a 3-fold or greater increase in lymph node cell proliferation are not associated with contact allergy in humans. Thus, induced proliferations up to a 3-fold increase can be considered as non-adverse effects regarding sensitization. 54
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The last presentation is concerned with endocrine disruption, an issue receiving great attention in recent years. This topic was presented by DR. PAUL FOSTER:
Endocrine active agents: Implications of adverse and non-adverse changes “The USEPA is currently in the process of developing screening and testing methodologies for the assessment of agents that may possess endocrine-like activity – the so-called endocrine disruptors. Moreover, the Agency has signaled its intention of placing information arising from such studies on the worldwide web. This has created significant interest in how such information may be used in risk assessment and by policymakers and the public in the potential regulation or deselection of specific chemical agents. The construction of lists of endocrine disruptors whilst fulfilling the requirements of some parties is really of little use when the nature of the response, the dose level employed and the lifestage of the test species used are not given. Thus we have already seen positive in vitro information available on the interaction with a receptor being used as a key indicator when the results of large, high quality in vivo studies showing no adverse changes have been ignored. Clearly a number of in vitro systems are available to ascertain chemical interaction with specific (mainly steroid) hormone receptors including a number of reporter gene assays. In the main these assays only provide indicators of potential problems and should not be, in isolation, indicators of toxicity. Likewise short-term in vivo screens such as the uterotrophic and Hershberger studies are frequently conducted in castrated animals and thus indicate the potential for a pharmacological response in vivo rather than an adverse effect. A number of new end points have been added to standard rodent testing protocols in the belief of providing more sensitivity to detect endocrine related changes. These include the measurement of anogenital distance (AGD), developmental landmarks (vaginal opening (VO), preputial separation (PPS)) and in some studies the counting of nipples and areolae on males. AGD, VO and PPS are all affected by the size of the pup in which they are measured and should always be compared using bodyweight as a co-variate. The historical control database for such changes is gradually growing, albeit that if pups are not individually identified it becomes problematic to associate any change with a specific malformation or to assess whether a delay or advance in, for example, developmental landmarks is biologically significant. Agents that significantly reduce AGD in males (it is an androgen dependent variable) frequently have other more adverse changes associated with this end point (e.g. reproductive tract malformations), but a 2-3% change in AGD although measurable is unlikely to be biologically of importance and in isolation would not necessarily be considered adverse. Retention of thoracic nipples in male rat pups is also an indicator of impaired androgen status. Recent studies have also shown that this
retention for some endocrine active chemicals is permanent. Thus the presence of a permanent structural change that is rarely found in adult control animals could be considered a malformation and therefore a developmental adverse effect on which risk assessment decisions could be made. The advent of multigeneration reproduction studies as the definitive studies for the assessment of the dose-response relationships and risk assessment for endocrine disruptors has shown that current testing protocols may be inadequate to reliably detect the adverse effects of concern as only 1 adult/sex/litter is examined. A number of the effects on reproductive development whilst due to an in utero exposure will not be manifest until after puberty or at adulthood. The use of only a limited number of animals to examine such changes, particularly for weaker acting materials indicates that some agents may have been examined in well conducted, modern protocols, but have insufficient power to detect low incidence phenomena (e.g. a 5% incidence of malformations).” Almost all effects of endocrine disruptors on hormone-sensitive organs are considered adverse, but even this heterogeneous group of causative compounds can lead to non-adverse effects, such as 1) transient retention of areolae/nipples in males, 2) up to 3% reduction of anogenital distance as isolated finding, 3) effects on anogenital distance, vaginal opening or preputial separation, secondary to reduced pup size/ weight (delayed development). Many in vitro and some manipulated in vivo experiments, such as the uterotrophic or Hershberger studies in
castrated animals, show effects which find no parallel in the relevant non-castrated animals, at least at lower dose levels. While it is important to identify potential endocrine disruptors using sensitive models, the identification of adverse effects to be used for risk assessment should be done in relevant animal models.
Concluding remarks In this session, concentrating on non-adverse effects, we dealt with definitions and general aspects, the liver, the immune system, and hormone-sensitive organs. We could not cover all organ systems, but selected the most important ones. All speakers emphasized the need to consider in each case of hazard identification all other related relevant effects, before deciding that an effect is non-adverse; each case has to be dealt with individually, considering its special circumstances. This session was the first of its kind before such a large international forum, and we realize that the results will need adjustments and additions. We hope we were able to give some guidance to those who have to make decisions on hazard identification for the benefit of producers and users alike.
Reference: Proceedings with complete manuscripts of all presentations, including references, is scheduled to be published in Toxicologic Pathology 2002.
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