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Hazard and risk based on in vitro test data
Toxic. in Vitro Vol. 4, No. 4/5, pp. 694-697, 1990 Printed in Great Britain.All rights reserved
0887-2333/90$3.00+ 0.00 Copyright © 1990PergamonPressplc
H A Z A R D A N D R I S K BASED O N I N VITRO TEST D A T A M. CHAMBERLAINand W. E. PARISH Environmental Safety Laboratory, Unilever Research, Colworth House, Sharnbrook, Bedford MK44 ILQ, UK
Introduction The prime objective of toxicology tests is to identify the nature of any hazard that may be associated with a substance and to use the evidence as part of an assessment of risk for man. It is necessary to define the terms 'hazard' and 'risk', since they often mean different things to different people. For example, the so-called R phrases used in the EEC to denote particular risks associated with substances in fact refer to hazards. For the purposes of this paper, the definitions of hazard and risk that will be used are consistent with the U K Control of Substances Hazardous to Health Regulations (1988). Hazard is defined as the potential to cause 'injury' and is determined by the intrinsic properties of a substance. Therefore, the hazard of a substance can be expressed in terms of the nature of an adverse effect resulting from exposure to it. Thus, we would recognize a substance as being, for example, an irritant or corrosive hazard. It is convenient to regard hazard as being without dimensions. Most toxicology tests are designed to identify hazard. Risk is defined as the likelihood of an injury occurring in man, animals or the ecosystem, as relevant under a given set of conditions. The assessment of risk is not based solely on the potential of a substance to induce hazard in toxicological tests, but also on the known activity of similar substances, physical state, concentration, route and frequency of exposure and several other parameters. Thus, the severity of a response in a test is a measure of the risk in that experimental system under a given set of conditions and is not a direct measure of risk for man. Application of in vitro tests In in vivo experiments, the test substance is presented to a myriad of cell types, organs and physiological systems in which a hazard may be expressed. In contrast, in vitro test systems are usually confined to a single type of primary or established cell derived from one organ, or possibly a representative mixture of cells from an organ, for example macrophages and lymphoid cells from a lymph node. The identification of hazard is often restricted to a few features of cell function or cell damage. Nevertheless, the use of cell cultures for predictive toxicology is already well established. Most of the routine tests to detect genotoxic hazards are in vitro culture systems using a Abbreviation: CAM = chorioallantoic membrane.
diversity of cells (bacteria, yeasts, or cell lines or primary cells from animals or man). From experience of tests for genotoxicity, valuable principles have been established, which include: (1) Indicator cells need not be identical to the cells at risk (target cells); (2) The change to be identified is some alteration in chromosome DNA, though the expression of that change, the endpoint determined in the test, may differ widely for each technique; (3) Where specific metabolism is an essential step in the toxic process, it is necessary to duplicate that metabolism in vitro.
The magnitude of response in an Ames test can vary, depending on the amount of S-9 fraction used (see, for example, Green, 1980). Similarly, a pre-incubation assay may be required for optimal detection of the genotoxic activity of azo dyes (Prival and Mitchell, 1982). These examples illustrate that the magnitude of response in an in vitro system may depend on several parameters, which may vary from one experiment (or laboratory) to another. It follows that the magnitude of response in an in vitro assay should not be taken automatically to indicate the magnitude of risk to man. This is because there are limitations to repeating in vitro what are multistage processes in vivo. The hazards of irritation and corrosion Several of the test procedures on animals required by regulatory authorities are designed to detect hazard, and they give little indication of risk for man. Skin corrosion
Corrosion is characterized by immediate killing of cells in contact with a substance and this is reflected by the determination of depth of necrosis and irreversibility after short periods of application (e.g. OECD, 1981). An in vitro rat epidermal slice technique has been demonstrated to detect reliably substances corrosive to rat skin in vivo (Oliver et al., 1986). Extension of the in vitro technique by making use of human cadaver skin has demonstrated that some chemicals that are corrosive for rat skin are not so for human skin. Therefore, the logic is that if there is no corrosive hazard for human skin there will be no risk of corrosion: this has been confirmed in human trials for a few chemicals (Oliver et al., 1989). Thus, this example demonstrates the virtue of distinguishing between hazard and risk. Skin irritation
The standard in vivo test for irritation is a 4-hr application under a semi-occlusive patch to the skin 694
Hazard and risk based on in vitro test data of the rabbit (OECD, 1981). The results form a basis of regulatory classification for warning in case of human exposure. This procedure detects the potential of irritants to induce the hazard of inflammation in skin with a vascular system: the histopathological features of inflammation are very similar in rabbit and man. However, the procedure provides very little evidence of risk for man. The rabbit is more susceptible than man to irritation, even when exposure in man is 48 hr compared with the rabbit 4-hr exposure with similar conditions of test. Substances labelled as irritant based strictly on the results of current standard toxicological tests, may not be a significant risk for man. The warning of a hazard for man that is embodied in the classification of irritant is derived from the rabbit test. This is based on evidence by analogy. Once the concept of analogy (i.e. classification of hazard not based on human experience) is accepted, application of the several in vitro systems discussed elsewhere in these proceedings is acceptable. An in vitro system using slices of skin, or cell monolayers, cannot reproduce the complex cascade of mediator release and integrated cell activation described in Session One. However, each in vitro test provides evidence of one or more features occurring during tissue damage, which may be used as some indication of the irritation hazard. As the evidence from any one test is limited, two or more procedures are likely to be required to obtain evidence of different features of inflammation, which together may be used to assess potential for irritancy (Parish, 1986; Pemberton et al., 1989). Eye irritation
The eye has attracted much research to find an in vitro system as an alternative to in vivo tests, or as a preliminary screen so that no harmful substance is tested in vivo: any in vivo test has to satisfy regulatory requirements that the substance is harmless. The development of eye irritation tests shows a progressive evolution from in vivo, to an in vitro corneal injury system using the whole eye (Burton et al., 1981) to cell culture methods (Jackson et al., 1988; Wallin et al., 1987), possibly supported by a physicochemical procedure, for example EYTEX (Gordon and Bergman, 1987). The in vitro eye organ procedure, in which eyes removed from freshly-killed rabbits and mounted in chambers maintain their physiological properties for at least 4 hr, is an effective means of detecting irritants. By prolonging the contact time of the substance with the eye, it is possible to identify and compare even very mild irritants (York et al., 1982). The primary physiological and histological responses in vivo and in vitro are similar (Parish, 1985). However, there are limitations to the routine use of the method. Rabbits have to be killed to obtain the eyes, and no account is taken of recovery from insult or of possible effects on the conjunctiva. An expensive slit lamp is required for accurate determination of corneal swelling. Despite these drawbacks, the use of eyes in vitro has proved to be an effective alternative method for many years and still provides control positive test data for comparison with results from other in vitro methods.
695
The results of the in vitro eye organ technique apply to damage to the cornea. Though it is unlikely that significant damage to the cornea would occur independently of damage to the conjunctiva and underlying mucosa, attempts were made to use the chorioaUantoic membrane (CAM) of the hen's egg embryo to simulate the vascular system of the conjunctiva. Of the two techniques proposed, one results in focal necrosis (Leighton et al., 1985), the other in vascular endothelial changes and haemorrhage (Luepke, 1985). However, the hen's egg system is too underdeveloped at 14 days to resemble the conjunctival response, lacking the neutrophils that are a major feature of an acute inflammatory response (Friend et al., 1989). In our experience, the CAM necrosis technique (Leighton et al., 1985) and the rapid vascular response (Luepke, 1985) are not reliable in identifying eye irritants (Lawrence et al., 1986 and 1989) and have not been shown to confer any advantage over tests on cell monolayers. Improvements in techniques have supported further refinements of cell culture methods to identify irritant hazard to the eye, and a battery of simple tests should eventually provide the basis of routine methods to detect potential eye irritants, each identifying features or intensity of potential damage. F o r example: (1) A cell monolayer to detect substances causing any damage. Unprotected cultured cells are very susceptible to contact with toxic substances. The origin or particular line of cells is not important, and the endpoint to detect cell damage is not important, though it is preferable not to use very sensitive methods that may not allow suflicie.nt discrimination between substances. Different laboratories could use different systems, provided that within each laboratory the chosen method is consistent and standards are available for interlaboratory comparison. (2) The agarose technique (Wallin et al., 1987), in which a monolayer of cells is cultured beneath a layer of agarose.. The test substance diffuses through the agarose, so the monolayer is protected from the sudden shock of exposure to the test substance; incompatible pH and non-isotonic solutions may become adjusted before contact with the cells, and binding of some chemicals to the agarose may simulate binding to ocular secretions, preventing contact with the cornea or diluting the chemical. (3) Denaturation of protein or giycoprotein as detected by the turbimetric changes in a defined substrate (e.g. the EYTEX technique; Gordon and Bergrnan, 1987). This may represent the ability of chemicals to induce opacity of the cornea in more severe responses. Even if this triad of tests is found to be inadequate, their consideration shows the evolution of method development until a cell culture system together with physicochemical techniques may become a feasible regimen to identify potential irritants without recourse to in vivo experiments. However, it should be emphasized that these refinements are aimed at improving identification of hazard.
The importance of validation Before any in vitro test can be used with any degree of reliability, a validation exercise has to be undertaken. Usually, in vitro data are compared with in vivo data derived from animal experiments. The work of
696
M. CHAMBERLAINand W. E. PARISH
Oliver et al. (1989) serves to remind us to be more critical in the selection of the in vivo database used in such validation exercises. If the laboratory animal models are not good indicators of hazard for man, then any in vitro method developed against that model may be of limited value. This point is illustrated by the work of Elcombe (1985) and Elcombe et al. (1985) in the case of trichloroethylene, which induces tumours in the livers of mice. Several substances, such as clofibric acid, diethylhexyl phthalate and trichloroethylene, induce tumours in the livers of mice in long-term studies (see review by Reddy and Lalwani, 1983). Although the mechanism of action of these substances as carcinogens is not known, considerable attention has been paid to their ability to induce peroxisome proliferation both in vivo and in hepatocytes in vitro. Elcombe et al. (1985) demonstrated that trichloroethylene, which does not induce tumours in rat livers, does not induce peroxisome proliferation in the rat. Further investigations (Elcombe, 1985) revealed that trichloroethylene does not induce peroxisome proliferation in cultured h u m a n hepatocytes. On this basis, it was proposed that trichloroethylene did not represent a hepatocarcinogenic hazard to man. In the absence of such a hazard, no risk of liver tumours would be posed to man. Conclusions
In consideration of the wider use of in vitro cultures to predict hazard, several principles should be applied: (1) It is essential to define the particular feature occurring in vivo that is represented by the change in the in vitro system. In vitro assays based on a relevant mechanism of action have more credibility than those assays in which a precise mechanism is not defined. (2) Depending upon the feature being examined, consideration should be given to using cells derived from a relevant target organ. Hepatocytes or a liver enzyme system may be essential in order to detect toxins for liver, but a wide range of cell types may be acceptable for detection of irritant or corrosive substances. (3) Culture systems may be used to detect the potential of substances to induce selective effects in a particular organ. However, distinction should be made between selective effects and non-specific effects resulting from overt toxicity. As stated at the outset, the concepts of hazard and risk are central to toxicology studies: the logic is that hazard identification is followed by risk assessment. M a n y in vitro assays have been developed solely against the backdrop of laboratory animal data, without the necessary consideration of the relevance of the animal data to assessing risk for man.
Therefore, it is recommended that more consideration be given to the selection of an appropriate in vivo database when developing in vitro systems. M a x i m u m use should be made of h u m a n data when they are available. While in vitro systems cannot reproduce the complexities that determine risk for man, the use of h u m a n tissue in relevant experiments should be a better way of detecting hazard for man: where there is no hazard it follows that there is no risk.
REFERENCES
Burton A. B. G., York M. and Lawrence R. S. (1981) The in vitro assessment of severe eye irritants. Fd Cosmet. Toxicol. 19, 471-480. Control of Substances Hazardous to Health Regulations 1988. (1988) SJ. No. 1657. HMSO, London. Elcombe C. R. (1985) Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazard assessment. Archs Toxicol. Suppl. 8, 6-17. Elcombe C. R., Rose M. S. and Pratt I. S. (1985) Biochemical, histological, and ultrastructural changes in rat and mouse liver following the administration of trichloroethylene: possible relevance to species differences in hepatocarcinogenicity. Toxic. appl. Pharmac. 79, 363-376. Friend J. V., Crevel R. W. R., Williams T. C. and Parish W. E, (1989) Immaturity of the inflammatory response of the chick chorioallantoic membrane (CAM). In Vitro Toxic. In press. Gordon V. C. and Bergman H. C. (1987) EYTEX, An in vitro method for evaluation of ocular irritancy. In In Vitro Toxicology-Approaches to Validation. Alternative Methods in Toxicology, Vol. 5. pp. 293-302. Edited by A.
M. Goldberg. Mary Ann Liebert Inc., New York. Green M. H. L. (1980) The scientific basis for shortterm tests for carcinogenicity: non-mammalian systems. In Molecular and Cellular Aspects of Carcinogen Screening Tests. Edited by R. Montesano, H. Bartsch, L. Tomatis and W. Davis. pp. 155-167. IARC Seient. Publ. no. 27. International Agency for Research on Cancer, Lyon. Jackson E. M., Hume R. D. and Wallin R. F. (1988) The agarose diffusion method for ocular irritancy screening; cosmetics products, Part II. J. Toxicol. cut. ocular Toxicol. 7, 187-194. Lawrence R. S., Ackroyd D. M. and Williams D. L. (1990) The chorioallantoic membrane in the prediction of eye irritation potential. Toxic. in Vitro. 4, 321-323. Lawrence R. S., Groom M. H., Ackroyd D. M. and Parish W. E. (1986) The chorioallantoic membrane in irritation testing. Fd Chem. Toxic. 24, 497-502. Leighton J., Nassauer J. and Tchao R. (1985) The chick embryo in toxicology: an alternative to the rabbit eye. Fd Chem. Toxic. 23, 293-298. Luepke N. P. (1985) Hen's egg chorioallantoic membrane test for irritation potential. Fd Chem. Toxic. 23, 287-291. Oliver G. J. A., Pemberton M. A. and Leeser J. (1989) In vivo validation of an in vitro skin corrosivity test in human volunteers. Toxicologist 9, 260. Oliver G. J. A., Pemberton M. A. and Rhodes C. (1986) An in vitro skin corrosivity test--modifications and validation. Fd Chem. Toxic. 24, 507-512. OECD (1981) Acute dermal irritation/corrosion. In OECD Guidelines for Testing of Chemicals. Section 4, No. 404. OECD, Paris. Parish W.. E. (1985) Ability of in vitro (corneal injury-eye organ and chorioallantoic membrane) tests to represent histopathological features of acute eye inflammation. Fd Chem. Toxic. 23, 215-227. Parish W. E. (1986) Evaluation of in vitro predictive tests for irritation and allergic sensitization. Fd Chem. Toxic. 24, 481-494. Pemberton M. A., Oliver G. J. A., Pate I., Rhodes C., Barlow A., Doe J. E. and Botham P. (1989) Initial validation of an in vitro technique for the assessment of skin irritant chemicals. Toxicologist 9, 260. Prival M. J. and Mitchell V. D. (1982) Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver $9. Mutation Res. 97, 103-116.
Hazard and risk based on in vitro test data Reddy J. K. and Lalwani N. D. (1983) Carcinogenesis by hepatic peroxisome proliferators: evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. ToxicoL 12, 1-58. Wallin R. F., Hume R. D. and Jackson E. M. (1987) The agarose diffusion method for ocular irritancy screening:
697
cosmetics products, Part I. J. Toxicol. cut. ocular Toxicol. 6, 239-250. York M., Lawrence R. S. and Gibson G. B. (1982) An in vitro test for the assessment ofeye irritancy in consumer products---preliminary findings. Int. J. cosmet. Sci. 4, 223-234.
0887-2333/90$3.00+ 0.00 Copyright © 1990PergamonPressplc
H A Z A R D A N D R I S K BASED O N I N VITRO TEST D A T A M. CHAMBERLAINand W. E. PARISH Environmental Safety Laboratory, Unilever Research, Colworth House, Sharnbrook, Bedford MK44 ILQ, UK
Introduction The prime objective of toxicology tests is to identify the nature of any hazard that may be associated with a substance and to use the evidence as part of an assessment of risk for man. It is necessary to define the terms 'hazard' and 'risk', since they often mean different things to different people. For example, the so-called R phrases used in the EEC to denote particular risks associated with substances in fact refer to hazards. For the purposes of this paper, the definitions of hazard and risk that will be used are consistent with the U K Control of Substances Hazardous to Health Regulations (1988). Hazard is defined as the potential to cause 'injury' and is determined by the intrinsic properties of a substance. Therefore, the hazard of a substance can be expressed in terms of the nature of an adverse effect resulting from exposure to it. Thus, we would recognize a substance as being, for example, an irritant or corrosive hazard. It is convenient to regard hazard as being without dimensions. Most toxicology tests are designed to identify hazard. Risk is defined as the likelihood of an injury occurring in man, animals or the ecosystem, as relevant under a given set of conditions. The assessment of risk is not based solely on the potential of a substance to induce hazard in toxicological tests, but also on the known activity of similar substances, physical state, concentration, route and frequency of exposure and several other parameters. Thus, the severity of a response in a test is a measure of the risk in that experimental system under a given set of conditions and is not a direct measure of risk for man. Application of in vitro tests In in vivo experiments, the test substance is presented to a myriad of cell types, organs and physiological systems in which a hazard may be expressed. In contrast, in vitro test systems are usually confined to a single type of primary or established cell derived from one organ, or possibly a representative mixture of cells from an organ, for example macrophages and lymphoid cells from a lymph node. The identification of hazard is often restricted to a few features of cell function or cell damage. Nevertheless, the use of cell cultures for predictive toxicology is already well established. Most of the routine tests to detect genotoxic hazards are in vitro culture systems using a Abbreviation: CAM = chorioallantoic membrane.
diversity of cells (bacteria, yeasts, or cell lines or primary cells from animals or man). From experience of tests for genotoxicity, valuable principles have been established, which include: (1) Indicator cells need not be identical to the cells at risk (target cells); (2) The change to be identified is some alteration in chromosome DNA, though the expression of that change, the endpoint determined in the test, may differ widely for each technique; (3) Where specific metabolism is an essential step in the toxic process, it is necessary to duplicate that metabolism in vitro.
The magnitude of response in an Ames test can vary, depending on the amount of S-9 fraction used (see, for example, Green, 1980). Similarly, a pre-incubation assay may be required for optimal detection of the genotoxic activity of azo dyes (Prival and Mitchell, 1982). These examples illustrate that the magnitude of response in an in vitro system may depend on several parameters, which may vary from one experiment (or laboratory) to another. It follows that the magnitude of response in an in vitro assay should not be taken automatically to indicate the magnitude of risk to man. This is because there are limitations to repeating in vitro what are multistage processes in vivo. The hazards of irritation and corrosion Several of the test procedures on animals required by regulatory authorities are designed to detect hazard, and they give little indication of risk for man. Skin corrosion
Corrosion is characterized by immediate killing of cells in contact with a substance and this is reflected by the determination of depth of necrosis and irreversibility after short periods of application (e.g. OECD, 1981). An in vitro rat epidermal slice technique has been demonstrated to detect reliably substances corrosive to rat skin in vivo (Oliver et al., 1986). Extension of the in vitro technique by making use of human cadaver skin has demonstrated that some chemicals that are corrosive for rat skin are not so for human skin. Therefore, the logic is that if there is no corrosive hazard for human skin there will be no risk of corrosion: this has been confirmed in human trials for a few chemicals (Oliver et al., 1989). Thus, this example demonstrates the virtue of distinguishing between hazard and risk. Skin irritation
The standard in vivo test for irritation is a 4-hr application under a semi-occlusive patch to the skin 694
Hazard and risk based on in vitro test data of the rabbit (OECD, 1981). The results form a basis of regulatory classification for warning in case of human exposure. This procedure detects the potential of irritants to induce the hazard of inflammation in skin with a vascular system: the histopathological features of inflammation are very similar in rabbit and man. However, the procedure provides very little evidence of risk for man. The rabbit is more susceptible than man to irritation, even when exposure in man is 48 hr compared with the rabbit 4-hr exposure with similar conditions of test. Substances labelled as irritant based strictly on the results of current standard toxicological tests, may not be a significant risk for man. The warning of a hazard for man that is embodied in the classification of irritant is derived from the rabbit test. This is based on evidence by analogy. Once the concept of analogy (i.e. classification of hazard not based on human experience) is accepted, application of the several in vitro systems discussed elsewhere in these proceedings is acceptable. An in vitro system using slices of skin, or cell monolayers, cannot reproduce the complex cascade of mediator release and integrated cell activation described in Session One. However, each in vitro test provides evidence of one or more features occurring during tissue damage, which may be used as some indication of the irritation hazard. As the evidence from any one test is limited, two or more procedures are likely to be required to obtain evidence of different features of inflammation, which together may be used to assess potential for irritancy (Parish, 1986; Pemberton et al., 1989). Eye irritation
The eye has attracted much research to find an in vitro system as an alternative to in vivo tests, or as a preliminary screen so that no harmful substance is tested in vivo: any in vivo test has to satisfy regulatory requirements that the substance is harmless. The development of eye irritation tests shows a progressive evolution from in vivo, to an in vitro corneal injury system using the whole eye (Burton et al., 1981) to cell culture methods (Jackson et al., 1988; Wallin et al., 1987), possibly supported by a physicochemical procedure, for example EYTEX (Gordon and Bergman, 1987). The in vitro eye organ procedure, in which eyes removed from freshly-killed rabbits and mounted in chambers maintain their physiological properties for at least 4 hr, is an effective means of detecting irritants. By prolonging the contact time of the substance with the eye, it is possible to identify and compare even very mild irritants (York et al., 1982). The primary physiological and histological responses in vivo and in vitro are similar (Parish, 1985). However, there are limitations to the routine use of the method. Rabbits have to be killed to obtain the eyes, and no account is taken of recovery from insult or of possible effects on the conjunctiva. An expensive slit lamp is required for accurate determination of corneal swelling. Despite these drawbacks, the use of eyes in vitro has proved to be an effective alternative method for many years and still provides control positive test data for comparison with results from other in vitro methods.
695
The results of the in vitro eye organ technique apply to damage to the cornea. Though it is unlikely that significant damage to the cornea would occur independently of damage to the conjunctiva and underlying mucosa, attempts were made to use the chorioaUantoic membrane (CAM) of the hen's egg embryo to simulate the vascular system of the conjunctiva. Of the two techniques proposed, one results in focal necrosis (Leighton et al., 1985), the other in vascular endothelial changes and haemorrhage (Luepke, 1985). However, the hen's egg system is too underdeveloped at 14 days to resemble the conjunctival response, lacking the neutrophils that are a major feature of an acute inflammatory response (Friend et al., 1989). In our experience, the CAM necrosis technique (Leighton et al., 1985) and the rapid vascular response (Luepke, 1985) are not reliable in identifying eye irritants (Lawrence et al., 1986 and 1989) and have not been shown to confer any advantage over tests on cell monolayers. Improvements in techniques have supported further refinements of cell culture methods to identify irritant hazard to the eye, and a battery of simple tests should eventually provide the basis of routine methods to detect potential eye irritants, each identifying features or intensity of potential damage. F o r example: (1) A cell monolayer to detect substances causing any damage. Unprotected cultured cells are very susceptible to contact with toxic substances. The origin or particular line of cells is not important, and the endpoint to detect cell damage is not important, though it is preferable not to use very sensitive methods that may not allow suflicie.nt discrimination between substances. Different laboratories could use different systems, provided that within each laboratory the chosen method is consistent and standards are available for interlaboratory comparison. (2) The agarose technique (Wallin et al., 1987), in which a monolayer of cells is cultured beneath a layer of agarose.. The test substance diffuses through the agarose, so the monolayer is protected from the sudden shock of exposure to the test substance; incompatible pH and non-isotonic solutions may become adjusted before contact with the cells, and binding of some chemicals to the agarose may simulate binding to ocular secretions, preventing contact with the cornea or diluting the chemical. (3) Denaturation of protein or giycoprotein as detected by the turbimetric changes in a defined substrate (e.g. the EYTEX technique; Gordon and Bergrnan, 1987). This may represent the ability of chemicals to induce opacity of the cornea in more severe responses. Even if this triad of tests is found to be inadequate, their consideration shows the evolution of method development until a cell culture system together with physicochemical techniques may become a feasible regimen to identify potential irritants without recourse to in vivo experiments. However, it should be emphasized that these refinements are aimed at improving identification of hazard.
The importance of validation Before any in vitro test can be used with any degree of reliability, a validation exercise has to be undertaken. Usually, in vitro data are compared with in vivo data derived from animal experiments. The work of
696
M. CHAMBERLAINand W. E. PARISH
Oliver et al. (1989) serves to remind us to be more critical in the selection of the in vivo database used in such validation exercises. If the laboratory animal models are not good indicators of hazard for man, then any in vitro method developed against that model may be of limited value. This point is illustrated by the work of Elcombe (1985) and Elcombe et al. (1985) in the case of trichloroethylene, which induces tumours in the livers of mice. Several substances, such as clofibric acid, diethylhexyl phthalate and trichloroethylene, induce tumours in the livers of mice in long-term studies (see review by Reddy and Lalwani, 1983). Although the mechanism of action of these substances as carcinogens is not known, considerable attention has been paid to their ability to induce peroxisome proliferation both in vivo and in hepatocytes in vitro. Elcombe et al. (1985) demonstrated that trichloroethylene, which does not induce tumours in rat livers, does not induce peroxisome proliferation in the rat. Further investigations (Elcombe, 1985) revealed that trichloroethylene does not induce peroxisome proliferation in cultured h u m a n hepatocytes. On this basis, it was proposed that trichloroethylene did not represent a hepatocarcinogenic hazard to man. In the absence of such a hazard, no risk of liver tumours would be posed to man. Conclusions
In consideration of the wider use of in vitro cultures to predict hazard, several principles should be applied: (1) It is essential to define the particular feature occurring in vivo that is represented by the change in the in vitro system. In vitro assays based on a relevant mechanism of action have more credibility than those assays in which a precise mechanism is not defined. (2) Depending upon the feature being examined, consideration should be given to using cells derived from a relevant target organ. Hepatocytes or a liver enzyme system may be essential in order to detect toxins for liver, but a wide range of cell types may be acceptable for detection of irritant or corrosive substances. (3) Culture systems may be used to detect the potential of substances to induce selective effects in a particular organ. However, distinction should be made between selective effects and non-specific effects resulting from overt toxicity. As stated at the outset, the concepts of hazard and risk are central to toxicology studies: the logic is that hazard identification is followed by risk assessment. M a n y in vitro assays have been developed solely against the backdrop of laboratory animal data, without the necessary consideration of the relevance of the animal data to assessing risk for man.
Therefore, it is recommended that more consideration be given to the selection of an appropriate in vivo database when developing in vitro systems. M a x i m u m use should be made of h u m a n data when they are available. While in vitro systems cannot reproduce the complexities that determine risk for man, the use of h u m a n tissue in relevant experiments should be a better way of detecting hazard for man: where there is no hazard it follows that there is no risk.
REFERENCES
Burton A. B. G., York M. and Lawrence R. S. (1981) The in vitro assessment of severe eye irritants. Fd Cosmet. Toxicol. 19, 471-480. Control of Substances Hazardous to Health Regulations 1988. (1988) SJ. No. 1657. HMSO, London. Elcombe C. R. (1985) Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazard assessment. Archs Toxicol. Suppl. 8, 6-17. Elcombe C. R., Rose M. S. and Pratt I. S. (1985) Biochemical, histological, and ultrastructural changes in rat and mouse liver following the administration of trichloroethylene: possible relevance to species differences in hepatocarcinogenicity. Toxic. appl. Pharmac. 79, 363-376. Friend J. V., Crevel R. W. R., Williams T. C. and Parish W. E, (1989) Immaturity of the inflammatory response of the chick chorioallantoic membrane (CAM). In Vitro Toxic. In press. Gordon V. C. and Bergman H. C. (1987) EYTEX, An in vitro method for evaluation of ocular irritancy. In In Vitro Toxicology-Approaches to Validation. Alternative Methods in Toxicology, Vol. 5. pp. 293-302. Edited by A.
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