Archive) of
Arch. Toxicol. 38, 135--140 (1977)
TOXICOLOGY 9 by Springer-Verlag 19 7 7
Mutagenieity Testing in Industry* D. Lorke Institute of Toxicology, Bayer AG, D-5600 Wuppertal, Federal Republic of Germany
Summary. The aims of authorities, university and industrial scientists are outlined. The prime feature remains: How important are the achieved results for estimating the mutagenic risk to humans? The various methods available for the testing of chemical substances for mutagenicity are compared. Their usefulness to estimate the mutagenic risk are considered and the disadvantages and advantages of such methods are discussed. The industrial toxicologist must apply those methods which permit the most accurate conclusions to be made. Further, the toxicologist has to develop new methods which, based on his own experience are more suitable for making more accurate statements. The results of methods carried out on the germ cell of living mammals permit more accurate statements to be made than investigations on isolated cell cultures and somatic cells. Thus, the dominant lethal test on male and female mice and the spermatogenial test on Chinese hamsters are considered suitable methods for estimating the possible dangers to man. Key words: Mutagenicity testing in industry - Relevance of test methods -Basis of risk estimation.
Zusammenfassung. Die Ziele von Beh/Srden, Universit~itswissenschaftlern und Wissenschaftlern in der Industrie werden herausgestellt. Oberster Gesichtspunkt bleibt: Wie bedeutsam sind die erzielten Ergebnisse zur Absch~itzung des mutagenen Risikos f/Jr den Menschen? Die allgemein bekannten verschiedenen Untersuchungsmethoden zur Testung yon chemischen Substanzen auf Mutagenit~it werden auf ihre Aussagef~ihigkeit zur Absch~itzung des Risikos/iberpr/ift und die Vorteile und Nachteile dieser Methoden erfrtert. * Presented at the 3rd Meeting of the Gesellschaft fiir Umwelt-Mutationsforschung e. V., D-8042 Neuherberg, July i - 2 , 1976
136
D. Lorke Der Toxikologe in der Industde hat diejenigen Methoden anzuwenden, die die grfBte Aussagekraft besitzen. Er hat dariiber hinaus auch neue Methoden zu entwickeln, wenn er aufgrund seiner Erfahrungen diese f/Jr aussagekr~iftiger h~ilt. Methoden an den Keimzellen lebender S/iugetiere besitzen eine gr613ere Aussagekraft als Untersuchungen an isolierten Zellkulturen und an somatischen Zellen. Daher ziihlen der Dominant-Letaltest an der m/innlichen und weiblichen Maus und der Spermatogonientest am Chinesischen Hamster zu den geeigneten Methoden, um eine evtl. Gefiihrdung ffir den Menschen abzusch/itzen.
From the title of this lecture one must assume that the determination of mutagenic effects in industry differs from other mutagenicity studies and how do they differ from those carded out in industry? The scientific basis for mutagenicity tests-has been developed by the universities and public research centers. The authorities have laid claim to this knowledge, used it to develop guidelines and the industry try to put both into practice and convincingly prove that a chemical correctly used, whether as a drug, a pesticide or another chemical substance, does not represent a mutagenic danger to the public. You perhaps may say, these are also the intentions of the authorities, where is the difference? In the event of a possible danger, the authorities must act immediately. It is their task to show the public that they are doing all they can to avoid any danger, or at least reduce the dangers to a minimum. As a result, the authorities are obliged to act prematurely. This, however, usually without a great deal of information being available and automatically results in an unsatisfactory situation arising. This knowledge has caused several countries to categorically refuse to lay down recommendations, guidelines or requirements for toxicological investigations. After the thalidomide tragedy different authorities immediately issued directives on the testing of the teratogenic effect of substances. Fortunately, no directives have been implemented even after it became apparent that substances had to be tested to avoid the risk of a mutagenic effect. With the exception of Japan (1974), I know of no other country which had issued exact requirements for testing the mutagenicity of chemical substances. Most authorities, quite rightly, ask what facts can one produce to prove that a mutagenic risk appears impossible or even can be excluded. Let us now turn our attention to the universities. The university scientists have a specific aim in mind. They occupy themselves with one aspect of the problem and are often only interested in researching that scientific sector. Basic scientific problems are studied without giving primary attention to the practical aspects of the problem. As a result, different scientists may have opposing views. Toxicologists employed in industry must think differently. They are faced with the task of determining, according to latest scientific aspects, whether a chemical substance used as directed creates any mutagenic hazards. They cannot spend years researching to develop a method which might or might not be adequate. Their maxim must be: The investigations on substances X, Y, and Z for mutagenic effects must start now.
Mutagenicity Testing in Industry
137
To overcome this problem industrial toxicologists will test all known methods and the relevance they may have to accurately estimate the risk to man. It is understandable that opinions may differ with regard to which method is best suited to estimate the risks open to man. Be it as it may, each industrial toxicologist will start with known methods which have been used manifold to study substances and thus gain experience. At scientific meetings such as this one in Munich different points of view are discussed and ideas are exchanged. Thus forming a broad mutual consensus which, in value, may vary slightly even among industrial toxicologists. Today, when I speak about mutagenicity testing in industry I would like to emphasize that to a great extent I am voicing my own opinions. As it is practically impossible to give a completely objective picture, would you please forgive me when I also express my personal opinions. I should like to classify the known methods used to establish a mutagenic effect as follows: I. methods using isolated cell cultures, II. methods using somatic cells of mammals, III. methods of determining mutagenic damage to germ cells of mammals. Let us, first of all, turn to the methods on isolated cell cultures. Their advantage is that the substance to be tested is directly applied to the mitotic cell and point mutations may be determined. The disadvantages are the absence of important host functions such as absorption, metabolism, distribution and excretion. These disadvantages are generally accepted and as a result all changes to these methods are directed at overcoming them. I should like to mention a few: 1. Best known is the host-mediated assay (Garbridge and Legator, 1969). 2. The addition of homogenised mammal tissue (liver, kidney) to the isolated cell cultures (Ames et al., 1973, 1975). 3. The intraveneous administration of cell cultures and their investigation after removal from the liver or other organs (Mohn and Ellenberger, 1973). The isolated cell cultures have always been the main target of research and not the important reproductive genital organs of the mammals. Would I not be more logical and justifiable to examine the germ cells themselves? With the methods using somatic cells it is assumed that mutagens cause impairment to those cell systems which are continually dividing. When a mutagenic effect is detectable then the germ cells are also mutagenically damaged. Since the study is carried out on living mammals, absorption, metabolism, distribution, and excretion will already have been taken into consideration. Why does one then study somatic systems such as bone marrow and lymphocytes instead of studying the germ cell directly? The answer to this question brings us directly to the methods of Group III. When it is apparent that such a test is required to estimate the risk of mutagenicity, then it should be carried out. With this mind we developed the spermatogonial test on Chinese hamsters (Machemer and Lorke, 1975). We were able to demonstrate that the sensivity of the spermatogonial test is sufficient to detect mutagenic effects. The second, very well known test in this group is the dominant-lethal test on
138
D. Lorke
male or female mice and rats (R6hrborn, 1970). It is very often said that this test is not sensitive enough i.e. only strong mutagens show an effect; only the specific effect of the death of the germ cells is tested and a 100% effect cannot be achieved unless special investigations are carried out, i.e. the method which was developed by Kratochvil (1975) to differentiate between genetic and unspecific toxic preimplantative lethal causes. Here, I should like to ask a heretical question: Is that not nature's way of protecting the generative system? Have we not here established the fact that nature alone can survive even under the permanent influence of mutagenic hazards, e.g. cosmic rays? The dominant-lethal test is not too insensitive, but rather the genetic system itself is insensitive. If it were not so insensitive perhaps there would be no mammals on this planet. As I said at the beginning, the most important criterion of an animal experimental method is, how can it be used to estimate the risk to humans. Here, I would like to mention two epidemiological studies which have been carried out in USA. The first was presented by the American Society of Anesthesiologists (1974). Infante et al. (1976) reported the second one. These studies were carried out on families of anaesthetists and on vinyl chloride workers with the result that in families where only the husbands were exposed but the wives had no contact with hospitals or were not exposed to vinyl chloride the number of stillbirths and/or the number of congenital malformations were significantly higher.
The Consequence? 1. The significantly higher number of stillbirths complies exactly with that of the dominant-lethal test. 2. The simultaneously observed significant rise, the increase of stillbirths and the number of malformations shows that the sensitivity of both parameters does not differ greatly. In other words, the dominant-lethal test on mammals is an adequate method of estimating the mutagenic risks to humans. From my lecture I should like to draw the following conclusion: The two methods which I prefer for estimating the risks to humans have the disadvantage that they do not include point mutations. From these endeavours and efforts to determine point mutations on living mammals, the specific locus test was developed (Russel, 1951). In this test, like all other test systems, the mutation rate of the untreated animal is compared with that of the treated animal. Therefore, sufficient offspring must be present to be able to prove an assumed effect such as doubling the spontaneous mutation rate. Now, spontaneous mutation is a rare occurrence, and as a result many individuals must be present. Let me do a rough calculation for you. The spontaneous mutation rate is given as 10-5-10 -8 per gen (Kaplan, 1967). Let us take a value between which is 10-7, this means that for every 10 million offsprings, a certain point mutation can occur spontaneously. If after treatment, due to one mutagen, the spontaneous rate doubled then we should be able to statistically differentiate between 1 : 10million and
Mutagenicity Testing in Industry
139
2 : 10 million. I am sure, it is quite apparent that this method is enormously costly and would only show an effect with very strong mutagens. Also the increasing of the observed specific loci would not make this method suitable for the day-to-day testing of the mutagenicity of a substance. The great advantage of the test on isolated cultures is the low costs and time required to carry out the test. Thus it is~ often said that it is possible to test many substances early enough and at low costs so that very early in the developmental stage dangerous substances can be discarded. Those considerations are based on the following: 1. More and more effective substances are available. 2. The mutagenic effects of such drugs vary markedly. Both are very rare if not practically never the case. Of 10,000 new substances only about 10 show an interesting effect. In other words, it is relatively seldom that one finds a new substance which is highly effective or has new potential characteristics. When, however, such a group has been found, then it is very frequently observed that the toxic effects of the individual derivatives of the group do not vary greatly. Greater difficulties arise, however, when one is concerned e.g. with antimycotics or antibiotics, because the desired effect is the damage of microorganisms. The application of microorganisms for mutagenicity testing, therefore, is in these cases very questionable. Since the frequency of new substances which have new and interesting effects are a rare find, no company would discard such substances when a test system shows a hazard probability of 80%. Further, at such an early stage nothing final can be said about the dose to be given (therapeutic dose) or about the actual advantages of such a new substance. To throw out a new, effective substance there must be a marked disproportion between toxicity and efficacy. No one will let a chance go until it has been established beyond doubt that the expected damage will prevent the further development of the substance. The industrial toxicologist can not present "ifs" and "whens" as arguments, but rather he must present conclusive proof in the form of manifold concurring test results on the dangers of using a new substance. And here in vitro tests are inadequate, as the answer to the question whether or not to develop the valuable drug must be decided with a straight-forward yes or no. In special cases tests on isolated cell cultures and on somatic systems may be of great help. To dispense with them for basic research in mutagenesis would be unthinkable. However, the best suited methods for estimating a mutagenic or genetic risk to humans are the dominant-lethal test and cytogenetic investigations on the generative cells of mammals.
References American Society of Anesthesiologists: Occupational disease among operating room personal. A national study. Anesthesiology 41, 321-340 (1974) Ames, B. N., Durston, W. E., Yamasaki, E., Lee, F. D.: Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. nat. Acad. Sci. (Wash.) 70, 2281-2285 (1973)
140
D. Lorke
Ames, B. N., McCann, J., Yamasaki, E.: Methods for detecting carcinogens and mutagens with the SalmoneUa/mammalian-microsome mutagenicity test. Mutation Res. 31, 347-364 (1975) Garbridge, M. G., Legator, M. S.: A host-mediated microbial assay for the detection of mutagenic compounds. Proc. Soc. exp. Biol. (N.Y.) 130, 831--834 (1969) Infante, P. F., Wagoner, J. K., Waxweiler, R. J.: Carcinogenic, mutagenie and teratogenic risks associated with vinyl chloride. Mutation Res. 41, 131--142 (1976) Japan: Tentative standard for investigation of genetic safety of food additives and others. Ministry of Health and Welfare, August 1974 Kaplan, R. W.: Molekulare Meehanismen der Mutationsprozesse. In: Molekularbiologie (Th. Wieland, G. Pfleiderer, Eds.), pp. 69--81. Frankfurt: Umschau 1967 Kratochvil, J.: Pr~iimplantativer Verlust dominanter Letalmutationen naeh Behandlung der m~innlichen M~iuse mit Methylmethansulfonat. GSF-Bericht B 565, 5-17 (1975) Machemer, L., Lorke, D.: Method for testing mutagenic effects of chemicals on spermatogonia of the Chinese hamster. Results obtained with cyclophosphamide, saccharin, and cyclamate. ArzneimittelForsch. (Drug. Res.) 25, 1989--1996 (1975) Mohn, G., EUenberger, J.: Mammalian blood-mediated mutagenicity tests using a multipurpose strain of Escherichia coli K-12. Mutation Res. 19, 257--260 (1973) R6hrborn, G.: The dominant lethals: Method and cytogenitic examination of early cleavage stages. In: Chemical mutagenesis in mammals and man (F. Vogel, G. R6hrborn, Eds.), pp. 148-161. BerlinHeidelberg-New York: Springer 1970 Russel, W. L.: X-ray-induced mutations in mice. Cold Spr. Harb. Symp. quant. Biol. 16, 327-336 (1951)
Received December 21, 1976
Arch. Toxicol. 38, 135--140 (1977)
TOXICOLOGY 9 by Springer-Verlag 19 7 7
Mutagenieity Testing in Industry* D. Lorke Institute of Toxicology, Bayer AG, D-5600 Wuppertal, Federal Republic of Germany
Summary. The aims of authorities, university and industrial scientists are outlined. The prime feature remains: How important are the achieved results for estimating the mutagenic risk to humans? The various methods available for the testing of chemical substances for mutagenicity are compared. Their usefulness to estimate the mutagenic risk are considered and the disadvantages and advantages of such methods are discussed. The industrial toxicologist must apply those methods which permit the most accurate conclusions to be made. Further, the toxicologist has to develop new methods which, based on his own experience are more suitable for making more accurate statements. The results of methods carried out on the germ cell of living mammals permit more accurate statements to be made than investigations on isolated cell cultures and somatic cells. Thus, the dominant lethal test on male and female mice and the spermatogenial test on Chinese hamsters are considered suitable methods for estimating the possible dangers to man. Key words: Mutagenicity testing in industry - Relevance of test methods -Basis of risk estimation.
Zusammenfassung. Die Ziele von Beh/Srden, Universit~itswissenschaftlern und Wissenschaftlern in der Industrie werden herausgestellt. Oberster Gesichtspunkt bleibt: Wie bedeutsam sind die erzielten Ergebnisse zur Absch~itzung des mutagenen Risikos f/Jr den Menschen? Die allgemein bekannten verschiedenen Untersuchungsmethoden zur Testung yon chemischen Substanzen auf Mutagenit~it werden auf ihre Aussagef~ihigkeit zur Absch~itzung des Risikos/iberpr/ift und die Vorteile und Nachteile dieser Methoden erfrtert. * Presented at the 3rd Meeting of the Gesellschaft fiir Umwelt-Mutationsforschung e. V., D-8042 Neuherberg, July i - 2 , 1976
136
D. Lorke Der Toxikologe in der Industde hat diejenigen Methoden anzuwenden, die die grfBte Aussagekraft besitzen. Er hat dariiber hinaus auch neue Methoden zu entwickeln, wenn er aufgrund seiner Erfahrungen diese f/Jr aussagekr~iftiger h~ilt. Methoden an den Keimzellen lebender S/iugetiere besitzen eine gr613ere Aussagekraft als Untersuchungen an isolierten Zellkulturen und an somatischen Zellen. Daher ziihlen der Dominant-Letaltest an der m/innlichen und weiblichen Maus und der Spermatogonientest am Chinesischen Hamster zu den geeigneten Methoden, um eine evtl. Gefiihrdung ffir den Menschen abzusch/itzen.
From the title of this lecture one must assume that the determination of mutagenic effects in industry differs from other mutagenicity studies and how do they differ from those carded out in industry? The scientific basis for mutagenicity tests-has been developed by the universities and public research centers. The authorities have laid claim to this knowledge, used it to develop guidelines and the industry try to put both into practice and convincingly prove that a chemical correctly used, whether as a drug, a pesticide or another chemical substance, does not represent a mutagenic danger to the public. You perhaps may say, these are also the intentions of the authorities, where is the difference? In the event of a possible danger, the authorities must act immediately. It is their task to show the public that they are doing all they can to avoid any danger, or at least reduce the dangers to a minimum. As a result, the authorities are obliged to act prematurely. This, however, usually without a great deal of information being available and automatically results in an unsatisfactory situation arising. This knowledge has caused several countries to categorically refuse to lay down recommendations, guidelines or requirements for toxicological investigations. After the thalidomide tragedy different authorities immediately issued directives on the testing of the teratogenic effect of substances. Fortunately, no directives have been implemented even after it became apparent that substances had to be tested to avoid the risk of a mutagenic effect. With the exception of Japan (1974), I know of no other country which had issued exact requirements for testing the mutagenicity of chemical substances. Most authorities, quite rightly, ask what facts can one produce to prove that a mutagenic risk appears impossible or even can be excluded. Let us now turn our attention to the universities. The university scientists have a specific aim in mind. They occupy themselves with one aspect of the problem and are often only interested in researching that scientific sector. Basic scientific problems are studied without giving primary attention to the practical aspects of the problem. As a result, different scientists may have opposing views. Toxicologists employed in industry must think differently. They are faced with the task of determining, according to latest scientific aspects, whether a chemical substance used as directed creates any mutagenic hazards. They cannot spend years researching to develop a method which might or might not be adequate. Their maxim must be: The investigations on substances X, Y, and Z for mutagenic effects must start now.
Mutagenicity Testing in Industry
137
To overcome this problem industrial toxicologists will test all known methods and the relevance they may have to accurately estimate the risk to man. It is understandable that opinions may differ with regard to which method is best suited to estimate the risks open to man. Be it as it may, each industrial toxicologist will start with known methods which have been used manifold to study substances and thus gain experience. At scientific meetings such as this one in Munich different points of view are discussed and ideas are exchanged. Thus forming a broad mutual consensus which, in value, may vary slightly even among industrial toxicologists. Today, when I speak about mutagenicity testing in industry I would like to emphasize that to a great extent I am voicing my own opinions. As it is practically impossible to give a completely objective picture, would you please forgive me when I also express my personal opinions. I should like to classify the known methods used to establish a mutagenic effect as follows: I. methods using isolated cell cultures, II. methods using somatic cells of mammals, III. methods of determining mutagenic damage to germ cells of mammals. Let us, first of all, turn to the methods on isolated cell cultures. Their advantage is that the substance to be tested is directly applied to the mitotic cell and point mutations may be determined. The disadvantages are the absence of important host functions such as absorption, metabolism, distribution and excretion. These disadvantages are generally accepted and as a result all changes to these methods are directed at overcoming them. I should like to mention a few: 1. Best known is the host-mediated assay (Garbridge and Legator, 1969). 2. The addition of homogenised mammal tissue (liver, kidney) to the isolated cell cultures (Ames et al., 1973, 1975). 3. The intraveneous administration of cell cultures and their investigation after removal from the liver or other organs (Mohn and Ellenberger, 1973). The isolated cell cultures have always been the main target of research and not the important reproductive genital organs of the mammals. Would I not be more logical and justifiable to examine the germ cells themselves? With the methods using somatic cells it is assumed that mutagens cause impairment to those cell systems which are continually dividing. When a mutagenic effect is detectable then the germ cells are also mutagenically damaged. Since the study is carried out on living mammals, absorption, metabolism, distribution, and excretion will already have been taken into consideration. Why does one then study somatic systems such as bone marrow and lymphocytes instead of studying the germ cell directly? The answer to this question brings us directly to the methods of Group III. When it is apparent that such a test is required to estimate the risk of mutagenicity, then it should be carried out. With this mind we developed the spermatogonial test on Chinese hamsters (Machemer and Lorke, 1975). We were able to demonstrate that the sensivity of the spermatogonial test is sufficient to detect mutagenic effects. The second, very well known test in this group is the dominant-lethal test on
138
D. Lorke
male or female mice and rats (R6hrborn, 1970). It is very often said that this test is not sensitive enough i.e. only strong mutagens show an effect; only the specific effect of the death of the germ cells is tested and a 100% effect cannot be achieved unless special investigations are carried out, i.e. the method which was developed by Kratochvil (1975) to differentiate between genetic and unspecific toxic preimplantative lethal causes. Here, I should like to ask a heretical question: Is that not nature's way of protecting the generative system? Have we not here established the fact that nature alone can survive even under the permanent influence of mutagenic hazards, e.g. cosmic rays? The dominant-lethal test is not too insensitive, but rather the genetic system itself is insensitive. If it were not so insensitive perhaps there would be no mammals on this planet. As I said at the beginning, the most important criterion of an animal experimental method is, how can it be used to estimate the risk to humans. Here, I would like to mention two epidemiological studies which have been carried out in USA. The first was presented by the American Society of Anesthesiologists (1974). Infante et al. (1976) reported the second one. These studies were carried out on families of anaesthetists and on vinyl chloride workers with the result that in families where only the husbands were exposed but the wives had no contact with hospitals or were not exposed to vinyl chloride the number of stillbirths and/or the number of congenital malformations were significantly higher.
The Consequence? 1. The significantly higher number of stillbirths complies exactly with that of the dominant-lethal test. 2. The simultaneously observed significant rise, the increase of stillbirths and the number of malformations shows that the sensitivity of both parameters does not differ greatly. In other words, the dominant-lethal test on mammals is an adequate method of estimating the mutagenic risks to humans. From my lecture I should like to draw the following conclusion: The two methods which I prefer for estimating the risks to humans have the disadvantage that they do not include point mutations. From these endeavours and efforts to determine point mutations on living mammals, the specific locus test was developed (Russel, 1951). In this test, like all other test systems, the mutation rate of the untreated animal is compared with that of the treated animal. Therefore, sufficient offspring must be present to be able to prove an assumed effect such as doubling the spontaneous mutation rate. Now, spontaneous mutation is a rare occurrence, and as a result many individuals must be present. Let me do a rough calculation for you. The spontaneous mutation rate is given as 10-5-10 -8 per gen (Kaplan, 1967). Let us take a value between which is 10-7, this means that for every 10 million offsprings, a certain point mutation can occur spontaneously. If after treatment, due to one mutagen, the spontaneous rate doubled then we should be able to statistically differentiate between 1 : 10million and
Mutagenicity Testing in Industry
139
2 : 10 million. I am sure, it is quite apparent that this method is enormously costly and would only show an effect with very strong mutagens. Also the increasing of the observed specific loci would not make this method suitable for the day-to-day testing of the mutagenicity of a substance. The great advantage of the test on isolated cultures is the low costs and time required to carry out the test. Thus it is~ often said that it is possible to test many substances early enough and at low costs so that very early in the developmental stage dangerous substances can be discarded. Those considerations are based on the following: 1. More and more effective substances are available. 2. The mutagenic effects of such drugs vary markedly. Both are very rare if not practically never the case. Of 10,000 new substances only about 10 show an interesting effect. In other words, it is relatively seldom that one finds a new substance which is highly effective or has new potential characteristics. When, however, such a group has been found, then it is very frequently observed that the toxic effects of the individual derivatives of the group do not vary greatly. Greater difficulties arise, however, when one is concerned e.g. with antimycotics or antibiotics, because the desired effect is the damage of microorganisms. The application of microorganisms for mutagenicity testing, therefore, is in these cases very questionable. Since the frequency of new substances which have new and interesting effects are a rare find, no company would discard such substances when a test system shows a hazard probability of 80%. Further, at such an early stage nothing final can be said about the dose to be given (therapeutic dose) or about the actual advantages of such a new substance. To throw out a new, effective substance there must be a marked disproportion between toxicity and efficacy. No one will let a chance go until it has been established beyond doubt that the expected damage will prevent the further development of the substance. The industrial toxicologist can not present "ifs" and "whens" as arguments, but rather he must present conclusive proof in the form of manifold concurring test results on the dangers of using a new substance. And here in vitro tests are inadequate, as the answer to the question whether or not to develop the valuable drug must be decided with a straight-forward yes or no. In special cases tests on isolated cell cultures and on somatic systems may be of great help. To dispense with them for basic research in mutagenesis would be unthinkable. However, the best suited methods for estimating a mutagenic or genetic risk to humans are the dominant-lethal test and cytogenetic investigations on the generative cells of mammals.
References American Society of Anesthesiologists: Occupational disease among operating room personal. A national study. Anesthesiology 41, 321-340 (1974) Ames, B. N., Durston, W. E., Yamasaki, E., Lee, F. D.: Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. nat. Acad. Sci. (Wash.) 70, 2281-2285 (1973)
140
D. Lorke
Ames, B. N., McCann, J., Yamasaki, E.: Methods for detecting carcinogens and mutagens with the SalmoneUa/mammalian-microsome mutagenicity test. Mutation Res. 31, 347-364 (1975) Garbridge, M. G., Legator, M. S.: A host-mediated microbial assay for the detection of mutagenic compounds. Proc. Soc. exp. Biol. (N.Y.) 130, 831--834 (1969) Infante, P. F., Wagoner, J. K., Waxweiler, R. J.: Carcinogenic, mutagenie and teratogenic risks associated with vinyl chloride. Mutation Res. 41, 131--142 (1976) Japan: Tentative standard for investigation of genetic safety of food additives and others. Ministry of Health and Welfare, August 1974 Kaplan, R. W.: Molekulare Meehanismen der Mutationsprozesse. In: Molekularbiologie (Th. Wieland, G. Pfleiderer, Eds.), pp. 69--81. Frankfurt: Umschau 1967 Kratochvil, J.: Pr~iimplantativer Verlust dominanter Letalmutationen naeh Behandlung der m~innlichen M~iuse mit Methylmethansulfonat. GSF-Bericht B 565, 5-17 (1975) Machemer, L., Lorke, D.: Method for testing mutagenic effects of chemicals on spermatogonia of the Chinese hamster. Results obtained with cyclophosphamide, saccharin, and cyclamate. ArzneimittelForsch. (Drug. Res.) 25, 1989--1996 (1975) Mohn, G., EUenberger, J.: Mammalian blood-mediated mutagenicity tests using a multipurpose strain of Escherichia coli K-12. Mutation Res. 19, 257--260 (1973) R6hrborn, G.: The dominant lethals: Method and cytogenitic examination of early cleavage stages. In: Chemical mutagenesis in mammals and man (F. Vogel, G. R6hrborn, Eds.), pp. 148-161. BerlinHeidelberg-New York: Springer 1970 Russel, W. L.: X-ray-induced mutations in mice. Cold Spr. Harb. Symp. quant. Biol. 16, 327-336 (1951)
Received December 21, 1976
