From the German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area of the Deutsche Forschungsgemeinschaft

Recommendations for the categorization of germ cell mutagens#

I.-D. Adler1, (* ), U. Andrae2, P. Kreis3, H.-G. Neumann4, R. Thier5 and D. Wild6


1, (* ) GSF - National Research Center for Environment and Health, Institute of Mammalian Genetics, D-85764 Neuherberg, Germany, FAX: +49-89-3187 2210, e-mail: adler@

2GSF - National Research Center for Environment and Health, Institute of Toxicology, D-85764 Neuherberg, Germany

3Institute of Toxicology and Environmental Hygiene, Technical University of Munich, D-80636 Munich, Germany

4Institute of Toxicology and Pharmacology, University of Würzburg, D-97078 Würzburg, Germany

5Institute for Occupational Physiology at the University of Dortmund, D-44139 Dortmund, Germany

6Federal Center of Meat Research, Institute of Microbiology and Toxicology, D-95326 Kulmbach, Germany

# These recommendations were published in German in Arbeitsmedizin, Sozialmedizin, Umweltmedizin (1999) 34: 400-403


Germ cell mutagens are currently classified into three categories in the German List of MAK and BAT Values (DFG 1999). These categories have been revised and extended in analogy to the new categories for carcinogenic chemicals. Germ cell mutagens produce heritable gene mutations, and heritable structural and numerical chromosome aberrations in germ cells. The original categories 1 and 2 for germ cell mutagens remained unchanged. Two new categories 3 A and 3 B are proposed for chemicals which are suspected to be germ cell mutagens. A new category 5 is proposed for germ cell mutagens with low potency which contribute negligibly to human genetic risk provided the MAK value is observed.

Key words: germ cell mutagens, List of MAK and BAT Values, genetic risk


I. Introduction

Germ cell mutagens produce heritable gene mutations, and heritable structural and numerical chromosome aberrations in germ cells. The consequences of germ cell mutations in subsequent generations include genetically determined phenotypic alterations without signs of illness, or reduction in fertility, or embryonic or perinatal death, more or less severe congenital malformations, or genetic diseases with various degrees of health impairment. The term ‘germ cell mutagenicity’ refers specifically to mutagenicity in male and female germ cells and is distinguished from mutagenicity in somatic cells which can initiate cancer.

The existence of biological processes resulting in the elimination of damaged germ cells, for example, during meiosis or as programmed cell death (apoptosis), has been demonstrated in animal studies. After fertilization has taken place, genetic damage is eliminated if it causes death of the embryo or foetus so that the child with an inherited disease is never born. The human genome is thus protected to some extent from the sequelae of germ cell mutations. During the long process of evolution of the species, the interaction of mutation and selection has resulted in the present species populations with their well-adapted, balanced gene pools. This also applies for man.

The balance of the gene pool can be disturbed in two ways. Firstly, it is assumed that exposure to mutagenic chemicals and ionizing radiation can increase the mutation frequency. Secondly, selection pressures have been reduced, for example, by the therapeutic capabilities of modern medicine and by the increased mobility of man. An increase in the incidence of the genetic component of diseases with multifactorial genesis is favoured especially by decreased selection pressure on the human population. As a consequence, such diseases with partially genetic origins are becoming more frequent. It is probable that mutagenic substances contribute to this increase although to date this could not be demonstrated in population studies. Even the demonstration that an increase in the frequency of monogenic diseases has been caused by exposure to ionizing radiation or mutagenic chemicals is very difficult.

II. Epidemiological Methods and their Limitations

A causal relationship between exposure of a population to chemicals or ionizing radiation and the occurrence of inherited diseases has not yet been demonstrated. One of the reasons for this is that exposure to a certain mutagen does not induce a specific and therefore conspicuous disorder like the non-genetic malformations seen after thalidomide or the forms of cancer associated with certain workplace exposures. Because of the nature of the genetic code, DNA damage and the resulting mutations are distributed more or less randomly in the genome and therefore can cause the one or other heritable disease at random.

Even among the progeny of the Japanese parents exposed to radiation in the towns of Hiroshima and Nagasaki, a significant increase in the incidence of genetic damage could not be demonstrated. These results appear to be in contradiction to the finding that an increase in the incidence of structural chromosome aberrations was found in the germ cells of men exposed voluntarily to radiation in the USA (Brewen and Preston 1975; Martin et al. 1986). In Japan, however, it was not inherited diseases in the narrow sense that were studied in the progeny but rather indicators with a genetic component, namely embryonal and neonatal mortality including congenital malformations, survival of the children to age 17, and alterations in the sex ratio (Neel et al. 1974; Schull et al. 1966). Only later were more specifically genetic end points added to the studied parameters, such as structural and numerical chromosomal anomalies (balanced translocations, inversions, trisomy 21 and aneuploidy of the sex chromosomes), mutations affecting protein charge and enzyme activity and certain childhood cancers resulting from germ cell mutations (Awa et al. 1987; Neel et al. 1982, 1988; Schull et al. 1981a, 1981b, 1982; Yoshimoto et al. 1991). But even that did not change the conclusion that a statistically significant increase in inherited diseases after exposure to radiation could not be demonstrated. The reason, however, does not lie in the lack of effectiveness of ionizing radiation but in the difficulty of detecting germ cell mutations with epidemiological methods. The especially critical factors are the determination of exposure levels, the size of the sample population and an adequate control population, as well as appropriate indicators of genetic damage (Sankaranarayanan 1988, 1993).

Of the multitude of genetic diseases, few are suitable for investigation in epidemiological studies, namely dominantly inherited diseases which are manifested during childhood. In addition, familial occurrence of an inherited disease must be differentiated from newly occurring mutations. Although a doubling of the mutation frequency is to be regarded as a marked effect, it cannot be expected that doubling of the incidence of such rare events would be recognized in population samples of a few hundred to a few thousand children. In Hungary, a programme for assessing the mutation frequencies for 25 exemplary dominantly inherited diseases (sentinel phenotypes) has been in operation for some years (Mulvihill and Czeizel 1983). This study was possible because Hungary has a registry of inherited diseases and malformations. On the basis of the Hungarian data it has been calculated that, for example, 47500 live-born children would have to be examined for the presence of the 25 inherited diseases in order to detect a doubling of the mutation rate with 95 % confidence (Czeizel and Kis-Varga 1987). This study can provide an appropriate basis for future epidemiological investigations.

It is still too early for an estimation of the genetic consequences of the reactor accident in Chernobyl, but it is to be expected that the application of appropriate genetic epidemiological methods will provide new insights.

In summary it is concluded that the probability of demonstrating mutagenic effects of a substance in human germ cells after workplace exposures is very small, even if the appropriate population studies are carried out optimally. The classification of substances as germ cell mutagens must therefore be based on experimental data from in vivo studies with appropriate animal models and relevant exposures.

III. Categories for Germ Cell Mutagens

The categories for germ cell mutagens have been established in analogy to the categories for carcinogenic chemicals at the workplace (Adler et al. 1999).

1. Germ cell mutagens which have been shown to increase the mutant frequency in the progeny of exposed  humans

2. Germ cell mutagens which have been shown to increase the mutant frequency in the progeny of exposed  mammals

3ASubstances which have been shown to induce genetic damage in germ cells of humans or animals, or  which are mutagenic in somatic cells and have been shown to reach the germ cells in their active forms

3BSubstances which are suspected of being germ cell mutagens because of their genotoxic effects in mammalian somatic cells in vivo or, in exceptional cases, in the absence of in vivo data if they are clearly   mutagenic in vitro and structurally related to in vivo mutagens

4. Not applicable *

5. Germ cell mutagens, the potency of which is considered to be so low that, provided the MAK value is observed, their contribution to genetic risk is expected not to be significant

* Category 4 carcinogenic substances are those with non-genotoxic mode of action. By definition, germ cell mutagens are genotoxic. Therefore, a Category 4 for germ cell mutagens cannot exist. Depending on future research results, a category 4 could be defined at a later time for genotoxic substances with targets other than DNA (i.e. pure aneugens).

IV. Classification of Germ Cell Mutagens

1. Germ cell mutagens which have been shown to increase the mutant frequency in the progeny of exposed humans

In Section II it was explained why epidemiological studies have to date not been able to prove that the exposure of a particular human population to a particular substance has resulted in an increase in the incidence of inherited mutations. This is true both for ionizing radiation and chemical mutagens. And even if epidemiological methods are improved further, it is unlikely that such proof will be available in the foreseeable future. Category 1 will therefore probably remain without any entries.


2. Germ cell mutagens which have been shown to increase the mutant frequency in the progeny of  exposed mammals

In this situation, the results of animal studies must be given particular attention. It is necessary to assume that human germ cells react to the mutagenic effects of chemicals in the same way as do those of animals. There are several reasons for this assumption and they have been discussed in detail (Favor 1989). The most important is the universality of the genetic code for the information carried in the DNA. The effects of changes in the genetic information in the germ cells are largely similar in experimental animals and man. In addition, the course of the biological and physiological processes in the various phases of germ cell development is the same for all mammals including man. Finally, the spectrum of mutants known for the mouse includes several which can serve as models with which the development and effects of human inherited diseases can be studied (Bedell et al. 1997). Many homologies in the genomes of mouse and man have been identified (Serikawa et al. 1998; Seldin 1997).

To be classified in Category 2 are substances which increase the incidence of genetically modified live progeny in animal studies, for example, in the specific locus test (Ehling et al. 1985) or in the test for heritable translocations (Adler 1984a). Likewise, in Category 2 substances should be classified which increase the incidence of embryos which die in utero, for example, in the dominant lethal test (Ehling 1977). The dominant lethal test detects progeny which are not viable because of chromosomal damage. The viability of foetuses with chromosomal damage is very different in experimental animals and man: human foetuses with unbalanced chromosome complements are more likely to survive until birth and beyond than are those of the mouse (Schinzel 1984). Therefore an increase in dominant lethal mutations in an animal study is considered relevant for the classification of a substance in Category 2.


3A. Substances which have been shown to induce genetic damage in germ cells of humans or animals,  or which are mutagenic in somatic cells and have been shown to reach the germ cells in their active forms

For many substances data appropriate for classification in Category 2 are not available but the substances have been tested with methods which detect genetic changes in germ cells. These methods include tests for genotoxicity in germ cells of experimental animals, such as tests for induction of structural chromosomal changes in spermatogonia or spermatocytes (Adler 1984b), for sister chromatid exchange in spermatogonia (Allen and Latt 1976), for micronuclei in round spermatids (Tates et al. 1983), for numerical chromosome changes in secondary spermatocytes (Pacchierotti et al. 1983) or in spermatozoa (Lowe et al. 1995), for DNA single strand breaks (Sega and Generoso 1988; Anderson et al. 1997) and for repair synthesis (Sega et al. 1976) or for covalent binding to the DNA (Sega and Owens 1978). Also relevant are the observations obtained from exposed human populations which provide evidence for structural or numerical chromosome changes in spermatozoa of exposed persons (Robbins et al. 1997). The development of new methods, especially molecular genetic methods for the detection of gene mutations in germ cells is to be expected. Substances which yield positive results in tests with germ cells are classified in Category 3 A.

Also taken into account are clearly positive results from in vivo tests for mutagenicity in somatic cells, for example, chromosomal aberrations or micronuclei in bone marrow cells (Adler et al. 1971; Schmid 1975), somatic mutations in the mammalian spot test (Fahrig 1977) or in transgenic animals (Gossen and Vijg 1993), provided that it has been demonstrated that the active substance or an active metabolite reaches the germ cells after relevant exposure of the experimental animals. Such substances are suspected of being mutagenic in germ cells too. Therefore they are classified in Category 3 A.


3B. Substances which are suspected of being germ cell mutagens because of their genotoxic effects in mammalian somatic cells in vivo or, in exceptional cases in the absence of in vivo data, if they are clearly mutagenic in vitro and structurally related to in vivo mutagens

If the available data are not sufficient for classification in Category 3 A but the substance is clearly genotoxic in somatic cells of exposed animals or man, it is suspected that the substance is also mutagenic in germ cells. Substances which have yielded positive results in one or several in vitro mutagenicity tests are generally not classified in Category 3 B. An exception is made for substances for which there are no relevant in vivo data but which are clearly genotoxic in vitro and also structurally related to substances known to be genotoxic in vivo. Such substances raise concern and are classified in Category 3 B.


5. Germ cell mutagens, the potency of which is considered to be so low that, provided the MAK value is  observed, their contribution to genetic risk is expected not to be significant

In analogy to the categorization of carcinogenic substances, also for germ cell mutagens a category has been formed which takes into account the potency of the mutagenic effect. For substances classified in Category 5 it is considered that no significant contribution to genetic risk is to be expected for man provided the MAK value is observed. For classification in this category, information on the spectrum of effects and their dose-dependence, and toxicokinetic data for species comparison are required. Biochemical and biological end points can be used to characterize the contribution to genetic risk.

The contribution to genetic risk is considered not to be significant after exposure at the workplace if the internal exposure level of the substance or its biomarker is in the range of the background levels in a not specifically exposed reference population.

n Under workplace conditions the levels of biochemical effect markers such as DNA and protein adducts are not significantly increased above the background levels.

n Physiological-toxicokinetic model calculations based on animal data do not reveal a significant genetic risk for man.


V References

Adler ID (1984a) Cytogenetic tests in mammals. in: Venitt S, Parry JM (eds) Mutagenicity testing. A practical approach. IRL Press, Oxford, 275–306

Adler ID (1984b) The heritable translocation test. BGA-Schriften 3: 291–298

Adler ID, Ramarao G, Epstein SS (1971) In vivo cytogenetic effects of trimethylphosphate and of tepa on bone marrow cells of male rats. Mutat Res 13: 263–273

Adler ID, Andrae U, Kreis P, Neumann HG, Thier R, Wild D (1999) Vorschläge zur Einstufung von                         Keimzellmutagenen. Arbeitsmed Sozialmed Umweltmed 34: 400--403

Allen JW, Latt SA (1976) Analysis of sister chromatid exchange formation in vivo in mouse spermatogonia as a new test system for environmental mutagens. Nature 260: 449–451

Anderson D, Dobrzy´nka MM, Jackson LI, Yu TW, Brinkworth MH (1997) Somatic and germ cell effects in rats and mice after treatment with 1,3-butadiene and its metabolites, 1,2-epoxybutene and 1,2,3,4-diepoxybutane. Mutat Res 391: 233–242

Awa AA, Honda T, Neriishi S, Sufuni T, Shimba H, Ohtaki K, Nakano M, Kodama Y, Itoh M, Hamilton HB (1987) Cytogenetic study of the offspring of atomic bomb survivors, Hiroshima and Nagasaki. in: Obe G, Basler A. (eds) Cytogenetics: Basis and applied aspects, Springer, Berlin Heidelberg, 166–183

Bedell MA, Largaespada DA, Jenkins NA, Copeland NG (1997) Mouse models of human disease. Part II: Recent progress and future directions. Genes and Development 11: 11–43

Brewen JG, Preston RJ (1975) Analysis of X-ray-induced chromosomal translocations in human and marmoset spermatogonial stem cells. Nature 253: 468–470

Czeizel A, Kis-Varga A (1987) Mutation surveillance of sentinel anomalies in Hungary, 1980–1984. Mutat Res 186: 73–79

Deutsche Forschungsgemeinschaft (DFG) (1999) List of MAK and BAT Values 1999, VCH Verlagsgesellschaft, Weinheim

Ehling UH (1977) Dominant lethal mutations in male mice. Arch Toxicol 38: 1–11

Ehling UH, Charles DJ, Favor J, Graw J, Kratochvilova J, Neuhäuser-Klaus A, Pretsch W (1985) Induction of gene mutations in mice: The multiple endpoint approach. Mutat Res 150: 393–401

Fahrig R (1977) The mammalian spot test (Fellfleckentest) with mice. Arch Toxicol 38: 87–98

Favor J (1989) Risk estimation based on germ-cell mutations in animals. Genome 31: 844–852

Gossen J, Vijg J (1993) Transgenic mice as model systems for studying gene mutations in vivo. Trends Genet 9: 27–31

Lowe X, Collins B, Allen J, Titenko-Holland N, Breneman J, van Beek M, Bishop J, Wyrobek AJ (1995) Aneuploidies and micronuclei in the germ cells of male mice of advanced age. Mutat Res 338: 59–76

Martin RH, Hildebrand K, Yamamoto J, Rademaker A, Barnes M, Douglas G, Arthur K, Ringrose T, Brown IS (1986) An increased frequency of human sperm chromosomal abnormalities after radiotherapy. Mutat Res 174: 219–225

Mulvihill JJ, Czeizel A (1983) Perspectives in mutation epidemiology, 6: A 1983 view of sentinel phenotypes. Mutat Res 123: 345–361

Neel JV, Kato H, Schull WJ (1974) Mortality in the children of atomic bomb survivors and controls. Genetics 76: 311–326

Neel JV, Schull WJ, Otake M (1982) Current status of genetic follow-up studies in Hiroshima and Nagasaki. in: Bora KC, Douglas GR, Nestmann ER (eds) Progress in Mutation Research, Volume 3, Elsevier Biomedical, Amsterdam, 39–51

Neel JV, Satoh C, Goriki K, Asakawa J, Fujita M, Takahashi N, Kageoka T, Hazama R (1988) Search for mutations altering protein charge and/or function in children of atomic bomb survivors: Final report. Am J Hum Genet 42: 663–676

Pacchierotti F, Bellincampi D, Civitareale D (1983) Cytogenetic observations, in mouse secondary spermatocytes, on numerical and structural chromosome aberrations induced by cyclophosphamide in various stages of spermatogenesis. Mutat Res 119: 177–183

Robbins WA, Meistrich ML, Moore D, Hagemeister FB, Weier HU, Cassel MU, Wilson G, Eskenazi B, Wyrobek AJ (1997) Chemotherapy induces transient sex chromosomal and autosomal aneuploidy in human sperm. Nature Genetics 16: 74–78

Sankaranarayanan K (1988) Invited review: Prevalence of genetic and partially genetic diseases in man and the estimation of genetic risks of exposure to ionizing radiation. Am J Hum Genet 42: 651–662

Sankaranarayanan K (1993) Ionizing radiation, genetic risk estimation and molecular biology: impact and inferences. Trends Genet 9: 79–84

Schinzel A (Ed.) (1984) Catalogue of unbalanced chromosome aberrations in man, Walter de Gruyter Berlin, New York 1984

Schmid W (1975) The micronucleus test. Mutat Res 31: 9–15

Schull WJ, Neel JV, Hashizume A (1966) Some further observations on the sex ratio among infants born to survivors of the atomic bombings of Hiroshima and Nagasaki. Am J Hum Genet 18: 328–338

Schull WJ, Otake M, Neel JV (1981a) Hiroshima and Nagasaki: a reassessment of the mutagenic effect of exposure to ionizing radiation. in: Hook EB, Porter IH (eds) Population and biological aspects of human mutation. Academic press, New York, London, Toronto, Sydney, San Francisco, 277–303

Schull WJ, Otage M, Neel JV (1981b) Genetic effects of the atomic bombs: a reappraisal. Science 213: 1220–1227

Schull WJ, Neel JV, Otake M, Awa A, Satoh C, Hamilton HB (1982) Hiroshima and Nagasaki: Three and a half decades of genetic screening. in: Sugimura T, Kondo S, Takebe H (eds) Environmental Mutagens and Carcinogens. Alan R. Liss, New York, 687–700

Sega GA, Generoso EE (1988) Measurement of DNA breakage in spermiogenic germ-cell stages of mice exposed to ethylene oxide, using an alkaline elution procedure. Mutat Res 197: 93–99

Sega GA, Owens JG (1978) Ethylation of DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of theses molecular targets to the induction of dominant lethals. Mutat Res 52: 87–106

Sega GA, Owens JG, Cumming RB (1976) Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl, ethyl, propyl, and isopropyl methanesulfonate. Mutat Res 36: 193–212

Seldin MF (1997) Genome surfing: using internet-based information tools toward functional genetic studies in mouse and humans. Methods 13: 445–457

Serikawa T, Cui Z, Yokoi N, Kuramoto T, Kondo Y, Kitada K, Guenet JL (1998) A comparative genetic map of rat, mouse and human genomes. Exp Anim 47: 1–9

Tates AD, Dietrich AJJ, de Vogel N, Neuteboom I, Bos A (1983) A micronucleus method for detection of meiotic micronuclei in male germ cells of mammals. Mutat Res 121: 131–138

Yoshimoto Y, Shull WL, Kato H, Neel JV (1991) Mortality among the offspring (F1) of atomic bomb survivors, 1946–1985. J Radiat Res 32: 327–351