Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Derivation of point of departure (PoD) estimates in genetic toxicology studies and their potential applications in risk assessment.

Genetic toxicology data have traditionally been employed for qualitative, rather than quantitative evaluations of hazard. As a continuation of our earlier report that analyzed ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) dose-response data (Gollapudi et al., 2013), here we present analyses of 1-ethyl-1-nitrosourea (ENU) and 1-methyl-1-nitrosourea (MNU) dose-response data and additional approaches for the determination of genetic toxicity point-of-departure (PoD) metrics. We previously described methods to determine the no-observed-genotoxic-effect-level (NOGEL), the breakpoint-dose (BPD; previously named Td), and the benchmark dose (BMD10 ) for genetic toxicity endpoints. In this study we employed those methods, along with a new approach, to determine the non-linear slope-transition-dose (STD), and alternative methods to determine the BPD and BMD, for the analyses of nine ENU and 22 MNU datasets across a range of in vitro and in vivo endpoints. The NOGEL, BMDL10 and BMDL1SD PoD metrics could be readily calculated for most gene mutation and chromosomal damage studies; however, BPDs and STDs could not always be derived due to data limitations and constraints of the underlying statistical methods. The BMDL10 values were often lower than the other PoDs, and the distribution of BMDL10 values produced the lowest median PoD. Our observations indicate that, among the methods investigated in this study, the BMD approach is the preferred PoD for quantitatively describing genetic toxicology data. Once genetic toxicology PoDs are calculated via this approach, they can be used to derive reference doses and margin of exposure values that may be useful for evaluating human risk and regulatory decision making.

Quantitative approaches for assessing dose-response relationships in genetic toxicology studies.

Genetic toxicology studies are required for the safety assessment of chemicals. Data from these studies have historically been interpreted in a qualitative, dichotomous "yes" or "no" manner without analysis of dose-response relationships. This article is based upon the work of an international multi-sector group that examined how quantitative dose-response relationships for in vitro and in vivo genetic toxicology data might be used to improve human risk assessment. The group examined three quantitative approaches for analyzing dose-response curves and deriving point-of-departure (POD) metrics (i.e., the no-observed-genotoxic-effect-level (NOGEL), the threshold effect level (Td), and the benchmark dose (BMD)), using data for the induction of micronuclei and gene mutations by methyl methanesulfonate or ethyl methanesulfonate in vitro and in vivo. These results suggest that the POD descriptors obtained using the different approaches are within the same order of magnitude, with more variability observed for the in vivo assays. The different approaches were found to be complementary as each has advantages and limitations. The results further indicate that the lower confidence limit of a benchmark response rate of 10% (BMDL(10) ) could be considered a satisfactory POD when analyzing genotoxicity data using the BMD approach. The models described permit the identification of POD values that could be combined with mode of action analysis to determine whether exposure(s) below a particular level constitutes a significant human risk. Subsequent analyses will expand the number of substances and endpoints investigated, and continue to evaluate the utility of quantitative approaches for analysis of genetic toxicity dose-response data.

Approaches to the risk assessment of genotoxic carcinogens in food: a critical appraisal.

The present paper examines the particular difficulties presented by low levels of food-borne DNA-reactive genotoxic carcinogens, some of which may be difficult to eliminate completely from the diet, and proposes a structured approach for the evaluation of such compounds. While the ALARA approach is widely applicable to all substances in food that are both carcinogenic and genotoxic, it does not take carcinogenic potency into account and, therefore, does not permit prioritisation based on potential risk or concern. In the absence of carcinogenicity dose-response data, an assessment based on comparison with an appropriate threshold of toxicological concern may be possible. When carcinogenicity data from animal bioassays are available, a useful analysis is achieved by the calculation of margins of exposure (MOEs), which can be used to compare animal potency data with human exposure scenarios. Two reference points on the dose-response relationship that can be used for MOE calculation were examined; the T25 value, which is derived from linear extrapolation, and the BMDL10, which is derived from mathematical modelling of the dose-response data. The above approaches were applied to selected food-borne genotoxic carcinogens. The proposed approach is applicable to all substances in food that are DNA-reactive genotoxic carcinogens and enables the formulation of appropriate semi-quantitative advice to risk managers.

DNA repair-deficient Xpa and Xpa/p53+/- knock-out mice: nature of the models.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in which repair of ultraviolet (UV)-induced DNA damage is impaired or is totally absent due to mutations in genes controlling the DNA repair pathway known as nucleotide excision repair (NER). XP is characterized, in part, by extreme sensitivity of the skin to sunlight, and XP patients have a more than 1000-fold increased risk of developing cancer at sun-exposed areas of the skin. To study the role of NER in chemical-induced tumorigenesis in more detail, the authors developed Xpa-/- homozygous knockout mice with a complete defect in NER (designated as Xpa mice or XPA model). Xpa mice develop skin tumors at high frequency when exposed to UV light, and as such, they mimic the phenotype of human XP. Moreover, the Xpa mice also appear to be susceptible to genotoxic carcinogens given orally. Based on these phenotypic characteristics, the Xpa mice were considered to be an attractive candidate mouse model for use in identifying human carcinogens. In an attempt to further increase both the sensitivity and specificity of the XPA model in carcinogenicity testing, the authors crossed Xpa mice with mice having a heterozygous defect in the tumor suppressor gene p53. Xpa/p53+/- double knockout mice develop tumors earlier and with higher incidences upon exposure to carcinogens as compared to their single knockout counterparts. Here the authors describe the development and features of the Xpa mouse and present some examples of the Xpa and Xpa/p53+/- mouse models' sensitivity towards genotoxic carcinogens. It appeared that the Xpa/p53+/- double knockout mouse model is favorable over both the Xpa and p53+/- single knockout models in short-term carcinogenicity testing. In addition to the fact that the double knockout mice respond more robustly to carcinogens, they also appear to respond in a very discriminative way. All compounds identified thus far are true (human) carcinogens, and, therefore, the authors believe that the Xpa/p53+/- mouse model is an excellent candidate for a future replacement of the chronic mouse bioassay, at least for certain classes of chemicals.

In vivo cytokinesis blocked micronucleus assay with carbendazim in rat fibroblasts and comparison with in vitro assays.

A successful in vivo application of the cytokinesis blocked micronucleus assay for the detection of aneuploidy induced by carbendazim (CARB) was carried out in the granuloma pouch assay. This was performed in two ways: (i) in vivo exposure of the skin fibroblasts to cytochalasin B (cytB) and CARB, by simultaneous injection of both substances into the pouch; (ii) in vivo exposure to CARB followed by in vitro culturing of the fibroblasts in the presence of cytB. Only the first assay was successful. Injection of cytB (with or without the test compound) into the pouch resulted in the induction of binucleate cells in vivo, up to a maximum of 5% at 1 mg cytB/pouch. After injection of CARB (0-50 or 0-10 mg/pouch) and cytB (1 mg) into the pouch, aneuploidy was determined in the isolated binucleate fibroblasts by fluorescence in situ hybridization with a general centromeric probe and combinations of chromosome-specific probes (19p + 19q, 4q + Yq). With all probes, the induction of chromosome loss and/or non-disjunction by CARB was very pronounced; at 10 mg CARB/pouch the total malsegregation frequency of chromosomes 4, 19 and Y was approximately 300/1000 binucleate cells. In an in vitro cytokinesis block assay with CARB (0-2.5 microg/ml) in primary skin fibroblasts the induced aneuploidy frequencies were as high as observed in the in vivo assay. The use of two probes for chromosome 19, which enabled the scoring of chromosome breaks in addition to aneuploidy, revealed no significant induction of chromosome breaks by CARB. The frequency of polyploid mononucleate and binucleate cells was decreased after CARB treatment, in both the in vivo and in vitro assays. However, in an additional in vitro assay without cytB a major induction of polyploidy from 2.5 microg/ml CARB and above was observed, showing that cytB may interfere with polyploidy induction.

Increased frequencies of diploid sperm detected by multicolour FISH after treatment of rats with carbendazim without micronucleus induction in peripheral blood erythrocytes.

The purpose of the present study was to determine the effect of a single oral dose of carbendazim (CARB) on the frequencies of numerical chromosome aberrations in sperm and on micronuclei in peripheral blood erythrocytes of rats. Dual colour FISH on epididymal sperm of rats treated 31 days before sacrifice (0, 50, 150, 450 and 800 mg/kg body wt CARB in corn oil), corresponding to exposure during late pachytene, revealed a clear induction of diploid sperm. Induction of aneuploid sperm was not observed. Although the absolute frequencies of diploidy were low, ranging from 0.03% in the control group to 0.22% in the highest dose group, the observed dose-response relationship was highly significant. In sperm of rats killed 50 days after treatment with CARB (corresponding to exposure of spermatogonial stem cells) the effect was no longer apparent. In a second experiment, in addition to more dose groups in the low dose range, the peripheral blood micronucleus assay was incorporated. Results of triple colour FISH on epididymal sperm of rats treated with CARB (0-800 mg/kg body wt) again showed induction of diploid, but not of aneuploid sperm. Induction was less prominent than in the first experiment, but the dose-response relationship for diploidy was again significant. In blood samples drawn from the tail vein 48 h after treatment with CARB induction of micronuclei in peripheral blood erythrocytes was not observed, whereas the micronucleus frequency was significantly increased after a single i. p. dose of mitomycin C (3 mg/kg body wt). In conclusion, the present results show that CARB induces diploidy in sperm, without an accompanying induction of micronuclei in erythrocytes. This finding suggests that in rats the peripheral blood micronucleus assay is a less sensitive indicator for the genotoxic potential of CARB than the epididymal sperm aneuploidy/diploidy assay.

Immunocytochemical identification of DNA adducts, O6-methylguanine and 7-methylguanine, in respiratory and other tissues of rat, mouse and Syrian hamster exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The present paper reports about an immunocytochemical inventory of the cell types involved in the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to a DNA methylating metabolite. The formation and distribution of the methylated DNA bases O6-methylguanine (O6-meGua) and 7-methylguanine (7-MeGua) were studied in respiratory tissues, oesophagus, liver, kidneys, pancreas, small intestine, colon and prostate of rat, mouse and hamster 6 h after treatment with a single dose of 30 mg NNK/kg. The tissue- and cell-specific distribution of O6-meGua- and 7-meGua-specific nuclear staining showed the same patterns and were remarkably similar in rat, mouse and hamster in spite of the diverging spectra of NNK-induced tumours in these species. In nasal tissue, a target for NNK-induced tumourigenesis in rat and hamster, but not in mouse, adduct-specific nuclear staining was observed in all three species in sustentacular cells, Bowman glands, respiratory epithelial cells and serous glands. Both methylated DNA bases were also observed in basal cells of the olfactory epithelium of rat and (occasionally) hamster, but not in those of the mouse. In the trachea, a target for NNK-induced tumourigenesis in hamster only, substantial adduct-specific nuclear staining was found in basal epithelial and glandular cells of the hamster; in the same cells of rat and mouse only a weak nuclear staining was found. In the lung, a common target for NNK-induced tumourigenesis, the formation of O6-meGua and 7-meGua was restricted predominantly to bronchial and proximal bronchiolar epithelium. Nuclear staining in the rat was occasionally found in alveolar cells and was also observed in hepatocytes. In the three species investigated, O6-meGua- and 7-MeGua-specific nuclear staining was found in target and non-target tissues. Apparently, and in analogy with results obtained in other studies, the species-specific organotropy for tumour formation of NNK is not exclusively determined by DNA methylation. Expanding methylation data with literature data on factors considered to be involved in tumour formation, namely proliferation, toxicity and DNA repair among others, still did not lead to a satisfactory explanation for the species-specific organotropy observed. Additional factors (yet to be identified), need to be taken into account in order to explain (and predict) tumourigenic effects induced by monofunctional methylating agents.

Accumulation of O6- and 7-methylguanine in DNA of N-nitroso-N-methylbenzylamine-treated rats is restricted to non-target organs for N-nitroso-N-methylbenzylamine-induced carcinogenesis.

The distribution and accumulation of O6-methylguanine (O6-meGua) and 7-methylguanine (7-meGua) was investigated immunocytochemically in target and non-target tissues of rats injected twice weekly with 0.5 mg N-nitroso-N-methylbenzylamine (NMBzA)/kg for 16.5 weeks. Seventy two hours after every two or three doses, two NMBzA-treated rats and one control rat were killed. Tissue-specific cell proliferation was investigated after two injections of bromodeoxyuridine (BrdU) to rats unexposed to NMBzA. Neither O6-meGua- nor 7-meGua-specific immunostaining could be observed in the target tissues for tumor induction, i.e. esophagus, tongue and forestomach. Accumulation of both O6-meGua and 7-meGua was found, however, in nasal, tracheal and bronchiolar epithelia and glands--tissues for which tumor induction by NMBzA has not been reported. An explanation for this phenomenon might be the relatively low levels of cellular proliferation we observed in the latter epithelia. The present results support the hypothesis that the tumorigenic organotropism of NMBzA is determined both by the level of DNA methylation and the proliferative capacity of the methylated cells.

Immunocytochemical localization of DNA adducts induced by a single dose of N-nitroso-N-methylbenzylamine in target and non-target tissues of tumor formation in the rat.

The formation and short-term persistence of O6-methylguanine (O6-meGua) and 7-methylguanine (7-meGua) in individual cells of various target and non-target tissues for tumor induction in rats were examined after a single dose of N-nitroso-N-methylbenzylamine (NMBzA). In the principal target organ, the esophagus, both adducts were observed at 6 h after 0.5, 1.0 and 2.5 mg NMBzA/kg in a dose-dependent manner in nuclei of epithelial cells only. Nuclear staining in this organ had apparently declined by 72 h and modified nuclei were found in the more differentiated cells located closer to the lumen. In epithelial cells of the tongue, another target organ of NMBzA, methylation at 6 h was also dose dependent. At 72 h nuclear staining was lower and again largely located in differentiated cells. In the liver, a non-target organ, O6-meGua was not detectable and 7-meGua-specific staining was weak, being only observed at 6 h after the highest dose. Dose-dependent DNA methylation was seen, both at 6 and 72 h, in other non-target organs such as lung (bronchiolar epithelial cells), trachea (epithelial and glandular cells) and nasal cavity (respiratory epithelial cells, ductal cells of the respiratory lamina propria and cells of Bowman glands of the olfactory lamina propria); the nuclei of the glandular cells were highly methylated. Visual inspection of lung, trachea and nasal cavity indicated no or only minor losses of O6-meGua and 7-meGua between 6 and 72 h. Microdensitometric determination of the nuclear staining at 6 and 72 h indicated that the promutagenic O6-meGua was partially lost from cells of the tongue epithelium but did persist in esophageal epithelial cells; 7-meGua was lost to a substantial extent from both tongue and esophagus. The present results imply that the organotropism of NMBzA is not uniquely determined either by the initial level or the short-term persistence of DNA methylation.

Localization of DNA adducts formed in the nasal cavity of the rat by the tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK).

The tissue localization of the DNA adducts O6- and 7-methylguanine induced in the nasal cavity by the nicotine-derived carcinogen 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK, 30 mg/kg intraperitoneally) has been investigated immunocytochemically in male Sprague-Dawley rats. Adduct-specific nuclear staining, indicative of the metabolic activation of NNK to a methylating compound, was observed in both respiratory and olfactory mucosa. In the respiratory epithelium, strong staining was generally observed in areas devoid of goblet cells. Less intense staining was observed both in the serous gland cells and their efferent ducts in the respiratory submucosa, whereas the mucous gland cells were unstained. In the olfactory mucosa, the sustentacular and basal cells of the olfactory epithelium were moderately stained; staining varied substantially from site to site. No DNA adduct was detected in the olfactory cells. Strong nuclear staining, similar to that in the respiratory mucosa, was observed in the cells of the Bowman glands of the olfactory submucosa. A similar distribution of methylated DNA bases in nasal tissues has been observed in rats after exposure to other N-nitrosamines and in Syrian hamsters after exposure to NNK. This finding may indicate that in man the same cell types undergo DNA adduct formation after exposure to NNK and other N-nitrosamines.

Immunocytochemical analysis of in vivo DNA modification.

In the past decades a large number of DNA adducts induced in the intact animal by alkylating agents have been identified. The formation and repair of these adducts are important determinants, not only of mutagenesis, tumor initiation and DNA-mediated toxicity but probably also of tumor progression. Most studies on in vivo DNA modification have been performed on isolated bulk DNA. More recently, methods have been developed to study the distribution of DNA adducts at the level of either the individual gene or the individual cell. This paper reviews immunocytochemical methods to study the formation and repair of DNA adducts and other DNA modifications at the level of the individual cell. DNA modifications induced by alkylating agents and a variety of other agents including ultraviolet radiation, aromatic amines, polycyclic aromatic hydrocarbons and platinum anti-cancer drugs will be discussed. Up to now, immunocytochemical analysis of in vivo modified DNA has largely concentrated on experimental animals. These studies have revealed striking heterogeneities with regard to formation and/or repair of DNA adducts in tissues from rat, hamster and mouse. Immunocytochemical adduct analysis can be used to identify in a convenient, fast and detailed way cell types, cell stages and sites in which biological effects of the adducts might be expressed. More recently, immunocytochemical analysis of DNA adducts also proved to be feasible on in situ exposed human samples. A number of existing and potential applications in the field of chemical carcinogenesis, experimental chemotherapy and molecular epidemiology are discussed.

Improved immunocytochemical staining of carcinogen-DNA adducts by a capillary slot block system.

We developed an immunocytochemical protocol in which incubation occurs in a capillary slot instead of the conventional horizontal drop. Slots of constant width were formed by placing slides on top of each other with parafilm spacer layers in between. Cryostat or semi-thin plastic-embedded sections were cut from organs of carcinogen-treated experimental animals. Carcinogen-DNA adducts were visualized in the affected nuclei by a double peroxidase-antiperoxidase method using rabbit antisera specific for certain DNA adducts formed. The staining in capillary slot blocks offered better staining reproducibility than the conventional method. This is particularly important when the staining intensity must be quantified. In addition, handling of the blocks was substantially less laborious than the individual treatment of slides, making this protocol especially suitable for larger series of slides. Other applications for the capillary slot block protocol should be enzyme histochemistry and in situ hybridization.

Intercellular variation in levels of adducts of aflatoxin B1 and G1 in DNA from rat tissues: a quantitative immunocytochemical study.

Adducts between aflatoxin B1 and G1 and DNA have been visualised and quantified in various rat tissues by a sensitive immunocytochemical approach. The quantitative validity of this assay has been examined by comparison with experiments using radioactively labelled aflatoxin. Rats were exposed to single and multiple doses of aflatoxin and a marked intercellular variation in adduct levels was observed in kidney and lung, in contrast to the liver, where binding was more homogeneous. No adducts were detected in the oesophagus, forestomach, colon, spleen or testes (detection limit approximately 300 pg aflatoxin/mg DNA). The DNA adduct data are discussed in relation to the carcinogenicity of aflatoxin B1 and G1.

The influence of light of different wavelengths on the methylating capacity of the pineal gland of male golden hamsters in relation to reproduction.

In the present experiments the influence of light of different wavelengths on pineal indole metabolism in relation to reproduction was studied. Therefore, during autumn and winter male golden hamsters were kept under natural conditions but for the sunlight which was filtered exposing the hamsters to either normal (control), red or blue light. During the gradually shortening photoperiod at the start of the experiments under normal light conditions, a marked decrease of FSH and LH plasma content as well as testicular weight was found, indicating the onset of gonadal atrophy. During this period a high synthesis of 5-methoxytryptophan (MW) and 5-methoxytryptamine (MT) was determined. The synthesis of other 5-methoxyindoles (MI) was low, while O-acetyl-5-methoxytryptophol (aML) synthesis even markedly decreased. Red and blue light did not cause significant changes in MI synthesis. As long as MT synthesis is high (under blue light), there is no increase in FSH content and testes weight is still decreasing. This influence of blue light confirms the putative antigonadotropic properties of MT. The increase of FSH content at week 9 was the first indication that recrudescence had started. At week 19, this recrudescence was also manifested in the increasing testes weight. The synthesis of melatonin (aMT), 5-methoxytryptophol (ML), 5-methoxyindole-3-acetic acid (MA) and aML increased whereas the production of MT decreased. Blue light exposure caused a significantly higher increase of synthesis of ML, MA, aML and, not-significantly, of aMT, whereas red light caused a significantly lower synthesis of MA. It was concluded that MT, a putative antigonadotropic, and aML, a putative counter-antigonadotropic, are probably important pineal compounds that transduce the photoperiodic messages, which cause either gonadal atrophy or recrudescence. The effect of blue light on indole metabolism and the reproductive cycle was more clear than that of red light. From the present results of blue light on indole metabolism, it was suggested that blue light delayed gonadal atrophy and stimulated gonadal growth, compared to red light. An opposite effect of red light was less obvious.

Single cell analysis of DNA modifications induced by chemical carcinogens and cytostatic drugs.

Antibodies recognizing specific DNA modifications allow the immunocytochemical visualization and quantification of these modifications at the level of the individual cell. Thus, the formation and repair of DNA adducts induced by chemical mutagens and carcinogens and by cytostatic drugs can be studied in very small samples in relation to e.g. cell type and tissue localization. A number of existing and potential applications in the fields of chemical carcinogenesis, chemical mutagenesis, experimental chemotherapy and molecular epidemiology are illustrated.

Immunocytochemical localization of DNA adducts in rat tissues following treatment with N-nitrosomethylbenzylamine.

Immunocytochemical visualization of O6-methylguanosine (meGua) and 7-meGua shows that DNA methylation by N-nitrosomethylbenzylamine(NMBzA) occurs not only in the target organs for tumour induction by this nitrosamine, the oesophagus and (occasionally) the tongue, but also in other tissues (liver, lung, trachea, tracheal glands and nasal cavity) for which no tumour induction by NMBzA has been reported. Thus, the organotropic carcinogenic action of NMBzA cannot be exclusively ascribed to differences in levels of DNA methylation. Additional determinants of the cancer risk in extra-oesophageal tissues could be the small size of the NMBzA-activating target cell population and a low proliferative activity.

Immunocytochemical analysis of DNA adducts in single cells: a new tool for experimental carcinogenesis, chemotherapy and molecular epidemiology.

The immunocytochemical staining of carcinogen-DNA adducts by a double peroxidase-anti-peroxidase (PAP) method is critically described. It is a powerful new tool for the investigation of the initial processes of chemical carcinogenesis--such as metabolic activation of carcinogens, and modification/repair of DNA--at the level of individual, putative target cell types. It is the method of choice if the cell populations are too small for determination of adducts in isolated DNA, or if information on the tissue distribution of DNA damage is needed. Advantages of the peroxidase staining endpoint over immunofluorescence are its stability on storage of slides and the possibility of evaluation by a conventional microscope. First attempts to quantify staining intensity by microdensitometric equipment are described.