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.

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.

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.

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.

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.

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.

Day and night rhythms in the methylation of N-acetylserotonin/5-hydroxytryptophol in the pineal gland of males rats of different ages.

Next to a night rhythm of the methylation of N-acetylserotonin/5-hydroxytryptophol the presence of a daytime rhythm could also be established. Rhythmicity was studied in May during the night and in June during daytime in 21, 42 and 70 days old male Wistar rats. In 21 days old rats, moderate HIOMT activity was observed from 12 p.m.--4 a.m. In rats aged 42 and 70 days HIOMT activity was increased showing a peak at 4 a.m. In September this night maximum is observed at 12 p.m. in rats, aged 42 days. This points to the presence of a seasonal change in HIOMT rhythmicity during the night. During daytime a moderate HIOMT activity is present, which reaches a maximum at 2 p.m. in the 21 and 42 day old animals, while in the adult 70 days old rats activity starts to increase at 2 p.m. probably reaching a maximum at 6 p.m.

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.

The effect of different photoperiods on the methylating capacity of the pineal gland of adult, male golden hamsters, with special reference to 5-methoxyindoles.

Testes weight, plasma FSH and LH concentration and pineal methylating capacity were compared in hamsters housed under either long (LD14:10) or short (LD8:16) photoperiods. Hamsters housed for 14 weeks under short photoperiod showed gonadal atrophy, which was complete after 6 weeks. Also plasma FSH and LH concentration showed a marked decline after transfer to short photoperiod. However, after 14 weeks the concentration of FSH and LH as well as testes weight increased again. Under both photoperiods day/night rhythms in plasma FSH and LH concentration were measured. Under both light regimes the concentrations did not show significant differences. Under long as well as short photoperiods in the pineal gland of animals no significant differences were found in the daily synthesis of various MI tested. Only the synthesis of ML was significantly higher in the pineal of hamsters housed under short photoperiod. The function of this higher synthesis of ML remains unknown. Although the maxima of the rhythm for the various MI found under different LD regimes did not differ in magnitude or duration, their location in respect to the onset of darkness was different. It is suggested that this specific location is of more physiological importance than the quantity or duration of synthesis, concentration or release of MI. At the moment the day/night rhythms were determined there were indications that recrudescence of the testes had already started. It is suggested that this recrudescence is responsible for the fact that no differences in the synthesis of MI were found comparing the influence of both photoperiods. After 14 weeks of exposure to short photoperiod, aML synthesis was, in contrast to the synthesis of the other MI, (not significantly) higher under LD8:16. Moreover, opposite results for aMT and aML synthesis during darkness were found. It is suggested that the ratio of synthesis of these compounds is of physiological significance.

[Retention of indoles in the pineal organ of the bird (Melopsittacus undulatus, Shaw) during its preparation for radioautographic study]. / Rétention des indoles-[3H] dans l'organe pinéal d'oiseau (Melopsittacus undulatus, Shaw) au cours de sa préparation pour l'étude radioautographique.

Since high-resolution radioautography (dipping technique) might be very useful for the study of indole metabolism in the pineal cells, the retention of [3H]-indoles has to be examined during the preparation of specimens for electron microscopy (EM). The pineal organ of the parakeet (Melopsittacus undulatus) was used in the present work. 1) Indole metabolism: following uptake of [3H]-5-hydroxytryptophan ([3H]-HW) in vivo and [3H]-5-hydroxytryptamine ([3H]-HT) in vitro in similar seasonal and nycthemeral conditions--all the known pineal indolic metabolites were recovered by thin layer chromatography. [3H]-5-methoxyindoles were also formed from [3H]-melatonin ([3H]-aMT). 2) The radioactivity of fluids used in the processing of pineal organs in EM was determined by liquid scintillation counting: (a) no exogenous [3H]-indoles could be revealed in EM solutions after [3H]-HW in vivo uptake. (b) 8.8 to 13.4% of [3H]-indoles were washed out by glutaraldehyde after [3H]-HT in vitro uptake. (c) most of the 5-methoxyindoles after in vitro uptake of [3H]-aMT were lost in glutaraldehyde. Our chromatography procedures did not permit the identification of [3H]-indoles extracted by the glutaraldehyde fixative. In previous experiments, [3H]-HW and [3H]-HT uptake showed the presence of selective radioautographic reactions in the cells of the receptor line; however, silver grains were scarce and diffusely distributed in the pineal parenchyma after [3H]-aMT uptake.

The influence of GABA on the synthesis of N-acetylserotonin, melatonin, O-acetyl-5-hydroxytryptophol and O-acetyl-5-methoxytryptophol in the pineal gland of the male Wistar rat.

The influence of GABA on the synthesis of N-acetylserotonin, melatonin, O-acetyl-5-hydroxytryptophol and O-acetyl-5-methoxytryptophol has been investigated using different experimental procedures. It was demonstrated that when GABA and an acetyl donor were added to the incubation medium together, a significant increase in synthesis of the N-acetylated products occurred during the night. Moreover there was a large increase in N-acetylserotonin synthesis at 15(00) hrs although none was observed in the control experiments. However, when GABA was added 20 min before the acetyl donor, synthesis of the N-acetylated products was significantly less. The opposite effect was observed for the O-acetylated indoles. These results confirm the proposal by Ebadi et al. (1982) that GABA, like norepinephrine, may be a regulator of melatonin synthesis. As melatonin is implicated in the regulation of reproduction it may be that GABA is equally significant in this regulatory effect.

Estimation of the methylating capacity in the pineal gland of the rat with special reference to the methylation of N-acetylserotonin and 5-hydroxytryptophol separately.

In the present paper, an extension is presented of an earlier described method, by which the methylating capacity of the pineal gland can be determined. Supplementary to the earlier method, the synthesis of melatonin and 5-methoxytryptophol can now be qualified and quantified separately.

Seasonal variations in HIOMT activity during the night in the pineal gland of 21 day old male Wistar rats.

In the pineal of 21-day old male Wistar rats hydroxyindole-O-methyltransferase (HIOMT) activities involved in the synthesis of several 5-methoxyindoles were determined during the night in April, June, October and January. A high HIOMT activity for the synthesis of melatonin/5-methoxytryptophol was determined in the months of January and April. In June and October a decrease was observed. The activity maxima coincide with peaks of activity found for the synthesis of 5-methoxytryptophan. Synthesis of 5-methoxytryptamine occurred only in June and October, whereas the synthesis of 5-methoxyindole-3-acetic acid occurred only in January. From these results it may be concluded that January and April are the most active months of those tested for the melatonin/5-methoxytryptophol synthesis in the rat pineal gland. A possible physiological role of the 5-methoxyindoles other than melatonin and 5-methoxytryptophol is discussed.

In vitro uptake and metabolism of [3H]indole compounds in the pineal organ of the pike. I. A radiochromatographic study.

Thin layer chromatography analysis of [3H]serotonin and [3H]melatonin metabolites synthetized in vitro by the pineal organ of the pike was performed. After a 10-min pulse, [3H]serotonin was mainly converted into [3H]-5-hydroxyindoleacetic acid (37%), [3H]-5-hydroxytryptophan and [3H]-5-methoxytryptophan (12 to 14%), and [3H]-5-hydroxytryptophol and [3H]-5-methoxytryptophol (3.5 and 9%) at the onset of darkness. When the pulse was followed by postincubations (in a cold medium) of increasing duration (15, 30, and 60 min), it appeared that the amount of [3H]-5-hydroxyindoleacetic acid decreased, that of [3H]-5-hydroxytryptophol decreased faster than that of [3H]-5-methoxytryptophol, and the amounts of [3H]-5-hydroxy- and [3H]-5-methoxytryptophan increased. [3H]-N-acetylserotonin, [3H]melatonin, and [3H]-5-methoxytryptamine were found in very low amounts. At the beginning of the photophase or at the onset of darkness, the uptake and metabolism of [3H]melatonin (after a 10-min pulse followed by a 10-min incubation in cold medium) resulted mainly in the formation of [3H]-5-methoxytryptophol (23 to 43%) and of [3H]-5-methoxytryptamine (6 to 12%). These results show that the pike pineal organ can synthesize all indoles that are known in the pineal gland of higher vertebrates. Usual, but also unusual, pathways of the indole metabolism were found that will need further clarification. Among these are the possible carboxylation of serotonin and deacetylation of melatonin (leading to the synthesis of 5-methoxytryptophol). Altogether, the results obtained suggest that the indole metabolism might be more complex than what has already been described in vertebrates.

Rhythmic synthesis of various 5-methoxyindoles in the pineal gland of male adult golden hamsters, kept under the same artificial conditions throughout the year.

Until now the day/night and seasonal rhythmicity in the synthesis of 5-methoxyindoles (MI) is thought to be regulated by environmental factors, especially photoperiod and temperature. Endogenous factors are also implicated in the generation of N-acetyltransferase and hydroxyindole-O-methyltransferase activity rhythms. In the present experiments seasonal rhythmicity in the synthesis of MI in the pineal gland was investigated in hamsters kept under the same artificial conditions throughout the year. Though the environmental conditions were the same, day/night and seasonal rhythmicity in the production of MI in the pineal were observed indicating the existence of endogenous factors influencing the rhythmicities. In November, most of the MI showed the highest synthesis, MA and ML excepted, which were especially produced in July and September. The results obtained sustain the hypothesis that aMT is synthesized from MT rather than from aHT. Moreover, the rhythmicities in aMT synthesis are not identical to those found in aMT concentration as described in the literature. This indicates that synthesis and concentration of a compound are not comparable. At the end of the light period, when aMT injections have an antigonadotropic effect, a peak of aMT synthesis was always present. Although MI synthesis showed seasonal rhythmicity, no reproductive cycle occurred in the hamsters. At present, the concept that the pro- and/or antigonadal effects of the pineal are mediated by aMT seems to be the most acceptable. The present results, however, indicate that aMT and perhaps other MI, often regarded as factors influencing gonadal growth in golden hamsters, are not the only factors involved.