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Recently, immune checkpoint inhibitors have been drawing much attention as cancer immunotherapy, but it has been shown that various immune-related adverse events (irAEs) are induced by immune checkpoint inhibitors in various organs, which has become one of the serious issues at present. A 58-year-old Japanese male with malignant melanoma was treated with nivolumab and/or ipilimumab. During the period of treatment, he suffered from various irAEs. Firstly, about 1 month after starting nivolumab monotherapy, destructive thyroiditis was induced, and so we started replacement therapy with levothyroxine. Secondly, about 1 month after starting nivolumab and ipilimumab combination therapy, aseptic meningitis was induced. We stopped both drugs and started steroid therapy with prednisolone. Finally, about 9 months after restarting nivolumab, isolated adrenocorticotropic hormone (ACTH) deficiency was induced, and so we started replacement therapy with hydrocortisone. Taken together, we should bear in mind the possibility of a variety of irAEs when we use immune checkpoint inhibitors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Katakura, Kimura, Kusano, Tatsumi, Iwamoto, Sanada, Fushimi, Shimoda, Kohara, Nakanishi, Kaku, Mune and Kaneto.)

To identify long non-coding RNAs (lncRNAs) and their potential roles in hepatic fibrosis in rat liver issues induced by CCl4, lncRNAs and genes were analyzed in fibrotic rat liver tissues by RNA sequencing and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Differentially expressed (DE) lncRNAs (DE-lncRNAs) and genes were subjected to bioinformatics analysis and used to construct a co-expression network. We identified 10 novel DE-lncRNAs that were downregulated during the hepatic fibrosis process. The cis target gene of DE-lncRNA, XLOC118358, was Met, and the cis target gene of the other nine DE-lncRNAs, XLOC004600, XLOC004605, XLOC004610, XLOC004611, XLOC004568, XLOC004580 XLOC004598, XLOC004601, and XLOC004602 was Nox4. The results of construction of a pathway-DEG co-expression network show that lncRNA-Met and lncRNAs-Nox4 were involved in oxidation-reduction processes and PI3K/Akt signaling pathway. Our results identified 10 DE-lncRNAs related to hepatic fibrosis, and the potential roles of DE-lncRNAs and target genes in hepatic fibrosis might provide new therapeutic strategies for hepatic fibrosis., Competing Interests: The authors have declared that no competing interests exist.

Immunotherapy with immune checkpoint inhibitor (ICI) monoclonal antibodies has shown to be an effective therapeutic alternative in several malignant tumors. However, adverse effects related to an activation of the immune system may accompany ICI therapy. Among the immune-related adverse events (irAEs) are autoimmune endocrine adverse effects, such as thyroiditis, and hypophysitis. Secondary adrenal insufficiency due to isolated ACTH deficiency (IAD) has also been recently reported to be associated with ICI antibodies. We carried out a systematic review of IAD cases induced by cancer immunotherapy published to date using PubMed's database. We selected 35 articles that reported 60 cancer patients diagnosed with IAD induced by ICI therapy. The prevalence was higher in men (ratio 1.6/1). Mean age at diagnosis was 63.2 ± 11.6 (range,30-87). Melanoma was the tumor most commonly reported (35%) followed by lung (28.3%) and kidney cancer (18.3%). The ICI monoclonal antibody most frequently associated was nivolumab in monotherapy (60%), followed by pembrolizumab (18.3%). Median (IQR) time to develop IAD after starting ICI therapy was 6 (4-8) months. The main symptoms at IAD diagnosis were fatigue (82.8%) and anorexia (67.2%). Hyponatremia (68%) and eosinophilia (31.8%) were the laboratory abnormalities most frequently associated with IAD. Pituitary magnetic resonance imaging (MRI) was normal in most patients (93%). Thyroiditis was the most prevalent (35%) endocrine irAE associated with IAD. In conclusion, ICI-induced IAD is a rare and potentially life-threatening condition that must be taken into account whenever treatment with immunotherapy in cancer patients is started due to their potential serious prognostic implications., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of aneugenic agents., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

The increased use of immune checkpoint inhibitors (ICIs) has led to the observation of a variety of immune-related adverse events (irAEs). These irAEs occur usually within the first months after ICIs onset and can involve theorically all organs. We describe two rare irAEs occurring in a 70-year-old caucasian man who was treated with nivolumab for an advanced urothelial cancer of the left kidney. He developed an isolated adrenocorticotropic hormone deficiency that was diagnosed at week 19 and a neurological complication that appeared at week 79 and initially confounded with a lumbar spinal stenosis. Diagnosis of Guillain-Barré syndrome was finally confirmed with the complete resolution of symptoms after 5 days of intravenous immunoglobulin and corticosteroids. We highlight the importance of quickly recognising these potential life-threatening irAEs such as cortisol insufficiency and neurologic adverse events whose initially presentation could be non-specific., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Therapeutic blocking antibodies against programmed death 1 (PD1) and cytotoxic T-lymphocyte antigen 4 (CTLA4) are applied for advanced cancer therapy, but induce a wide range of immune-related adverse events. In our recent case of a 52-year-old female doctor suffering from breast cancer having metastasized to the lung and liver, it was decided to use nivolumab to prevent the disease progressing after excisional surgeries and multiple chemotherapies. One month after completing the nivolumab course, fatigue, hypoglycemia and hypotension developed and isolated ACTH deficiency (IAD) was diagnosed. A further month later, under steroid supplementation, hyperglycemia emerged alongside thirst and polydipsia, prompting a diagnosis of fulminant type 1 diabetes (FT1D). Her susceptibility to type 1 diabetes was examined by HLA haplotype and CTLA4 gene polymorphism analyses. Polymorphisms CT60G>A and +49G>A in CTLA4 both generated a GG genotype. Our patient manifested one of the rarest combinations of autoimmune disease induced by nivolumab. Whereas the HLA haplotype was unsusceptible to autoimmune type 1 diabetes, polymorphisms of CTLA4, the antibody of which frequently causes hypophysitis, were susceptible to FT1D. Peripheral modulation of activated T cells, mainly by PD-1 antibodies, induced FT1D associated with IAD in patients with CTLA4 polymorphism. This case reveals hints of the T-cell etiology in T1D and evidence of CTLA4 involvement in IAD.

Introduction: Immune checkpoint inhibitors are a promising class of anticancer drugs. The clinical benefits afforded by immune checkpoint inhibitors can be accompanied by immune-related adverse events that affect multiple organs, and endocrine immune-related adverse events include thyroiditis and hypophysitis. Hypophysitis is less frequent and has a less severe clinical presentation in patients treated with other immune checkpoint inhibitors, such as nivolumab, pembrolizumab, and atezolizumab, than in those treated with ipilimumab. However, studies have described isolated adrenocorticotropic hormone deficiency cases associated with nivolumab, pembrolizumab, and atezolizumab therapy, most of which occurred during the course of immune checkpoint inhibitor therapy. We report a rare case of patient with isolated adrenocorticotropic hormone deficiency that occurred after nivolumab therapy., Case Presentation: A 69-year-old Japanese woman with advanced lung adenocarcinoma developed painless thyroiditis with transient elevations of serum thyroid hormones during 3 months of cancer treatment with nivolumab and began thyroid hormone replacement therapy for subsequent primary hypothyroidism. Four months after nivolumab therapy was discontinued, she developed isolated adrenocorticotropic hormone deficiency; corticosteroid replacement therapy relieved her secondary adrenal insufficiency symptoms, such as anorexia and fatigue. Human leukocyte antigen typing revealed the presence of DRB1*04:05-DQB1*04:01-DQA1*03:03 and DRB1*09:01-DQB1*03:03-DQA1*03:02 haplotypes, which increase susceptibility to autoimmune polyendocrine syndrome associated with thyroid and pituitary disorders in the Japanese population., Conclusions: Our patient developed thyroiditis during cancer treatment with nivolumab and subsequently exhibited isolated adrenocorticotropic hormone deficiency 4 months after discontinuing the drug. Administration of nivolumab in combination with a genetic predisposition to polyglandular autoimmunity probably caused both the thyroiditis and hypophysitis, resulting in primary hypothyroidism and isolated adrenocorticotropic hormone deficiency, respectively, in our patient. The present case highlights the need for physicians to be aware that endocrine immune-related adverse events, including hypophysitis, can occur more than several months after discontinuing a drug.

Background: Immune checkpoint inhibitors including nivolumab, an anti-programmed cell death protein 1 antibody, are recently developed cancer immunotherapy agents. Immune checkpoint inhibitors are known to cause autoimmune-related side effects including endocrine dysfunctions. However, there are few reports on late-onset isolated adrenocorticotropic hormone (ACTH) deficiency caused by nivolumab., Case Presentation: The patient was a 72-year-old female. When she was 64 years old, she was diagnosed with malignant melanoma of the left thigh accompanied by left inguinal lymph node metastases, and she received several courses of chemotherapy for malignant melanoma followed by the resection of these lesions. At 71 years of age, multiple metastases were found and treatment with nivolumab 2 mg/kg every 3 weeks was initiated. Six months later, replacement with levothyroxine was started because of hypothyroidism following mild transient thyrotoxicosis. Eleven months after the beginning of nivolumab, the treatment was discontinued because of tumor expansion. Four months after the discontinuation of nivolumab, general malaise and appetite loss worsened, and 2 months later, hyponatremia (Na; 120-127 mEq/L) and hypoglycemia (fasting plasma glucose; 62 mg/dL) appeared. Her ACTH and cortisol levels were extremely low (ACTH; 9.6 pg/mL, cortisol; undetectable). Challenge tests for anterior pituitary hormones showed that responses of ACTH and cortisol secretion to corticotropin-releasing hormone were disappeared, although responses of other anterior pituitary hormones were preserved. Thus, she was diagnosed with isolated ACTH deficiency. Her symptoms were improved after treatment with hydrocortisone., Conclusions: The present report showed a case of late-onset isolated ACTH deficiency accompanied by hyponatremia, which was diagnosed 6 months after the discontinuation of nivolumab. The effects of nivolumab last for a long time and the side effects of nivolumab can also appear several months after discontinuation of the drug. Repeated monitoring of serum sodium levels may be a beneficial strategy to find the unexpected development of adrenal insufficiency even after discontinuation of nivolumab.

Rationale: Nivolumab is a monoclonal IgG antibody blocking programmed death receptor-1 (PD1), leading to restoration of the natural T-cell-mediated immune response against the cancer cells. However, it also causes plenty of autoimmune-related adverse events, which often involves endocrine system., Patient Concerns: A 54-year-old male with renal clear cell carcinoma was treated with nivolumab intravenously. Routine monitoring showed elevated thyroid-stimulating hormone and low free thyroxine after the 6th administration of nivolumab. After the 12th administration, he developed general fatigue, recurrent hypoglycemia, and relative hypotension. Laboratory tests showed low sodium, low morning cortisol without correspondence increase of corticotrophin (ACTH). Other pituitary hormones were normal. MRI showed no space-occupying lesions, but heterogeneous enhancement of the pituitary gland., Diagnoses: Primary hypothyroidism and isolated ACTH deficiency. The etiologies were assumed to be nivolumab induced autoimmune lymphocytic thyroiditis and hypophysitis, respectively., Interventions: Hormone replacements with levothyroxine and acetate cortisone were given orally. Nivolumab was adjusted to lower dose and longer interval., Outcomes: The patient felt good after adequate replacement. Nivolumab was returned to routine dose and interval six months later. And the metastasis was not obviously progressed during this time., Lessons: The present report provides the first detailed presentation of combined hypothyroidism and isolated ACTH deficiency induced by nivolumab. Adrenal deficiency often develops insidiously. We suggest routine monitoring of fasting blood-glucose, blood pressure and serum sodium as well as thyroid function during nivolumab and other cancer immunotherapies. When unexpected fatigue, hypoglycemia, hypotension or hyponatremia appeared, adrenal deficiency should be taken into consideration.

Nivolumab has promising efficacy for treating various advanced malignant tumors, although it has been reported to induce a wide range of autoimmune adverse effects. We herein report the case of a patient with metastatic lung adenocarcinoma who developed adrenal insufficiency after 12 cycles of nivolumab treatment. Endocrine test results supported a diagnosis of isolated adrenocorticotropic hormone deficiency due to hypophysitis, and replacement therapy using hydrocortisone has been successful. Although hypophysitis is a rare immune-related adverse event that is associated with nivolumab therapy, clinical awareness is essential, as this condition can be life-threatening and requires prompt treatment.

Nivolumab, an anti-programmed death-1 antibody, is a breakthrough treatment for several malignancies. Its specific adverse effects caused by autoimmunity are termed immune-related adverse events, which involve several endocrine dysfunctions. Herein, we report two cases of isolated adrenocorticotropic hormone (ACTH) deficiency induced by nivolumab for the treatment of metastatic malignant melanoma. Case 1 was a 39-year-old man and Case 2 was a 50-year-old woman, both of whom presented with progressive melanoma. After 13 courses of nivolumab administration, both cases were diagnosed with adrenal insufficiency. Despite their basal serum ACTH and cortisol levels being low with little response to corticotropin-releasing hormone loading, other anterior pituitary hormone levels were preserved. Based on these endocrinological data, isolated ACTH deficiency was diagnosed. Magnetic resonance imaging showed normal pituitary glands, excluding hypophysitis. Finally, hydrocortisone replacement enabled the patients to continue nivolumab treatment. Therefore, it is important to consider isolated ACTH syndrome as a possible and potentially severe immune-related adverse event of nivolumab, even when head magnetic resonance imaging of affected cases does not show enlargement. We should not misdiagnose hidden immune-related adverse events behind general complaints of malignancies such as general malaise and appetite loss, to allow successful treatment using this beneficial immune checkpoint inhibitor., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A 73-year-old Japanese woman with untreated Graves' hyperthyroidism developed glucocorticoid-induced adrenal insufficiency (AI) after a supraphysiological dose of prednisolone therapy for bronchial asthma. Days later, she had high plasma adrenocorticotropic hormone (ACTH) levels and was expected to recover from glucocorticoid-induced AI. Her plasma ACTH levels remained high over 3 months during a physiological dose of hydrocortisone replacement. However, she suffered a further decrease in her serum cortisol level and was diagnosed with isolated adrenocorticotropin deficiency (IAD), in which bioinactive ACTH likely caused the high ACTH value. IAD should be considered as an unusual disorder associated with Graves' disease, especially in older patients.

Background: The surveillance of newborn infants exposed to potential teratogens often relies on the findings in routine physicians' examinations to identify malformations. Exposed newborn infants can have a wide variety of physical features, including malformations, birth marks, positional deformities, and minor anomalies. The routine physician's findings are not standardized. Some physicians record a wide variety of physical features and others do not. The purpose of this study was to develop criteria and definitions for identifying malformations and for identifying the more common and less severe physical features that would be excluded as not being malformations., Methods: The physical features recorded by the examining pediatricians were obtained from a review of the medical records of a consecutive sample of 1000 liveborn and stillborn infants and elective terminations for fetal anomalies., Results: A malformation, defined as a structural abnormality with surgical, medical or cosmetic importance, was present in 18 (2.8%) of the infants; 222 other recorded features were identified and excluded: malformations attributed to dominant or recessive genes (4) or chromosome abnormalities (6), minor anomalies and normal variations (65), birth marks (110), positional deformities (6), prematurity-related features (5), physiologic findings (4) and findings identified by prenatal ultrasound (but not by the examining pediatrician) (20), functional abnormalities (1) and findings in newborn screening (1)., Conclusions: Investigators should establish, in advance, the exclusion criteria to be used in programs, such as malformation surveillance programs or pregnancy registries, whose findings are based on a review of the routine examinations in medical records. It is essential that the same criteria be used in evaluating the drug-exposed and the unexposed comparison group., (Copyright © 2011 Wiley-Liss, Inc.)

Translocations have provided invaluable tools for identifying both cancer-linked genes and loci associated with heritable human diseases, but heritable human translocations are rare and few mouse models exist. Here we report progress on analysis of a collection of heritable translocations generated by treatment of mice with specific chemicals or radiation during late spermatogenic stages. The translocation mutants exhibit a range of visible phenotypes reflecting the disruption of coding sequences or the separation of genes from essential regulatory elements. The breakpoints of both radiation-induced and chemically induced mutations in these mice are remarkably clean, with very short deletions, duplications, or inversions in some cases, and ligation mediated by microhomology, suggesting nonhomologous end joining as the major path of repair. These mutations provide new tools for the discovery of novel genes and regulatory elements linked to human developmental disorders and new clues to the molecular basis of human genetic disease.

Heritable genetic alterations, although individually rare, have a substantial collective health impact. Approximately 20% of these are new mutations of unknown cause. Assessment of the effect of exposures to DNA damaging agents, i.e. mutagenic chemicals and radiations, on the integrity of the human genome and on the occurrence of genetic disease remains a daunting challenge. Recent insights may explain why previous examination of human exposures to ionizing radiation, as in Hiroshima and Nagasaki, failed to reveal heritable genetic effects. New opportunities to assess the heritable genetic damaging effects of environmental mutagens are afforded by: (1) integration of knowledge on the molecular nature of genetic disorders and the molecular effects of mutagens; (2) the development of more practical assays for germline mutagenesis; (3) the likely use of population-based genetic screening in personalized medicine.

Embryonic exposure to the endocrine disruptor vinclozolin at the time of gonadal sex determination was previously found to promote transgenerational disease states. The actions of vinclozolin appear to be due to epigenetic alterations in the male germline that are transmitted to subsequent generations. Analysis of the transgenerational epigenetic effects on the male germline (i.e. sperm) identified 25 candidate DNA sequences with altered methylation patterns in the vinclozolin generation sperm. These sequences were identified and mapped to specific genes and noncoding DNA regions. Bisulfite sequencing was used to confirm the altered methylation pattern of 15 of the candidate DNA sequences. Alterations in the epigenetic pattern (i.e. methylation) of these genes/DNA sequences were found in the F2 and F3 generation germline. Therefore, the reprogramming of the male germline involves the induction of new imprinted-like genes/DNA sequences that acquire an apparent permanent DNA methylation pattern that is passed at least through the paternal allele. The expression pattern of several of the genes during embryonic development were found to be altered in the vinclozolin F1 and F2 generation testis. A number of the imprinted-like genes/DNA sequences identified are associated with epigenetic linked diseases. In summary, an endocrine disruptor exposure during embryonic gonadal sex determination was found to promote an alteration in the epigenetic (i.e. induction of imprinted-like genes/DNA sequences) programming of the male germline, and this is associated with the development of transgenerational disease states.

The fetal basis of adult disease is poorly understood on a molecular level and cannot be solely attributed to genetic mutations or a single etiology. Embryonic exposure to environmental compounds has been shown to promote various disease states or lesions in the first generation (F1). The current study used the endocrine disruptor vinclozolin (antiandrogenic compound) in a transient embryonic exposure at the time of gonadal sex determination in rats. Adult animals from the F1 generation and all subsequent generations examined (F1-F4) developed a number of disease states or tissue abnormalities including prostate disease, kidney disease, immune system abnormalities, testis abnormalities, and tumor development (e.g. breast). In addition, a number of blood abnormalities developed including hypercholesterolemia. The incidence or prevalence of these transgenerational disease states was high and consistent across all generations (F1-F4) and, based on data from a previous study, appears to be due in part to epigenetic alterations in the male germ line. The observations demonstrate that an environmental compound, endocrine disruptor, can induce transgenerational disease states or abnormalities, and this suggests a potential epigenetic etiology and molecular basis of adult onset disease.

The ultimate goal of the Environmental Genome Project is the improvement of human health on the basis of information about the variations in certain genes. The first phase of the project will involve the selection of the human genes and characterization of the alleles occurring in the American population. However, intelligent use of this information will require analysis of the relevance of the allelic differences. Epidemiology alone will not solve the problem, and mechanistic studies will be required. Factors to be considered in the design of functional analyses are considered in this commentary.

This article reviews the effects of lead on genetic systems in the context of lead's various other toxic effects and its abundance and distribution in the environment. Lead is perhaps the longest used and best recognized toxic environmental chemical, yet it continued be used recklessly until only very recently. Lead is thus a lesson in the limitations and strengths of science, human conscience and common sense. Lead has been tested and found to be capable of eliciting a positive response in an extraordinarily wide range of biological and biochemical tests; among them tests for enzyme inhibition, fidelity of DNA synthesis, mutation, chromosome aberrations, cancer and birth defects. It reacts or complexes with many biomolecules and adversely affects the reproductive, nervous, gastrointestinal, immune, renal, cardiovascular, skeletal, muscular and hematopoietic systems as well as developmental processes. It is likely that lead is a selective agent that continues to act on and influence the genetic structure and future evolution of exposed plant and animal populations.

Reciprocal translocations have provided crucial tools for the localization of genes associated with a variety of human cancers and hereditary diseases. Although heritable translocations are relatively rare in humans, they can be easily induced in mice through exposure of male germ cells at specific spermatogenic stages to different types of radiation and chemicals. Mutagenesis schemes that produce translocations at high frequencies in the progeny of treated males are summarized, and the use of these valuable mutations for analyzing developmental consequences of partial aneuploidy, for identification of mutant genes, and for other purposes is reviewed. Preliminary studies of a large collection of translocation mutants, including several stocks that display dominantly or recessively inherited phenotypes caused by the disruption of critical genes are described. These combined studies demonstrate that several mutagenesis protocols can be used to generate easily mapped, novel mouse mutations with high efficiency and highlight the unique value of reciprocal translocations as tools for gaining access to the biological functions of mammalian genes., (Copyright 1997 Academic Press.)

Because genetically based diseases have a major impact on human health, the National Institute of Environmental Health Sciences (NIEHS) has conducted a research and testing program for more than a decade to address chemical induction of heritable genetic damage in the germ cells of mammals. Although most genetic disease results from preexisting mutations, a portion is due to the occurrence of new mutations. The supposition that exposure to mutagenic chemicals contributes to the occurrence of new mutations in the human population is strongly supported by the results from animal models. Such studies clearly demonstrate the potential of environmental chemicals to induce mutations in both somatic and reproductive cells of mammals. This NIEHS program has become a leader in the identification of genetic hazards in the environment and in the acquisition of animal model data used by regulatory agencies in assessing genetic risks to human health.

One of the interests of ICPEMC is to identify situations in which the possible induction of inherited defects in man by mutagen exposure could actually be studied. The large-scale use of mutagenic drugs in field programmes against schistosomiasis, mainly during the 1970's, was considered a possible case. An ICPEMC task group approached the problem by (1) updating the genetic toxicology data base for antischistosomal drugs, and (2) reviewing possible study areas. Expertise was combined from genetic toxicology, mutation epidemiology and tropical medicine. It was considered that: (a) if any, hycanthone would be the most appropriate candidate drug for study; (b) it would be virtually impossible to meet the basic requirements of an appropriate mutation epidemiology study, in endemic countries; (c) as more defined genetic endpoints would be selected (e.g. sentinel phenotypes) the required large sample sizes would seem prohibitive, since documentation on past programmes is limited and local demography would render the reliable tracking of substantial numbers of offspring of treated persons an almost impossible task; (d) in most endemic countries proper diagnosis and registration of inherited defects is largely lacking; (e) the problems encountered in demonstrating inherited effects in humans after heavy or chronic exposure to established animal mutagens such as ionizing radiation and cancer chemotherapy, in combination with the ambiguous nature of the animal germ cell data with hycanthone, do not particularly warrant large expectations; (f) since non-mutagenic antischistosomal drugs are now in use, the problem is academic and of low priority in the endemic countries whose medical and research resources are often limited. Thus, studying offspring of hycanthone-treated people to demonstrate the mutagenic potential of the drug in man is not a viable enterprise.

As a basis of the suggested test-system, the following conditions are observed: 1) the economy of fulfilment in a short time; 2) the analysis of gene and chromosome mutations in germ and somatic cells; 3) the evaluation of mutagenic effects of not only substance, but also of the products of its metabolism; 4) including in the system only the tests which give the minimal variability between separate experiments; 5) the evaluation of dose-effect relationship. The practical scheme of testing is divided into two parts: a screening and a complete one. The screening programme consists of two tests: a) a test on microorganisms with a metabolic activation in vitro; b) a cytogenetic analysis of bone marrow of mammals. The complete programme of testing includes 4 tests: a) a test on microorganisms with a metabolic activation in vitro and in vivo; b) a test of dominant lethal mutations on mammals; c) a cytogenetic analysis of bone marrow of mammals; d) a cytogenetic analysis in the culture of human lymphocytes. There are good reasons for the principles of selection of substance for testing according to the screening and complete programme: population occurence, economic (or medical) significance, information about relative chemicals showing mutagenic, carcinogenic and teratogenic effect. In the group of chemicals which are to be tested according to the screening programme, such ones can be included: industrial chemicals, phosphoorganic insecticides, drugs which are taken by a limited group of patients. The group of chemicals which are to be tested according to the complete programme consists of the following ones: pesticides, food additices, widespread drugs, the chemicals of the group 1, if during one of the tests of the screening programme a genetic effect is detected. At the genetic risk estimation it is advisable to keep to the following rule: a positive effect, identified in any object of the system must in the direct meaning extrapolate on men. The quantitative evaluation of the mutagenic danger of a chemical can be determined by the increase of the spontaneous level of mutations in the test-object on the basis of an average dose and exposition of the given chemical in the human population. Those chemicals are subject to the quantitative evaluation, which have shown a mutagenic activity during any of the test-objects; they are also widespread and because of their social or economic value can not be replaced or excluded from taking. From the point of view of genetics any substance with a mutagenic activity is dangerous and must be prohibited from using or replaced by any other non-mutagenic chemical, or limited by the contact of persons of non-reproductive age. As a temporary measure from a hygienic point of view, it is recommended to evaluate this chemical as especially mutagenic and prohibit or limit its using, when its average population dose produces 1/10 or more increase of the spontaneous level of mutations.