HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei.

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of "normal" values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin-B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5 per thousand and the interquartile range was between 3 and 12 per thousand. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14-24%). Statistical analyses were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.

Activities of the rodent carcinogens thioacetamide and acetamide in the mouse bone marrow micronucleus assay.

Thioacetamide is confirmed as active in the mouse bone marrow micronucleus assay, following single oral administration to both sexes of two different strains of mice (C57BL/6, BALB/c) in five separate experiments. Acetamide is shown to be consistently and clearly negative as a micronucleus-inducing agent in mouse bone marrow in four repeated assays using different sexes of two strains of mice (C57BL/6, CBA). The present findings support the adequacy of the limited micronucleus test protocol (male animals, two sampling times) for the efficient detection of genotoxic rodent carcinogens in this assay.

[The toxic hygiene problems of workers in the Dinamik automobile tire plant in Sofia]. / Toksikokhigienni problemi pri raboteshtite v zavod za avtomobilni gumi "Dinamik"--Sofiia.

Toxic hygienic investigations were carried out involving a representative group of 131 workers (50 females and 81 males) distributed into three main workshops--preparatory, confection, and vulcanization--at "Dynamic" automobile tire plant in Sofia. The majority of male and female workers were in the age range beyond 40 years, having a general occupational experience in excess of 10 years and specialized occupational experience from 10 to 20 years or more. The chemical hazard was among the leading ones in the occupational environment, differing in nature according to the technologies used. Included were various chemical substances and compounds: synthetic rubbers, fillers (soot, chalk, kaolin); softeners (mazut, paraffin, etc.); accelerants (mercaptothiazoles--captax and altax); dithiocarbamates (thiuram); vulcacides (diphenylguanidine); antiwear agents (antioxidants and antiozonators-isopropyl-phenyl-paraphenylene diamine, naphthyl-beta-naphthylamine); antiaccelerants (phthalic anhydride, ets.); organic solvents and others. The indicated chemical substances and compounds, though often found at concentrations below the mean-shift MACs, do produce health impacts by virtue of prolonged and combined exposures. Use was made of the questionnaire method. Also, hematologic, clinical laboratory, and toxic chemical testing was performed. Findings pointed to changes in hemopoiesis, deviations in hepatic functional state, while sulfate and glucuronide values confirmed the workers' high exposures.

[The cytogenetic effects (the frequency of micronuclei) in lymphocyte cultures from the peripheral blood of workers in automobile tire manufacture]. / Tsitogenetichni (chestota na mikronukleusi) v limfotsitni kulturi ot periferna kruv na rabotnitsite ot proizvodstvoto na avtomobilni gumi.

At the Preparatory Workshop of the Plant for Automobile Tires (PAT), Sofia, complex investigations were undertaken to reveal possible genotoxic exposure. The studies included chemical analyses for levels of identifiable human carcinogens in the occupational ambient air (benz(a)pyrene, mineral oils, 2-naphthylamine); special techniques--questionnaire investigations and cytogenetic analysis by cytokinesis-block micronucleus method in peripheral lymphocyte cultures from 23 workers in occupational groups at risk; and urinalysis for contents of mercapturic acids. An out-of-house control group of 13 nonexposed subjects was concurrently investigated. For contents of benz(a)pyrene and mineral oils exceeding 2.5 to 3.5 times the respective occupational environment MACs, evidence from cytogenetic analysis showed substantial, 4-fold, increase in indexes of genotoxic impairment (frequency of micronucleated-binucleated lymphocytes, number of micronuclei per 1000 binucleated lymphocytes) in the workers investigated. These results are an indicator of genotoxic exposure and point to an increased potential risk of cancer development in the group of workers studied.

[The genotoxicity and carcinogenic potential of gastrofenzin]. / Genotoksichnost i kantserogenen potentsial na gastrofenzin.

Genotoxicity of the Bulgarian drug gastrophensin was studied by using a battery of two genotoxicity assays "in vitro" - Salmonella/mutation assay and "in vivo" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" was tested in five mutant, histidine auxotrophic strains - TA 1535, TA 1537, TA 1538, TA 98 and TA 100 without and in the presence of metabolic activation (+/- S9) at concentration of 0.4, 2 and 10 mg center dot ml-1. Gastrophensin did not induce mutagenic response in the Salmonella/mutation assay in a range of tested concentrations in both series of assays (+/- S9). Gastrophensin did not induce micronuclei in bone marrow cells of male C57Bl6 mice at 24, 48 and 72 hours after single oral treatment with 236 mg center dot kg-1 (80% DL50 oral, mice) and 118 mg center dot kg-1 (40% DL50 oral, mice). Based on the present data a conclusion of the lack of mutagenicity and of carcinogenic potency of gastrophensin was made.

[An evaluation of the genotoxicity activity of the microbial preparation bulmoscide]. / Otsena na genotoksichnata aktivnost na mikrobniia preparat bulmostsid.

The genotoxic activity of the microbial preparation bulmoscid "in vivo" for somatic cells of mammals is studied by micronucleus method in marrow of mice. The experiments are performed on inbred mice C57BL6 of both sexes at unrepeated oral introduction of the preparation in two doses: 2200 mg/kg-1 (2.64.10(10) cells kg-1), and 1100 mg/kg-1 (1.32.10(10) cells kg-1) presenting 80% and respectively 40% of the unrepeated maximum tolerable dose of "Bulmoscid" for mice (2750 mg/kg-1). The genotoxic index, frequency of micronuclear polychromatophilic erythrocytes (MPE) in marrow is determined in dynamics on the 24, 48 and 72 hour after the introduction of the preparation. The data of the performed cytogenetic analysis in marrow of C57BL6 mice of both sexes point out that the preparation "Bulmoscid" possesses no clastogenic activity for mammals. The data received are extrapolated also in direction of absence of genotoxic potential for germinative cells.

Activity of the human carcinogens benzidine and 2-naphthylamine in triple- and single-dose mouse bone marrow micronucleus assays: results for a combined test protocol.

The activities of the human bladder carcinogens benzidine and 2-naphthylamine in the mouse bone marrow micronucleus assays using a limited test protocol (oral dosing to male mice, sampling 24 h later) have recently been established. As a contribution to the International Collaborative Study on the evaluation of the sensitivity of the triple-dose micronucleus test protocol it was decided to re-evaluate benzidine and 2-naphthylamine using a combined triple- and single-dose test protocol. Benzidine gave a clear positive response in male mice 24 h after 3 daily doses of 150 and 300 mg/kg. A single dose of 900 mg/kg of benzidine gave a weaker response 24 h after dosing. In the case of 2-naphthylamine a stronger positive response was observed 24 h after a single dose of 600 mg/kg as compared to 3 daily doses of 200 or 400 mg/kg. There was no significant difference in the increased positive response observed for a single dose of 30 mg/kg of cyclophosphamide compared with 3 successive daily doses of 10 mg/kg. Based on the present data the combined triple/single-dose micronucleus test protocol is strongly supported.

Activity of the human carcinogens benzidine and 2-naphthylamine in male mouse bone marrow micronucleus assays.

Conflicting reports exist in the literature regarding the activity of the human bladder carcinogens benzidine and 2-naphthylamine in the rodent bone marrow micronucleus assay. These chemicals have therefore been re-evaluated in mouse bone marrow micronucleus assays, and each gave a clear positive response in male mice 24 h after oral dosing of approximately 20% of the 4-day median lethal dose level. Experiments were repeated.

Species-specific response to the rodent carcinogens 1,2-dimethylhydrazine and 1,2-dibromo-3-chloropropane in rodent bone-marrow micronucleus assays.

1,2-Dimethylhydrazine is confirmed as active in the mouse bone-marrow micronucleus assay when administered as an aqueous solution via oral gavage to three different strains of mice. It is also shown to be inactive in a corresponding rat assay under similar conditions of test. The observations were independently repeated in two laboratories. In contrast, 1,2-dibromo-3-chloropropane showed the reverse profile, being active in the rat bone-marrow assay but inactive in the mouse; the latter observations were also made in two laboratories. The carcinogen procarbazine was active in both species. These findings are discussed within the context of the present GeneTox revision of the standard test protocol of the rodent bone-marrow micronucleus assay.

Relative distribution of mature erythrocytes, polychromatic erythrocytes (PE) and micronucleated PE on mouse bone-marrow smears: control observations.

Maps are presented showing the distribution of normal erythrocytes (NE), polychromatic erythrocytes (PE) and micronucleated PE (MPE) on 3 mouse bone-marrow smears. Adjacent areas of each slide were assessed along their whole length, and it was found that both the incidence of MPE and the ratio of PE:NE varied between different regions of the slide. These two variables were essentially independent of each other. The maps give the impression of clusters or islands of MPE on the slide, but these become less significant when the number of erythrocytes in those areas is taken into account. The present data confirm our previous conclusion that the resolving power of the bone-marrow micronucleus assay is related to the number of PE assessed for MPE per slide. Our data also illustrate that in instances where small departures from control values are observed, clarification/confirmation of the result can be obtained by extending the assessment of slides for MPE. It is proposed that the present recognition of the inherent sensitivity contained within this assay necessitates careful examination of the criteria for a positive response. The selection of PE suitable for assessment is discussed, and our recent 24-h oral corn oil control data are analysed.

The activity of MNNG in the mouse bone marrow micronucleus assay.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is established herein as reproducibly active in the mouse bone marrow micronucleus assay. Activity was evident in two strains of mice, using suspensions in water or corn oil, and using two routes of exposure (oral gavage and i.p. injection). Activity was greatest when using the i.p. injection route, but a weak positive response was evident following oral gavage dosing of MNNG as an homogenate in corn oil. The latter weak response was statistically significant and was confirmed by both extended assessment of the slides and by re-analysis of selected slides in an independent laboratory. However, only some of the animals responded, as observed by others earlier. It is concluded that several earlier literature reports of the inactivity of this agent in this assay must be due either to the hydrolysis of MNNG prior to dosing, or to inadequate methods of data acquisition.

Re-evaluation of 1,2-dimethylhydrazine in the mouse bone marrow micronucleus assay: observation of a positive response.

1,2-Dimethylhydrazine showed dose-related clastogenic activity in the mouse bone marrow micronucleus assay following intubation of an aqueous solution of the dihydrocholoride salt (10 and 50 mg/kg, 24 h sampling). These data counter earlier reports of the inactivity of this material in a rat and a mouse micronucleus assay, following its intraperitoneal injection. In the present study, 2,000 polychromatic erythrocytes were assessed per animal. However, had only 300 cells been assessed for micronucleated polychromatic erythrocytes, it is shown that a negative result could have been recorded. This may explain the earlier and negative results.

Re-evaluation of the need for multiple sampling times in the mouse bone marrow micronucleus assay: results for DMBA.

7,12-dimethylbenzanthracene (DMBA) is confirmed as active in the mouse bone marrow micronucleus assay 24 hr after dosing as a corn-oil homogenate via either oral gavage or intraperitoneal (ip) injection. These data are consistent with recent observations made by several investigators. However, when dosed via ip injection as a solution in DMSO, peak activity was evident 48 hr after dosing and a dramatic reduction in erythropoiesis was observed. The latter results are consistent with early observations made by Salamone and Heddle. The early observations of Salamone and Heddle were highlighted in the Gene-Tox review of this assay and led the OECD to recommend the use of 3 sampling times for this assay (between 12 and 72 hr). The present data indicate that this advice should be reviewed. In particular, it suggested that a maximum of two sampling times is adequate and that, as a consequence, the number of animals employed in the conduct of the test could be reduced with no loss of sensitivity. The present data also suggest that the use of a corn-oil homogenate of insoluble test agents may provide an efficient replacement for the use of ground suspensions or solutions in DMSO.

[Current methodologic approaches to the study of various delayed effects of pesticides]. / Suvremenni metodologichni podkhodi pri izuchavane niakoi otdalecheni efekti na pestitsidite.

Studies on the possibilities of origination of remote sequelae (embryotoxic and teratogenic, gonadotropic, mutagenic, carcinogenic, alterations in cardiovascular system, etc) under the effect of the wide application of pesticides in the practice is one of the most important aspects of the present-day toxicology. Numerous of our studies in that field are directed to the pesticides, to fungicides in particular, the greater part of them being with mechanisms of biological activity, triggering changes in DNA synthesis and disturbances in cellular respiration. The results from our experimental studies are presented in the communication, with an aspect of alterations in the gonads, embryotoxicity and teratogenicity, of representatives of various groups of chemical preparations for plant protection. The eventual use of those data in the prognosis of potential risk for humans is discussed. Model conditions are proposed for a more thorough assessment and more accurate determination of threshold levels of effect with a view to their utilization in hygienic standardization.

Prenatal toxicity of xylene.

Prenatal inhalation toxicity of xylene (industrial mixture of isomers) was studied in experiments of white Wistar rats exposed daily (6 hours a day, 5 days in a week) to concentrations of 10, 50 (MAC for xylene in the air of work environment in Bulgaria) and 500 mg.m-3 throughout the period of gestation from the first to the 21st day. Both routine teratological indices and biochemical and physiological methods of observation were used to evaluate the integrity of the individual organs - liver, brain, lungs and myocardium of the generation in the postnatal period of development. In concentrations of 50 and 500 mg.m-3, xylene exhibits pronounced embryotoxic and teratogenic effects. Postimplantation embryonal mortality increases, the process of physical development of the fetus is delayed, the incidence of induced anomalies of internal organs (hydrocephalus, microphthalmia, intracerebral haematomas, haemorrhages in the liver) is enhanced, the processes of ossification of the sternum and the skull are impaired. In concentrations of 50 and 500 mg.m-3, xylene causes disturbances in postnatal development of F1 generation. The concentration of 50 mg.m-3 is the threshold of the embryotropic effect of the solvent. Measures for the protection of women at work are proposed to reduce industrial hazard of developing antenatal pathology in the newborn of workwomen in xylene works.

[Evaluation of inhalation toxicity of organic solvent xylene in pregnant rats]. / Otseniavane inkhalatorna toksichnost na organichniia raztvoritel ksilol v opit vurkhu bremenni plukhove.

The objective of the present experimental study was the assessment of organism susceptibility to the toxic xylene effect during gestation period, with a view to elaborating recommendations for female work protection. The experiments were carried out in a comparative aspect in 60 pregnant and 60 virgin, sexually matured female albino rats, exposed to 1850 and 467.8 mg/m3 technical xylene in inhalation chambers--levels 40 and 10 times higher than MAC of the solvent, in the course of 21 days (period of gestation of rats), 5 hours daily, 5 times a week. The assessment of inhalation toxicity of xylene is based on the data from the complex biochemical investigations in serum, liver, brain, myocardium and aorta, supported by pathomorphological studies. Xylene disturbs the balance of serum lipid fractions with both levels of inhalation effect and has a manifested hepatotoxic and cardiovascular effect to pregnant animals. Less manifested disorders in serum lipid profile, liver and the brain of the female, non-pregnant rats, were established with the administration of high xylene concentrations only, suggesting the higher vulnerability of the pregnant organism to the toxic xylene effect. Recommendations for early labour readjustment of pregnant women engaged in xylene production are presented on the base of the data obtained about the disorders of vital functions of organism during gestation period, supported by epidemiological observations and the presence of experimentally established specific embryotoxic effect of xylene at MAC levels.

[Embryotropic action of the triazine herbicide polyzin 50]. / Vurkhu embriotropnoto deistvie na triazinoviia kherbotsid polizin 50.

The embryotropic effect of the new Bulgarian simtriazine herbicide polyzin 50 was studied in an experiment upon 90 pregnant albino rats - Wistar, via inhalation effect in concentrations 2 mg/m3 and 0.2 mg/m3, daily during the whole pregnancy and at a level of 2 mg/m3 during the first trimester of gestation. With both regimes, the preparation has a teratogenic and a more lightly manifested embryotoxic activity. In the absence of general toxic effect upon the pregnant organism, polyzin induces external malformations (hypognathus, oedema), anomalies in the development of the internal organs (hydrocephalus, intracerebral hematoma, hemorrhages in the abdominal cavity, infarctus-like foci in liver) and defects in the ossification of the cranial bones of fetus. The preparation has an embryotoxic activity (increased incidence of subcutaneous hematomas of the fetus), only with the daily penetration in organism of the pregnant animals in a concentration of 2 mg/m3. With both concentrations of antenatal effect of polyzin studied, a functional insufficiency of the liver of the progeny develops during the postnatal development period. On the grounds of the data obtained about the embryotoxic and teratogenic effect of polyzin with its inhalation effect in low concentrations - 2 mg/m3 and 0.2 mg/m3, a recommendation is proposed-prohibition of women's participation in its production at a fertile age.

Experimental studies on the chronic toxic effects of vinyl chloride in rats.

Male rats were exposed to vinyl chloride at the concentrations of 50, 500, and 20 000 ppm, 5 hours daily, 5 days a week for 10 months. Morphological lesions in the liver and the testes detected by light and electron microscope and depression in body weight increase intensified with the duration of exposure. Increased relative weights of some organs and slight hematological and biochemical changes in blood during the course of the experiment were also observed. Some toxic effects including morphological liver injuries arose at the smallest exposure level, i. e., 50 ppm. Assuming 50 ppm as the threshold concentration for rats, the 5 ppm level has been estimated as the safe exposure limit in industry in relation to systemic effects of vinyl chloride.