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20702232

Studies on variability of the micromass teratogen test.

The micromass assay is an in vitro test that can be used to predict the teratogenic potential pounds being developed in the pharmaceutical and chemical industries. The assay is currently undergoing validation by an interlaboratory blind trial at several national and international institutions. The present paper investigates some areas of the assay system where variability in technique may influence the reproducibility of the test results. The main areas identified are possible variations in the micromass preparation procedure (for example in terms of the precise source of tissue and the time taken), degrees of subjectivity in the endpoint measurement and possible variations in the experiment design. Control of these sources of variability increases the reproducibility of the results.

20702234

Seminal plasma superoxide dismutase activity following radiotherapy: Protection by the exogenous enzyme and effects of intracellular inhibition.

For referred subfertile, radiation-exposed and known fertile males seminal plasma superoxide dismutase activity did not correlate with sperm density, % sperm motility, sperm velocity or penetration in the sperm penetration assay (SPA), but was found to correlate positively with seminal plasma zinc. A study of seminal plasma superoxide dismutase in relation to post-irradiation clinical treatment time provided tentative evidence that following a radiation challenge, superoxide dismutase might be subject to induction. When sperm intracellular superoxide dismutase was inhibited by incubation with diethyldithiocarbamate (DDC) in defined culture medium, motility ceased within 30 min but there was no associated rise in lipid peroxidation. Sperm pretreatment with DDC at concentrations as low as 10 mum could abolish penetration in the SPA provided sperm were prepared by passive incubation. Cells electropermeabilized in the presence of calcium could e the DDC block. Exogenous addition of superoxide dismutase and catalase could provide effective protection to sperm against the effects of ultraviolet treatment.

20702235

The potential usefulness of a differentiating teratocarcinoma cell line in in vitro toxicity testing.

A number of in vitro systems have been put forward as potential alternative methods for testing chemicals for teratogenic potential. The most promising of these systems, for example mammalian whole embryo culture and the micromass technique, are currently undergoing further interlaboratory validation. However, such tests involve the use of a considerable number of animals. It was therefore decided to investigate the possible use of a permanent cell line that possessed many of the properties of embryonic cells, that is a differentiating cell line, F9 (derived from a mouse teratocarcinoma), in the development of an in vitro teratogenicity test. In a preliminary study, six chemicals were tested for their modulating effects on differentiation in undifferentiated, differentiating and differentiated F9 cells. These effects were assessed morphologically and by measuring the production of laminin (a biochemial marker of F9 differentiation). The use of the F9 cell line in in vitro teratogenicity testing shows promise, but further work is necessary before its potential can be fully evaluated.

20702236

Changes in visceral yolk sac ultrastructure after exposure of rat embryos to selected teratogens in vitro.

It has been determined that a number of teratogens alter the osmotic environment around the rat embryo, an effect that is associated with abnormal fluid accumulation (and ultimately abnormality) in the embryo. At least one of these teratogens, trypan blue, changes lysosomal structure in the visceral yolk sac (VYS), an extra-embryonic membrane that envelops the extra-embryonic partment. The osmotic and ultrastructural effects parable in the in vivo and in vitro rat embryo. In the present study, the effects of other osmotic teratogens on VYS ultrastructure were investigated in rat whole embryo culture. Leupeptin (10 mug/ml) and E-64 (10 mug/ml) both caused a marked increase in the size of VYS lysosomes. Both chemicals inhibit cysteine proteinases, which are abundant in lysosomes. Suramin (750 mug/ml), an inhibitor of a number of lysosomal hydrolases, caused vacuolization of large areas of VYS cells. Ethylenethiourea (120 mug/ml) produced no marked ultrastructural changes, although the endocytotic apparatus of VYS cells appeared to have increased electron density, an effect that was also observed after treatment with the other teratogens. These results indicate that teratogens which alter embryonic osmotic balance also affect structures involved in endocytosis or lysosomal degradation of material by VYS cells.

20702237

Bovine serum: An alternative to rat serum as a culture medium for the rat whole embryo culture.

The results reported here demonstrate the ability of supplemented bovine serum to serve as a culture medium for rat whole embryos. After 48 hours' culture in bovine serum supplemented with Tyrode's buffer and methionine, 9.5-day-old rat embryos were at a stage of parable with that of embryos cultivated in homologous serum, although some deficiency in the formation of haemoglobin in embryonic blood cells could be observed. However, supplementation of the culture medium with haemoglobin overcame this deficiency. The procedure used for preparing the culture medium is described in detail and some advantages of bovine serum are discussed.

20702238

Embryonic micromass limb bud and midbrain cultures: Different cell cycle kinetics during differentiation in vitro.

Rat embryo micromass limb bud (LB) and midbrain (CNS) cultures have been proposed as a screening system for teratogenic potential as well as a model system for differentiation. To further characterize these in vitro differentiating cultures, their population kinetics have been examined. Cellular proliferation, cell cycle kinetics and cell differentiation were monitored at days 1, 2 and 5 in culture. For cell cycle analysis, cellular DNA was stained with 4,6-diamidino-2-phenyl indole and nuclei were analysed by flow cytometry. Cell viability was assessed using trypan blue and differentiation was monitored using haematoxylin (CNS) or alcian blue (LB) stain. CNS cultures underwent 2-3 cell doublings during the 5 days of culture. There was a gradual accumulation of cells in the partment (54% +/- 10%, day 1 to 78% +/- 2%, day 5) (P < 0.05, chi(2) test). This accumulation appeared to occur at the expense of the S and G(2) + partments since both decreased over this same period of time. LB cultures underwent 1-2 cell doublings during the same culture period. In contrast to the CNS cultures, the limb bud cultures exhibited only minimal changes in the size and distribution of the cell partments during the 5 days in culture. Characterization of the cellular kinetics of these two widely used embryo cell culture systems should help to delineate potential sites and mechanisms of developmental toxicity.

20702239

In vitro micromass teratogen test: Interpretation of results from a blind trial of 25 compounds using three separate criteria.

The results are presented on an interlaboratory study to validate the Micromass assay by pounds under code using a similar protocol, with an assessment of results for pounds tested without S-9 mix. Four of these were co-tested with S-9 mix. Three separate sets of criteria have been proposed (Flint, 1986 and 1987; Flint and Orton, 1984) for interpreting the results for teratogenic hazard from in vitro data using IC(50) values: (i) the '< 500 mug/ml rule', (ii) the '< 50 mug/ml rule' and (iii) the 'specific inhibition of cell differentiation 2-fold rule'. The data were decoded and assessed using the criteria for their sensitivity, specificity and accuracy. Using the '< 500 mug/ml rule' a higher sensitivity was obtained but at the expense of a high false positive frequency (50%). Conversely, the '2-fold rule' gave a high specificity with only 10% false positives. Diphenhydramine and furazolidone gave false positive responses; the teratogens beta-aminopropionitrile and methotrexate were not detected. Selective inhibition of cell differentiation was related to high potential teratogenicity. In the application of the test, it is suggested that if there was an indication of teratogenic hazard using the '2-fold rule' pound should be rejected without recourse to animal testing. In its present form the assay cannot be used to unequivocally identify non-teratogens.

20702242

Rat post-implantation culture: Production of 5-8-somite embryos by a time-controlled mating procedure.

The use of in vitro studies allows a reduction in the number of test animals needed to perform a study. In 24-hr post-implantation embryo culture (from day 10 to day 11 post-insemination) the 5-8 somite stage is considered to be the best starting point. A time-controlled mating procedure has been developed using a positive lordosis reaction and a short period for mating to synchronize pregnancy in rats. With this procedure, 243 hr post-mating 97% of embryos are in a stage of development ranging between 5 and 8 somites. The pregnancy rate (96%) and embryo viability rate (93.4%) are excellent. In these conditions only two or three females are needed to obtain 20 embryos at the required somitic stage. Compared monly used mating procedures, this technique reduces considerably the number of females needed for each experiment and workload involved. Furthermore, it allows better planning of whole embryo culture studies and can be easily adapted to obtain embryos at any somitic stage.

20702243

Data on development of post-implantation rat embryos in a 24-hr culture.

Cumulative data on growth, differentiation and development of control rat embryos over a 24-hr culture period (day 10-11 post-insemination) are presented. Cultures were performed in pure rat serum supplemented with 0.5% gelatin, with embryos at 5-8 somites at the start of the culture, according to New's technique. The embryos were evaluated according to the morphological scoring system of Brown and Fabro. Results are given, showing the repartition for each final somitic age, for the following parameters: yolk sac diameter, crown-rump length, head length, total score of development, and scores of development of 16 different territories. Viability of embryos, and the number and type of anomalies are also presented. Data were collected on 144 control embryos.

20702244

The micromass test: Is it subject to strain variation?

Strain difference in the teratogenic response in vivo is an important consideration in the design and interpretation of regulatory studies. The importance of strain difference in vitro has been examined using all trans-retinoic acid. Effects on development of the forelimb in vivo and on differentiation and survival of embryonic limb bud and midbrain cells in vitro pared for three strains of rat, Allen & Hanbury Albino (AHA), Random Hooded (RH) and Alderley Park (AP). Transformed dose-response curves for the incidence of shortened forelimb in vivo were parallel. Comparison of ED(50) values (the concentration at which 50% of embryos respond with the index abnormality) allowed the strains to be ranked in order of susceptibility in vivo: AHA > RH = AP (ED(50) values 93, 124 and 123 mg/kg, respectively). In vitro, there was little difference in cytotoxicity or in effects on midbrain cell differentiation between strains. However, marked differences for effects on limb bud cell differentiation allowed the three strains to be ranked as follows: AHA > RH > AP (IC(50) values 0.008, 0.09 and 0.25 mug/ml, respectively). These findings suggest that for retinoic acid, the micromass test is subject to strain variation, although this had no effect on the prediction of teratogenic potential. Furthermore, the order of susceptibility of the three strains examined was the same in vivo and in vitro. Thus, target tissue sensitivity may play an important part in strain variation in the teratogenic response, although differences in vitro were much more marked than those seen in vivo.

20702246

In vitro transformation of human skin epithelial cells: Role of RAS oncogene in malignant progression.

Immortalized human skin epithelial cell lines provide useful models to study progressive stages in human carcinogenesis. Alterations have been examined occurring with immortalization and malignant progression of the human keratinocyte cell line HaCaT, which developed spontaneously in a long-term culture from trunk skin keratinocytes. The cell line has maintained many features of epidermal growth and differentiation in vitro and has acquired clonogenicity. HaCaT cells exhibited a transformed phenotype (aneuploidy and clonogenicity in soft agar) but remained non-tumorigenic. On transplantation they formed normally structured and differentiating epithelia, but did not grow invasively. Following transfection with the c-Ha-ras oncogene (EJ) randomly selected (G418-resistant) clones exhibited different stages of tumour progression. They formed either (1) rapidly regressing cysts, as seen with the parental line, (2) slowly growing benign tumours or (3) progressively enlarging well differentiated carcinomas. Tumorigenic (benign and malignant) clones had higher levels of mRNA expression and produced mutated p21. However, no correlation existed between both parameters and malignant growth. Ras-transfected clones showed improved morphological differentiation in vitro, in transplants, and in tumours and expressed differentiation-specific keratins. Tumorigenic HaCaT-ras clones were clonogenic in serum-free medium but had lost their ability to grow in soft agar. Thus, c-Ha-ras oncogene expression initiated tumour progression in immortalized human keratinocytes by altering growth regulation in vitro and in vivo, but per se was insufficient for malignant transformation.

20702245

In vitro micromass teratogen test: Results from a blind trial of 25 compounds.

The results obtained by Huntingdon Research Centre participating in a blind trial of the micromass assay for the prediction of teratogenic potential are presented. Twenty-five pounds were tested without S-9 mix using a pre-agreed protocol; pounds were later tested with S-9. The data were assessed for sensitivity, specificity and accuracy using three separate sets of criteria based on either concentration (the <500 mug/ml rule (i) and the <50 mug/ml rule (ii)) or specific inhibition of cell differentiation at relatively non-cytotoxic concentrations (the 2-fold rule (iii)). The best in vivo/in vitro correlation was obtained using the 2-fold rule; the <500 mug/ml rule was the most sensitive but gave a high false positive rate and the <50 mug/ml rule was of low overall accuracy (60%). It is suggested that selective inhibition of differentiation of one cell type and cytotoxicity at low dose levels may also indicate risk of embryo-foeto toxicity, a factor to be considered with the pharmacokinetics of pound. The teratogens procarbazine, methotrexate and caffeine were not detected; diphenhydramine and furazolidone initially classified as non-teratogens in vivo, were predicted as teratogens by the micromass assay.

20702249

Studies of human bronchial epithelium in vitro: Changes of [Ca(2+)](i) in relation to injury, growth and differentiation.

The objectives of the present work included the study of the response of normal human bronchial epithelial (NHBE) cells to injury following treatment with several agents, especially those associated with tumour promotion. The effects of putative tumour promoters including 12-O-tetradecanoylphorbol acetate (TPA), a series of aldehydes, and H(2)O(2) on the regulation of cytosolic ionized calcium ([Ca(2+)](i)) pared with the effects of growth and squamous differentiation stimuli, including serum and transforming growth factor-beta. The cells studied included primary cultures of NHBE cells and the BEAS-2B cell line, created by transformation of NHBE cells with the adenovirus 12-Simian virus 40 hybrid. The results indicate that, with several stimuli, the onset of terminal squamous differentiation is preceded by a rise in [Ca(2+)](i). The results are interpreted in terms of a general hypothesis regarding the interaction between acute and chronic cell injury.

20702250

Induction of sister chromatid exchanges in NIH 3T3 cells transformed by DNA from mice given cyclophosphamide.

The rationale behind this experiment was to investigate the role of sister chromatid exchange (SCE) in the transformation process in vitro. High molecular weight DNA was extracted from bone marrow cells of mice given 20 mg cyclophosphamide/kg body weight. 30 mug of this DNA was used to transfect NIH 3T3 in Eagle's medium seeded at 0.7 x 10(6) cells/plate. Morphologically visible foci were picked up after 16-21 days. The foci were trypsinized, washed and allowed to grow in the presence of bromodeoxyuridine (25 mum) for 72 hr in the dark. Analysis of SCE per chromosome indicated a 20-fold increase in the frequency of exchanges pared with the induction seen in negative controls. These data suggest that the transformation process of NIH 3T3 cells by DNA from mice treated with cyclophosphamide is associated with an increased induction of SCEs.

20702251

Transformation of ras transfected BALB 3T3 clone (Bhas 42) by promoters: Application for screening and specificity of promoters.

BALB 3T3 cells transfected by v-Ha-ras (Bhas 42 clone) were found to be sensitive to contact inhibition, but were susceptible to drastic transformation by 12-O-tetradecanoylphorbol-13-acetate. These results indicate that Bhas 42 cells are the initiated cells in the two-stage transformation process. By plating Bhas 42 cells together with BALB 3T3 cells followed by treatment with known promoters, a transformation assay system was established for the detection of tumour promoters. The Bhas 42 system showed advantages over conventional chemically induced transformation assays at several points, but some promoters did not induce transformation of Bhas 42 cells.

20702252

DNA-binding studies with allylchloride and allylbromide using the isolated perfused rat liver technique.

The isolated perfused liver technique has been adapted for DNA-binding studies of pounds without the need for expensive radioactive pounds plex and protracted methods such as that including monoclonal antibodies. The technique was found to be easy to perform and gave reproducible results. The portioned administration of the pounds (200-600 mg) was less damaging to the liver than the addition in a single dose. The liver functions could be held at nearly physiological values over a period of 10 hr. Three allylguanine adducts (O(6)-allylguanine, N(2)-allylguanine, 7-allylguanine) and two adenine adducts (N(6)-allyladenine and 3-allyladenine) could be detected in the hydrolysed DNA obtained from isolated perfused livers. These adducts demonstrate the modifications of DNA in mammalian liver and that a potential cancer-initiating effect must be considered with pounds that possess alkylating activities.

20702254

Acceptance of in vitro testing by regulatory authorities.

National regulatory authorities have responsibility for taking decisions that possibly affect the health of whole populations and it is therefore to be expected that they will be reluctant to substitute alternative in vitro toxicity test methods for conventional animal studies unless the new procedures have been demonstrated to be reliable and have been adequately validated. Validation of in vitro methods presents particularly difficult problems because whilst they tend to produce consistent and objective results, the test systems used are incapable of mirroring plexity of the biochemical processes seen in animals. As a result, a single animal study would need a large battery of in vitro studies to replace it to cover the various endpoints that are involved in the in vivo study; each of these in vitro tests would need to be validated with regard to the specific endpoint that it is investigating. Although great advances have been made in recent years in the development of alternative methods, few have been validated to an extent that makes them acceptable to regulatory authorities as replacements for in vivo studies. Rather, they are largely seen and used as screening techniques whereby decisions can be taken on the value of further development of newly pounds, and as aids in the interpretation of animal studies and in their extrapolation to man, that is, they are of value in 'mechanism of action' studies. Nevertheless, certain in vitro procedures are already accepted by regulatory authorities and their use, for example, in 'screening pounds that have severe irritant properties, and in pounds with potential mutagenic and carcinogenic activity, has had a profound effect on both the number of animal studies carried out and on the welfare of those animals still used.

20702253

Strategic considerations in industry's use of in vitro toxicology.

Industrial concerns have a legal and ethical responsibility to provide information that allows products to be used safely. Current toxicological practice and legal requirements rely on animal experiments to provide much of this information on safety. Nevertheless, there is a clear role for in vitro methods in the overall development and testing of chemicals. During the early stages of development of a new chemical, in vitro tests provide information used in the selection of appropriate candidates from among the many that may be available. Such use of in vitro tests for screening of chemicals must be preceded by adequate validation. The interpretation of results of screening chemicals requires a detailed knowledge of the sensitivity and specificity of the test and of the structures of the chemicals under test. Once in vivo toxicological data are available, in vitro tests may have a key role in providing an understanding of species differences in toxic responses. Examples of the use of in vitro techniques to improve the specificity of animal studies are given and these include studies of trichloroethylene, di(2-ethylhexyl) phthalate, hexachlorobutadiene and a substituted triazole. In the overall process of risk assessment, extrapolation of animal data to the assessment of human hazard is a prerequisite. In vitro techniques can be used to assess species differences in transdermal absorption. They may also help in assessing quantitative differences in metabolic conversion between species. Examples of the use of in vitro techniques to improve the sensitivity of animal studies include the study of the toxicity of methylene chloride. In vitro techniques have developed rapidly over the last decade. Nevertheless, there is only one category of testing within regulatory guidelines that specifies in vitro methods, namely mutagenicity. At the moment, in vivo methods are considered to provide the best general information for risk assessment, with in vitro methods contributing as screening techniques and as adjuncts to improve the sensitivity and specificity of animal studies.

20702255

Computer modelling and in vitro tests in the safety evaluation of chemicals-Strategic applications.

The cytochromes P-450 detoxicate most chemicals, but activate carcinogens and other toxic chemicals by oxygenating them to reactive intermediates. A new strategy based on the P-450s has resulted in the development of puter program (COMPACT) and of a programme of enzyme induction studies, as short-term tests to predict the potential toxicity/carcinogenicity of chemicals. Most carcinogens are metabolically activated by P450 I (P-448); P450 IIE and P450 IV also mediate chemical toxicity by producing oxygen radicals. P450 IIB mostly results in detoxication, but where chemicals are oxygenated with difficulty (e.g. phenobarbitone), the cytochrome is induced and futile cycling and oxygen radical formation may result. Oxygen radicals are highly toxic, mutagenic and carcinogenic and mediate much chemical toxicity in small rodents. As P450 genes of man differ from those of rodents, the latter have limited value as surrogates for man in chemical safety evaluation; many short-term tests, such as the Ames test, although valuable in detecting genotoxic chemicals, are similarly limited by their rat liver microsomal activation (S-9) systems. COMPACT is able to e both of these limitations, requires no laboratory animals or tissues, and can be conducted from a knowledge of the structure of the chemical, even prior to its synthesis. As the rodent tumorigenicity assay and the Ames test are currently regarded as the standard procedures for safety evaluation, COMPACT was validated against these in a study of 100 miscellaneous chemicals and showed excellent correlations. A quantitative structure-activity relationship (QSAR) for a series of 14 methyl benzanthracenes shows good correlation of mutagenicity with the energy of the lowest empty molecular orbital [E(LEMO)] values of the chemicals. A strategy for COMPACT and P-450 induction studies in chemical safety evaluation is presented.

20702256

A quantitative structure-activity/dose relationship for contact allergenic potential of alkyl group transfer agents.

Since no in vitro model is available, or indeed likely, to predict or investigate skin sensitization potential of substances, an approach based on a model using physicochemical criteria is the most likely route to a reduced requirement for animal testing. As part of the investigation of structure-activity relationships in contact allergy, it was shown that methyl transfer agents are capable of acting as skin sensitizers. This work has now been extended to a more general examination of alkyl transfer reactions. In vivo dose responses to challenge and the patterns of cross reactivity between C(12), C(16), unsaturated C(16) and methyl transfer agents were examined to form the basis of the mathematical model. All alkyl transfer agents examined were potent sensitizers, with evidence of mutual cross reactivity between them. The sensitization data have been accurately modelled using a mathematical equation. Analysis in terms of a modified relative alkylation index model showed evidence of an overload effect.

20702257

Preliminary results from the Scandinavian multicentre evaluation of in vitro cytotoxicity (MEIC).

The multicentre evaluation study of in vitro cytotoxicity tests (MEIC) is organized by the Scandinavian Society of Cell Toxicology. All interested laboratories are invited to test a published list of 50 reference chemicals in their various in vitro assays with a bearing on general toxicity. Submitted results will be centrally evaluated for their relevance to human toxicity, including parison with the efficiency of conventional animal tests. This munication presents the very first preliminary results of the study, that is, prediction of human acute lethal toxicity for the first 10 MEIC chemicals by all the results submitted to date, that is, five in vitro cytotoxicity assays. As a baseline for judging the efficiency of the cytotoxicity tests, rat and mouse LD(50) values pared with human acute lethal dosage of the chemicals. Rat LD(50) prediction was relatively poor, but mouse LD(50) values correctly predicted the human lethal dose for six out of the 10 substances. A multivariate method parison including all cytotoxicity test results, predicted human lethal blood concentrations as well as the mouse LD(50) prediction of dosage. Since the blood concentrations used in parison were derived from human lethal dosage with the help of two simple pharmacokinetic factors (absorbed fraction in the intestine and distribution volume of chemicals), the cytotoxicity assays were found also to be able to predict human dosage, as well as did the mouse LD(50) prediction.

20702258

Validation of alternative toxicity tests: Principles, practices and cases.

In vitro toxicity tests must be properly developed and scientifically validated for relevance and reliability before they are independently evaluated for possible inclusion in toxicity testing schemes and promoted for regulatory and legal acceptance. Some lessons learned in the administration of the FRAME Alternative Test Validation Scheme are reported, using as an example correlations between the results obtained in cell growth inhibition tests, both with each other and with rat oral and mouse intraperitoneal LD(50) values. Some mendations are given for consideration for the design of future validation schemes.

20702260

Comparison of two in vitro and two in vivo methods for the measurement of irritancy.

A number of in vitro assays have recently been developed that need further validation in order to judge their value for local tolerance testing. For this purpose, results from the following experiments pared: (A) cytotoxicity testing in cell culture (neutral red assay); (B) tests with the chorioallantoic membrane of fertilized chicken eggs; (C) rabbit eye mucous membrane tests; and (D) occluded epicutaneous testing in human volunteers. The data presented indicate that in vitro testing with the first two methods yields reliable results with respect to the eye and human skin irritation data within homologous substance classes. A test procedure is proposed that parative testing of the chemicals with unknown irritant properties together with known weak and strong irritants from the same class of chemicals as standards in the test series. This procedure seems to be suitable as a preliminary screen for identifying severe irritants prior to the performance of any in vivo studies. It would reduce the number of animals to be used in vivo and lead to the avoidance of exposure of animals to harmful substances.