Early toxicity screening of new drugs is performed to select candidates for development. Many cell models are used to assess basic cytotoxicity and to show a good correlation with acute toxicity. However, their correlation with chronic in vivo exposure is inadequate. The new hepatoma cell line (HBG BC2) possesses the capacities of being reversibly differentiated in vitro, and of maintaining a relatively higher metabolic rate when in the differentiated phase (3 weeks) as compared to Hep G2 cells. MTT reduction was used to evaluate the toxicity of propranolol, perhexiline, aspirin and paracetamol, after both single and repeated treatments (three times a week for 2 weeks). Under conditions of repeated treatment, cytotoxicity was observed at lower doses when compared with single administration. Moreover, the first non-toxic doses were in the same range as plasma concentrations measured in humans during therapeutic use. Our results suggest that the new human hepatoma HBG BC2 cell line may be of interest for the evaluation of cell toxicity under repeated treatment conditions.

Many cell models that are used to assess basic cytotoxicity show a good correlation with acute toxicity. However, their correlation with the toxicity seen following chronic in vivo exposure is less evident. The new human hepatoma cell line HBG BC2 possesses the capacity of being reversibly differentiated in vitro and of maintaining a relatively higher metabolic rate when in the differentiated state (3 weeks) as compared to HepG2 cells, and thus may allow the conduct of repeated toxicity testing on cells in culture. In order to evaluate the genetic background of HBG BC2 cells, the expression of selected genes was analyzed in untreated cultures and, in addition, the behavior of HBG BC2 cultures under conditions of repeated treatment was studied with acetaminophen as a test substance and coupled with the use of standard staining techniques to demonstrate toxicity. Results showed that cultures of HBG BC2 cells retained a capacity to undergo apoptosis and proliferation, allowing probable replacement of damaged cells in the culture monolayer. MTT reduction was used to evaluate the toxicity of acetaminophen, acetylsalicylic acid, perhexiline, and propranolol, after both single and repeated (3 times/week for 2 weeks) administration. Under the conditions of repeated treatment, cytotoxicity was observed at lower doses as compared to single administration. In addition, the lowest nontoxic doses were in the same range as plasma concentrations measured in humans under therapeutic use. Our results suggest that the new human hepatoma HBG BC2 cell line is of interest for the evaluation of cell toxicity under conditions of repeated administration.

The cytochalasin B micronucleus test was performed in human peripheral lymphocyte cultures to assay the ability of hydroquinone and chloral hydrate to induce micronuclei, in the presence or absence of an exogenous metabolic activation system. Cultures and readings were performed in duplicate. No significant damage Was found after treatment with chloral hydrate in this test system, whereas hydroquinone induced a clear positive response in one culture in the presence of metabolic activation during the G1 phase. Isolated lymphocytes used as a test system provide information about the test compound itself without interference by blood components. Comparison of the two readers' data showed few marked discrepancies in the number of micronuclei recorded in binucleated cells. Strict criteria for data analysis are therefore necessary to avoid intra-assay or operator variability.

A multicentre validation study of the acute in vitro cytotoxicities of 31 liquid or solid chemicals was carried out by six laboratories, using primary rat hepatocyte cultures as a model system. We report here a comparison of neutral red uptake IC50 and LD50 values. Oral, i.p. and i.v. LD50 values were available for 27, 24 and 18 chemicals, respectively, and an IC50 value was obtained for 15, 14 and 11 of these compounds, respectively. A significant correlation was found only between IC50 and i.v. LD50 values.

A multicentre validation study of the acute in vitro cytotoxicities of 31 liquid or solid chemicals was carried out by six laboratories, using primary rat hepatocyte cultures as a model system. We report here a comparison of neutral red uptake IC50 and LD50 values. Oral, i.p. and i.v. LD50 values were available for 27, 24 and 18 chemicals, respectively, and an IC50 value was obtained for 15, 14 and 11 of these compounds, respectively. A significant correlation was found only between IC50 and i.v. LD50 values.

The in vitro micronucleus test was performed on isolated human lymphocytes using the cytokinesis-block technique with and without a rat liver metabolizing system. Positive control substances were used to evaluate this test: a direct agent (vincristine) requiring no metabolic activation, and three promutagens (cyclophosphamide, benzo[a]pyrene and dimethylbenz[a]anthracene). All of them, when compared with controls, caused a significant increase in micronucleus frequency, with a clear dose response. Five compounds were then tested in this in vitro micronucleus test: safrole, azathioprine, procarbazine, diethylstilbestrol and o-toluidine. The chemicals were examined with and without exogenous metabolic activation. Of these five compound, o-toluidine was found to be a marked direct genotoxic agent and azathioprine gave positive results with or without metabolic activation (a better response was noted without the addition of S9 mix). Diethylstilbestrol gave conflicting results and was considered inconclusive. Two chemicals, safrole and procarbazine, were found to be non-genotoxic in this test system, whatever the protocol used.

The cytotoxicity of 30 chemicals was assessed in rat hepatocyte primary cultures using four methods: lactate dehydrogenase release, neutral red uptake, the MTT assay, and measurement of total protein content. Comparison of the data obtained in vitro (IC50 values) and in vivo (LD50 values) resulted in a significant correlation (p<0.001) between IC50 values and intravenous LD50 values. The validity, as well as the predictability of the model, were determined by multivariate analysis (principal component analysis and correspondence analysis). The predictability area, expressed in IC50 values, was in the range of 0–l,500μg/ml and reached 95%, with a 75–100% confidence interval (p = 0.05). Assessment of the cytotoxicity of 54 additional chemicals would provide a more accurate predictability limit around l,500μg/ml and the estimated predictability confidence interval could be reduced to 90–100%.

4 presumptive clastogens (caffeine, dimethyl sulfoxide, diethylstilbestrol and p,p′-DDE) and 4 chemicals thought to induce chromosomal mutations in L5178Y mouse lymphoma cells (procarbazine · HCl, epichlorohydrin, hycanthone and iodomethane) were tested in the CHO/HGPRT gene mutation assay for the induction of 6-thioguanine-resistant (6TGR) mutants. Of the clastogens, p,p′-DDE was mutagenic at several concentrations and diethylstilbestrol (DES) increased the 6TGR mutant frequency over control levels at the 6.7 and 8.0 μg/ml doses, but the results for DES were neither convincing nor significant. Caffeine was not mutagenic although at very high concentrations (6667–8000 μg/ml) there was a slight elevation in mutant frequency over background. This was probably due to a selective effect of caffeine against the HGPRT+ phenotype, for 2 different HGPRT− cell lines were refractory to the toxic effects of caffeine at the highest test level (8000 μg/ml). All 4 ‘chromosomal mutagens’ produced dose-related increases in mutant frequencies at the HGPRT locus of these CHO cells. 6TGR colonies were generally uniform in size when normal precautions were taken to prevent the formation of satellite (secondary) colonies. Excepting DES, dimethyl sulfoxide, and caffeine, these data demonstrate that 5 of 8 clastogenic chemicals reproducibly induce mutations at the HGPRT locus of CHO cells which lack the small colony-forming potential of 3.7.2C L5178Y cells.

A multicentre validation study of the acute in vitro cytotoxicity of drugs involving six French laboratories from INSERM or pharmaceutical companies has been carried out. Thirty liquid or solid chemicals such as antibiotics, anticancer drugs and solvents were selected and incubated for 20 hr with normal rat hepatocytes and FaO hepatoma cells. Miniaturized and automated methods were defined for the evaluation of cytotoxic effects. Four endpoints were evaluated: the ratio of extracellular lactate dehydrogenase to total lactate dehydrogenase, total cellular protein content, reduction of a tetrazolium salt, and neutral red uptake. For each test IC50 values were calculated. A good interlaboratory reproducibility was demonstrated. The neutral red assay was found to be the most sensitive and the least reproducible endpoint. More compounds were shown to be cytotoxic to hepatocytes than to hepatoma cells (18 v. 12). On the basis of the IC50 values a few compounds were found to be much less cytotoxic than predicted from in vivo data, suggesting that a simple experimental protocol and non-specific cytotoxicity parameters are not sufficient to test certain drug families. However, such methods appear to provide a useful means of defining the concentration range of the drug that will be selected for further analysis using more specific tests.

Primary cultures of rat and human hepatocytes were used in our in vitro studies for investigating species differences in the response to a peroxisome proliferating benzofuran derivative, benzbromarone. Cyanide-insensitive palmitoyl coenzyme A oxidation (a marker of peroxisome fatty acid beta-oxidation) and electron microscopy were used to assess peroxisome proliferation. Hepatocytes were cultured essentially as described by Mitchell et al. (1984, Arch. Toxicol. 55, 239-246); clofibric acid and mono(2-ethylhexyl) phthalate (MEHP) were used as reference compounds, as they are well known to cause peroxisome proliferation in rat hepatocytes in primary culture. The benzofuran derivative, tested at drug concentrations ranging from 2.37 to 59.20 microM in rat hepatocyte primary cultures, induced, after 96 hr, a dose-related increase of the peroxisomal beta-oxidase activity correlated with an increased number of peroxisomes; this increase was much less marked than that obtained with clofibric acid or MEHP. By contrast, using the same range of concentrations, human hepatocytes in primary culture treated with benzbromarone revealed no enhancement of enzymatic activity and no concomitant statistically significant increase in the number of peroxisomes; the same observations were reported with clofibric acid and MEHP. These results demonstrate clearly that species differences in sensitivity to peroxisome proliferation with the benzofuran derivative do exist.

Recently, much attention has been given to the photogenotoxicity of bergamot oil with regard to its potential carcinogenic risk (Zajdela and Bisagni 1981); Cartwright and Walter 1983). Bergamot oil is widely used as a component in cosmetic formulae (sunscreen preparations, perfumes, eaux de toilette). Among its furocoumarin components, 5-methoxypsoralen (5-MOP) is the major photoactive agent (Ashwood-Smith et al. 1980). DNA photoreactivity of 5-MOP has been described in several in vitro biological systems, mainly yeasts (Averbeck et al. 1978) and bacterial and mammalian cell systems (Ashwood-Smith et al. 1982).