Determination of genetic toxicity and potential carcinogenicity in vitro--challenges post the Seventh Amendment to the European Cosmetics Directive.

Genetic toxicology and its role in the detection of carcinogens is currently undergoing a period of reappraisal. There is an increasing interest in developing alternatives to animal testing and the three R's of reduction, refinement and replacement are the basis for EU and national animal protection laws the Seventh Amendment to the EU Cosmetics Directive will ban the marketing of cosmetic/personal care products that contain ingredients that have been tested in animal models. Thus in vivo tests such as the bone marrow micronucleus test, which has a key role in current testing strategies for genotoxicity, will not be available for this class of products. The attrition rate for new, valuable and safe chemicals tested in an in vitro-only testing battery, using the in vitro tests currently established for genotoxicity screening, will greatly increase once this legislation is in place. In addition there has been an explosion of knowledge concerning the cellular and molecular events leading to carcinogenesis. This knowledge has not yet been fully factored into screening chemicals for properties that are not directly linked to mutation induction. Thus there is a pressing need for new, more accurate approaches to determine genotoxicity and carcinogenicity. However, a considerable challenge is presented for these new approaches to be universally accepted and new tests sufficiently validated by March 2009 when the animal testing and marketing bans associated with the Seventh Amendment are due to come into force. This commentary brings together ideas and approaches from several international workshops and meetings to consider these issues.

Survey of junior hospital doctors' attitudes to cardiopulmonary resuscitation.

Most cardiac arrest teams are made up of junior doctors. The stressful effect of cardiopulmonary resuscitation (CPR) on doctors has not previously been established. A questionnaire was sent to all 52 junior doctors who participated in the cardiac arrest team at a district general hospital. Forty one questionnaires were returned by 22 junior house officers, 12 senior house officers, and seven specialist registrars. The questionnaire was anonymous so non-responders could not be recontacted. Seventy three per cent found CPR stressful. The main reason for stress was the inappropriateness of CPR on the individual patient (12), poor outcome (13), no advanced life support (ALS) course (4), and the procedure itself (4). Fifty four per cent felt the number of inappropriate CPR had increased in the last six months with the main reason given (48%) being failure of senior staff to make "do not resuscitate" orders. Ninety seven per cent felt some CPRs were inappropriate; 70% felt a debriefing session should occur after CPR, while 88% reported not having one. Seventy six per cent felt competent at performing CPR, 22% felt incompetent of whom none had undergone ALS training. Fifty eight per cent found it difficult to discuss CPR with patients; 46% found it difficult to discuss CPR with relatives. Most junior doctors feel stress from CPR. Adequate review by senior doctors with documentation of do not resuscitate orders where appropriate, after discussion with patients, might be beneficial. Adequate training, improving communication skills, and support for junior doctors in the cardiac arrest team need to be reviewed since improvement in these areas may reduce stress.

Cryopreservation of rat hepatocyte monolayers: cell viability and cytochrome P450 content in post-thaw cultures.

Cryopreservation of primary hepatocyte monolayers may provide a means of long-term storage of the cells for in vitro studies of xenobiotic metabolism and toxicity. Rat hepatocytes can be stored at -70 degrees C as simple monolayers attached to collagen-coated dishes, and post-thaw cultures can be continued for up to 72 h. Throughout this post-thaw period viability of the cells was demonstrated by retention of intracellular fluorescence after exposure to carboxyfluorescein diacetate (CFDA) and examination by confocal laser scanning microscopy (CLSM). CLSM images revealed an uneven distribution of CFDA-derived fluorescence within hepatocytes post-thaw, particularly in Williams' E medium, indicating generation and retention of carboxyfluorescein within the intracellular organelles. The membranes of the intracellular organelles appear to be less sensitive to freeze/thaw damage than the cell membrane. Viability was not compromised with storage for up to 28 days at -70 degrees C. Cytochrome P450 content was retained in post-thaw culture to a similar extent as in non-frozen cultures. Cryopreserved rat hepatocyte monolayers may provide a useful in vitro model for studying xenobiotic metabolism and toxicity.

Development of a high throughput in vitro toxicity screen predictive of high acute in vivo toxic potential.

At an early stage of drug discovery high throughput screens are an invaluable tool to de-select compounds with undesirable properties. A high throughout in vitro toxicity screen has been developed and validated to identify compounds that have a high potential to be acutely toxic in vivo. This screen is based on treating Chinese hamster ovary (CHO) cells with test compounds for 24 h and then determining the degree of cytotoxicity by the reduction of Resazurin. Twenty-six structurally unrelated compounds were chosen that spanned a range of acute LD(50) values and mechanisms of toxicity. The acute LD(50) values (intraperitoneal and intravenous routes) from rat and mouse were taken from the RTECS database. Experimentally derived in vitro IC(35) results were compared to the 'most toxic' (lowest) LD(50) values for each compound. The resulting correlation was statistically significant (r=0.8475). However, due to the scatter of the data points, it was considered not appropriate to rank compounds according to their degree of in vivo toxicity on the basis of the in vitro result. However, by defining cut-off concentrations for both the in vivo (LD(50)) and the in vitro (IC(35)) values it was possible, using the in vitro result (IC(35) <10 microM), to identify compounds that had a high potential to be acutely toxic in vivo ('most toxic' LD(50) <25 micromol/kg). Further development led to a high throughput screen capable of giving a 'Yes', 'No' or 'Borderline' classification as to whether a compound has a high acute in vivo toxic potential. This screen is highly specific (no false positive classifications) and has a sensitivity of approximately 80%. This is deemed acceptable for a first tier toxicity screen at an early stage in the drug discovery process. Transfer of this screen from GlaxoSmithKline UK to sites in Italy, Spain and the USA resulted in very similar findings indicating the inter-laboratory robustness of this screen and therefore the ability to compare results across the GlaxoSmithKline sites.

The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures.

It is important to assess the usefulness of long-term in vitro liver models for studying chronic toxicity, since acute assays may not reflect the in vivo situation. A potential long-term hepatocyte culture (i.e. liver spheroids) was investigated and compared to primary rat hepatocyte monolayer cultures following exposure to methotrexate (MTX), a well-documented chronic hepatotoxin. Following up to 7 days' treatment with MTX, cultures were morphologically assessed and assayed for enzyme leakage, intracellular reduced glutathione (GSH) and adenosine triphosphate (ATP). Spheroids maintained higher concentrations of GSH over the 14-day culture and ATP was maintained, but at a concentration not significantly different from monolayer cultures. Treatment of monolayer cultures resulted in concentration-related decreases in GSH and ATP, accompanied by enzyme leakage. In contrast, only ATP was affected following treatment of spheroids for 7 days. Spheroids appeared to be less sensitive to exposure to MTX, when compared with monolayer cultures. This may result from the maintenance of cellular functions, or from the lack of compound penetration into the three-dimensional spheroid structure. Therefore, the usefulness of spheroids to chronic in vitro toxicity testing may be limited.

The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status.

In this report, metabolically competent in vitro systems have been reviewed, in the context of drug metabolising enzyme induction. Based on the experience of the scientists involved, a thorough survey of the literature on metabolically competent long-term culture models was performed. Following this, a prevalidation proposal for the use of the collagen gel sandwich hepatocyte culture system for drug metabolising enzyme induction was designed, focusing on the induction of the cytochrome P450 enzymes as the principal enzymes of interest. The ultimate goal of this prevalidation proposal is to provide industry and academia with a metabolically competent in vitro alternative for long-term studies. In an initial phase, the prevalidation study will be limited to the investigation of induction. However, proposals for other long-term applications of these systems should be forwarded to the European Centre for the Validation of Alternative Methods for consideration. The prevalidation proposal deals with several issues, including: a) species; b) practical prevalidation methodology; c) enzyme inducers; and d) advantages of working with independent expert laboratories. Since it is preferable to include other alternative tests for drug metabolising enzyme induction, when such tests arise, it is recommended that they meet the same level of development as for the collagen gel sandwich long-term hepatocyte system. Those tests which do so should begin the prevalidation and validation process.

Comparison of hepatocyte cultures and liver slices in in vitro toxicity testing.

The aim of this study was to compare the in vitro toxicities of two hepatotoxins in hepatocyte cultures and in liver slices from both rats and dogs. Hepatocytes and liver slices were pre-incubated for 2 hours and then exposed to galactosamine or paracetamol, both of which mainly induce liver necrosis in vivo. Following exposure to the compounds for 20 hours, neutral red uptake (NRU [hepatocyte cultures only]), MTT reduction, and reduced glutathione (GSH), adenosine triphosphate (ATP) and protein content, were used to measure the toxicity induced. In general, galactosamine and paracetamol exposure caused comparable levels of toxicity in hepatocyte cultures and in liver slices. For galactosamine, no consistent differences were seen between hepatocyte cultures and liver slices. With paracetamol, the toxic effects were generally slightly more pronounced in hepatocyte cultures than in liver slices, and the preparations from dog liver were more sensitive than those from rat liver to paracetamol exposure. These results are in agreement with previously described species differences in vitro. NRU and GSH content were more sensitive and more consistent endpoints than MTT reduction, ATP content or protein content. Liver slices appeared to lose viability over the 20 hours in culture. Therefore, it can be concluded that liver slices should only be used in relatively short-term investigations.

Preliminary Investigations into the use of a Human Bronchial Cell Line (16HBE14o-) to Screen for Respiratory Toxins In Vitro.

A transformed epithelial cell line derived from normal human bronchial epithelium (16HBE14o- cells) was used to assess the in vitro toxicity of six compounds. The compounds were sodium chloride and titanium dioxide (reference compounds) and sodium carbonate and silica (respiratory toxins). In addition, two compounds (compounds A and B) were tested which have been shown to induce respiratory toxicity in the rat during preclinical safety assessment. Confluent monolayers of 16HBE14o- cells were treated for 24hr with the test compounds and toxicity was assessed using two conventional cytotoxicity assays (neutral red uptake and MTT reduction). Transepithelial resistance (TER) was also measured throughout the treatment period as a possible alternative endpoint for toxicity measurement. Neither sodium chloride nor titanium dioxide caused toxicity in 16HBE14o- cells using any of the toxicity endpoints. With the exception of silica, all irritant compounds caused concentration-related cytotoxicity in 16HBE14o- cells. For each compound, when the three toxicity endpoints were compared, similar IC(50) values were obtained irrespective of the endpoint used. These initial results indicate that 16HBE14o- cells may be a suitable cell line for future use in development of in vitro assays for respiratory toxicity.

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations.

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and beta-oxidation (liver specific functions). Ethionine (0-30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10-30 mM ethionine and reduced after 20 h in cultured hepatocytes (18-30 mM). Protein synthesis was reduced and beta-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo.

Histamine concentrations and hemodynamic responses after remifentanil.

Remifentanil is a new potent opioid analgesic that undergoes rapid esterase metabolism. The purpose of this study was to investigate hemodynamic responses to 2-30 micrograms/kg remifentanil (escalating doses) injected as a bolus over 1 min during general anesthesia. After general anesthesia with endotracheal intubation, placement of a radial artery catheter, and pretreatment with glycopyrrolate, remifentanil 2, 5, 15, or 30 micrograms/kg (six patients, three male and three female per group) was administered over 1 min. Arterial blood pressure and heart rate were measured noninvasively before drug administration, after drug administration, and then every minute for 5 min. Arterial blood was taken for histamine determinations before drug administration and then at 1, 3, and 5 min after drug administration. Administration of remifentanil was associated with a reduction in systolic blood pressure from 134 +/- 18 to 91 +/- 16 mm Hg and heart rate from 99 +/- 20 to 69 +/- 21 bpm and was not associated with alterations in histamine concentration.

Fentanyl or alfentanil decreases the minimum alveolar anesthetic concentration of isoflurane in surgical patients.

The minimum alveolar anesthetic concentration (MAC) is an accepted potency measure for inhaled anesthetics. There is no generally accepted intraoperative measure of opioid potency, partly because of the difficulty in obtaining steady state biophase concentrations. We have studied the relative potency of fentanyl and alfentanil by using computer-assisted continuous infusions (CACI), in terms of reduction of isoflurane MAC. Data are presented from 79 patients in whom anesthesia was induced with thiopental and maintained with a CACI of fentanyl (set to achieve a plasma concentration of 0, 1, 3, 6, or 8 ng/mL) or alfentanil (0, 70, 210, 400, or 1000 ng/mL) with a predetermined end-tidal concentration of isoflurane. A determination of movement in response to skin incision was made. Without any opioid, the MAC of isoflurane was 1.25%. The maximum likelihood solution to a logistic regression model showed that fentanyl 0.5 ng/mL (95% confidence intervals [CI], 0-4.6 ng/mL) or alfentanil 28.8 ng/mL (95% CI, 0-70.9 ng/mL) resulted in a 50% isoflurane MAC reduction. In the logistic regression model, age or weight were not significant factors in the model. These results suggest that the comparative intraoperative potency ratio for alfentanil and fentanyl is 58:1, and that this methodology allows for direct intraoperative comparisons of opioid potency.

Investigations into the mechanisms of carcinogen-induced nuclear enlargement in HeLa S3 cells in vitro.

Carcinogen-induced nuclear enlargement has been reported both in vitro and in vivo, but the mechanism, and whether it is causally related to carcinogenesis, has not yet been established. This study was designed to investigate the role of increased DNA content, such as might occur in polyploidy, in induction of nuclear enlargement. The effects of two genotoxic carcinogens, N-methyl-N-nitrosourea and adriamycin, were compared with the effects induced by diethylstilboestrol, which is arguably a non-genotoxic carcinogen but is known to induce polyploidy. HeLa S3 cells were used as the model system for comparison with previous studies. N-methyl-N-nitrosourea and adriamycin both induced a concentration-related increase in nuclear size 24 to 72 hr after a 30 min pulse-treatment. This was accompanied by an increase in the proportions of cells in the G(2) + M stage of the cell cycle, possibly due to a G(2) block. There was some evidence of polyploidy with adriamycin but not with N-methyl-N-nitrosourea. The distributions of nuclear areas indicated that increases in ploidy contributed to, but did not totally account for, the nuclear enlargement. In contrast, diethylstilboestrol increased the range of nuclear areas and DNA content, to both less than and greater than that of control cells, but only after a prolonged exposure period of 48 hr. These data were consistent with diethylstilboestrol inducing spindle damage. These results demonstrate that carcinogen-induced nuclear enlargement is only partially explained by increased nuclear DNA content, and that certain classes of non-genotoxic carcinogen may produce a completely different pattern to that from genotoxic carcinogens.

Pharmacokinetics of remifentanil (GI87084B) and its major metabolite (GI90291) in patients undergoing elective inpatient surgery.

BACKGROUND: Remifentanil is a highly potent opioid with a rapid onset and a short duration of action due to its rapid hydrolysis by esterases in blood and tissues. The major metabolite of remifentanil, GI90291, is much less potent than remifentanil. METHODS: The pharmacokinetics of remifentanil and its major metabolite, GI90291, were determined in 24 patients undergoing elective inpatient surgery. Remifentanil was administered as a 1-min infusion (2, 5, 15, and 30 micrograms/kg) after the induction of anesthesia and tracheal intubation. Serial arterial blood samples were collected over 6 h and assayed for remifentanil and GI90291. RESULTS: The pharmacokinetics of remifentanil were described using a three-compartment model. Total clearance (250-300 l/h) of remifentanil was independent of dose and was approximately three to four times greater than the normal hepatic blood flow. Volume of distribution at steady state (25-40 l) also was independent of dose. The terminal half-life of remifentanil ranged from 10 to 21 min. Covariate analysis of remifentanil clearance and patient demographics showed that patient body weight, age, and gender did not influence total clearance. This suggests that remifentanil may not need to be dosed according to body weight in adult patients. A simulation was conducted to determine the time required for a 50% reduction in effect site concentration after an infusion designed to maintain a constant effect site concentration. The time required for a 50% reduction in the effect site concentration of remifentanil (3.65 min) was considerably less than that for sufentanil (33.9 min), alfentanil (58.5 min), and fentanyl (262 min). The pharmacokinetics of the major metabolite, GI90291, were independent of the dose of remifentanil. The mean terminal half-life of GI90291 ranged from 88 to 137 min. CONCLUSIONS: The pharmacokinetics of remifentanil are consistent with its rapid elimination by blood and tissue esterases; its major metabolite is eliminated more slowly but is not likely to make any significant contribution to the total effect because of its much lower potency. The rapid onset and short duration of action of remifentanil make it well suited for titration of dose (infusion rate) to the desired degree of effect.

Indirect memory during anesthesia. The effect of midazolam.

BACKGROUND: The preservation of implicit memory function during anesthesia is controversial, with conflicting results appearing in the literature. This study was designed to elucidate the effect of midazolam as part of an anesthetic technique on implicit memory function during anesthesia. Using a prospective randomized, double-blind study design, performance in three tasks (category generation, free association, and homophone spelling) was assessed. METHODS: Forty-eight consenting patients were assigned to two equal groups, to receive 2 mg intravenous midazolam or normal saline before induction of anesthesia. Anesthesia was induced with fentanyl and propofol and maintained with isoflurane 1.3 MAC until incision and isoflurane 1.0 MAC in 70% nitrous oxide thereafter. Fentanyl was used for supplementation of anesthesia. During anesthesia, one of two 50-min tapes containing the test material was played to each patient on a portable cassette player. In the postanesthesia care unit and 48 h after surgery, patients were engaged in three tasks by an observer unaware of the treatment group or tape. RESULTS: No significant main effect of priming or midazolam was observed in any of the tasks. In the word-association task, an interaction was observed between priming and treatment group (F = 9.62, P < .01) due to negative priming in the placebo group. CONCLUSIONS: The lack of a main effect of priming in any of the three tasks is consistent with the conclusion that indirect memory was not demonstrated for events occurring during the standard anesthetic conditions of this study. Further, midazolam appeared to have no effect.

D and C Red No. 9: genotoxic or non-genotoxic carcinogen?

The azo-compound, D and C Red No. 9 was assayed for genotoxicity in vivo using the rat micronucleus test and the rat ex vivo liver UDS assay. Uniformly negative results were obtained in both assays, even though large oral doses were used (2 g/kg). These results suggest that the tumorigenic effects of this compound in rats are mediated through non-genotoxic rather than a genotoxic mechanism. Further experiments using additional end-points such as 32P-post-labelling would further substantiate this conclusion.

In vivo genotoxicity studies with p-benzoquinone dioxime.

P-Benzoquinone dioxime (BQD) appears to be a sex-specific rat carcinogen inducing tumours of the urinary bladder in female rats. The present paper shows that BQD is a direct-acting mutagen in Salmonella typhimurium TA98, confirming published data. In contrast to this in vitro data, negative results were obtained after oral administration of BQD to female rats in both the bone marrow micronucleus test and the in vivo liver UDS test. BQD did, however, induce a marked effect upon S-phase synthesis in the livers of female rats between 14 and 48 hr after a single oral dose of 250 mg/kg. A similar effect was also observed in the livers of male rats. There was no evidence of hepatotoxicity (in terms of elevated liver enzyme levels) after treatment of female rats with the compound indicating that the increase in cell proliferation was due to a direct mitogenic effect of BQD in this organ. Some liver mitogens have been found to be liver carcinogens; this does not appear to be the case for BQD. Nevertheless, the mitogenic activity of this compound might play a contributory role to the induction of bladder cancer in rats if it also acted as a mitogen in this tissue. Further studies are indicated, measuring genotoxicity and cell-proliferative activity in the bladder in order to further elucidate the mechanism of action of this compound as a rodent carcinogen.

Evaluation of the in vivo genotoxicity of the structural analogues 2,6-diaminotoluene and 2,4-diaminotoluene using the rat micronucleus test and rat liver UDS assay.

The two structural isomers 2,4- and 2,6-diaminotoluene (DAT) differ in their carcinogenic properties; the 2,4-isomer is carcinogenic in rats and mice, whereas the 2,6-isomer has been reported to be non-carcinogenic. Both isomers were reported to be mutagenic in Salmonella typhimurium in the presence of S9, which was confirmed in the present study before in vivo assays were commenced. Both isomers were tested in the rat bone marrow micronucleus test and the rat liver UDS test to investigate how well these assays discriminate between the carcinogenic and the non-carcinogenic isomer. In the micronucleus test both isomers gave weakly positive results; however, with the carcinogen 2,4-DAT this weak effect was only detectable at very toxic doses and therefore the biological relevance of this result is questionable. Thus, the micronucleus test did not discriminate correctly between the carcinogen and the non-carcinogen. With the liver UDS test, discrimination was achieved but the positive effect seen for the carcinogenic isomer was weak and dependent on the method of preparation of the dosing suspensions. The results are discussed in relation to the carcinogenicity data on both compounds. It is concluded that although both isomers are potent genotoxins in vitro they exert their genotoxic potential only weakly in vivo and convincing discrimination between the carcinogenic and non-carcinogenic isomer was not demonstrated.

The differential clastogenicity of Solvent Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test (observations on species and tissue specificity).

Solvent Yellow 14 is carcinogenic in rats, inducing neoplastic nodules of the liver, but is non-carcinogenic in mice. The present paper shows that Solvent Yellow 14 induces micronuclei in the bone marrow of rats after a single oral dose of 250 mg/kg and above. In mice, however, there was no increased incidence of micronuclei after single oral doses of up to 2000 mg/kg Solvent Yellow 14, thus reflecting the species specific carcinogenic effect of the compound. The structurally related azo dye FD & C Yellow No. 6 is noncarcinogenic to rats and mice and gave a negative result in both rat and mouse bone marrow micronucleus tests after a single oral dose of up to 2000 mg/kg. The rat bone marrow micronucleus test is therefore capable of discrimination between the carcinogenic and the non-carcinogenic azo dye. A negative result was obtained for Solvent Yellow 14 in an in vivo liver unscheduled DNA synthesis assay after oral doses up to 1000 mg/kg. This result demonstrates the inability of the two in vivo assays used to predict target organ specificity seen in the cancer bioassay.

Effects of aniline hydrochloride in the mouse bone marrow micronucleus test after oral administration.

Mice are non-responsive to aniline-induced carcinogenicity. However, the present paper shows that aniline hydrochloride induces micronuclei in the bone marrow of male CRH mice 24 h after a single oral dose of 1000 mg/kg (in terms of free base) though not at the lower doses of 400 and 500 mg/kg. No micronucleus inductions was found 48 h after oral dosing. A positive response was also seen 24 h after a single i.p. dose of 380 mg/kg aniline. The high oral dose of aniline needed for micronucleus induction, the presence of micronuclei with abnormal morphology and the large inter-animal variability found within this group may possibly indicate a more complex mechanism of micronucleus induction than simple clastogenicity.

Activity of urethane in rat and mouse micronucleus tests after oral administration.

Urethane is readily detectable as an in vivo clastogen in the micronucleus test after oral administration to both rats and mice. Mice appear to be the more sensitive species. Positive results were obtained irrespective of the strain (CD1, CRH) or dosing regimen (single, triple-dose) used.