Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

A 90-day feeding study of the alkaloids of Lupinus angustifolius in the rat.

Groups of 20 Sprague-Dawley rats of each sex were fed diets containing lupin alkaloid at dose levels of 0, 100, 330, 1000 and 5000 ppm supplemented with maltodextrin to attain a level of 4.5%, for 13 wk (equivalent to average daily intakes of lupin alkaloid of approximately 0, 10, 30, 100 and 500 mg/kg body weight/day, respectively, over the course of the study). A further group of rats was fed control (basal) diet over the same period. All control and high-dose animals underwent an ophthalmological examination before the start of the study and before autopsy. Blood samples were collected from all rats prior to the start of treatment, during wk 6 and prior to autopsy for haematological and clinical chemistry examination. All animals were monitored daily for change in clinical condition, and body weight and food intake were measured twice weekly. A range of tissues were preserved for histological examination at autopsy. There was an initial drop in food intake by all rats in the 1000 and 5000 ppm groups and thereafter the intake was between 90% and 95% of that of the controls. In general, no other effects related to treatment were seen. On the basis of the lower body weights and food intakes of the groups fed the alkaloid at levels of 1000 and 5000 ppm, a no-observed-adverse-effect level (NOAEL) of 330 ppm is seen under the conditions of this study. It is likely that these effects are entirely due to the antipalatability effect of the lupin alkaloids. In view of the growth rates, haematology, clinical chemistry and histological findings, a speculative NOAEL of 1000 ppm may be more appropriate.

Effects on 4080 rats of chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine: a detailed dose-response study.

Four thousand eighty inbred rats were maintained from weaning on various different concentrations of N-nitrosodiethylamine (NDEA) or N-nitrosodimethylamine (NDMA). The principal aim was to characterize the dose-response relationship for the effects of these agents on esophageal cancer (NDEA) or on various types of liver cancer (NDEA and NDMA), although NDEA also caused a few tumors of the nasopharynx and NDMA also caused a few tumors of the lung. The numbers of tumors of mesenchymal and Kupffer cells in the liver were too few to allow easy characterization of the dose-response relationships, and although NDMA induced large numbers of bile duct neoplasms, NDEA did not. Thus, the four principal dose-response relationships studied were of NDEA on esophageal or liver cells and of NDMA on bile duct or liver cells. At doses sufficiently high for the median time to death from the disease of interest to be estimated, relationships were observed of the general form (Dose rate) x (median)n = constant where n was about 2.3 for the first three relationships and about 1 for the last one (NDMA on liver cell tumors). By contrast, at doses sufficiently low for longevity to be nearly normal (median survival about 2.5 years), there remained no material dependence on the dose rate of the age distribution of the induced neoplasms. At these low dose rates, the number of liver (but not of esophageal) neoplasms induced by treatment was simply proportional to the dose rate. This finding is not surprising, since the background incidence of liver (but not of esophageal) neoplasms was appreciable. The linear relationship observed at low dose rates (below 1 ppm) suggests that under these experimental conditions, among rats allowed to liver their natural life span, a dose of 1 ppm of NDEA or NDMA in the drinking water will cause about 25% to develop a liver neoplasm, a dose of 0.1 ppm will cause about 2.5% to do so, and a dose of 0.01 ppm will cause about 0.25% to do so, etc., with no indication of any "threshold." (At these low dose rates, the incidence of liver neoplasms appears likely to exceed greatly that of esophageal neoplasms.) In addition, even quite low dose rates of the test agents caused a variety of nonneoplastic liver abnormalities (e.g., hyperplastic nodules, or shrinkage of hepatocytes) at a frequency roughly proportional to the dose rate.

Dose and time relationships for tumor induction in the liver and esophagus of 4080 inbred rats by chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine.

A Weibull analysis is presented of the dose and time relationships for the effects on 4080 inbred rats of chronic ingestion in the drinking water of 16 different doses of N-nitrosodimethylamine (NDMA) or N-nitrosodiethylamine (NDEA). The sites chiefly affected were the liver (by both agents) and the esophagus (by NDEA only). Since the experiment continued on into extreme old age, effects became measurable at doses of only 0.01 to 0.02 mg/kg/day, which is an order of magnitude lower than previously achieved. (After only 2 years of treatment, however, the TD50 doses needed to halve the proportion of tumorless survivors would have been about 0.06 mg/kg/day of NDEA, or about 0.12 mg/kg/day of NDMA.) The general pattern of response was that the natural logarithm of the probability of remaining tumorless was given by the product of two terms, the first (the "Weibull b value") depending on the dose rate but not on the duration of exposure and the second depending not on dose at all but only on duration. For all types of tumor the dependence on duration was fairly similar (and for each the second term was taken to be -t7, where t = years of treatment), but for different types of tumor the dependence on dose rate was quite different. For esophageal tumors, the "Weibull b value" was approximately proportional to the cube of the dose rate of NDEA (males 21 d3, females 11 d3, where d = dose rate in mg/kg adult body weight/day), and the background incidence was unmeasurably low. For liver tumors induced by NDEA, the b value was approximately proportional to the fourth power of dose rate + 0.04 mg/kg/day [males, 19 (d + 0.04)4; females, 32 (d + 0.04)4], although the relationships were somewhat different for the different cell types of liver tumor. This one formula implies both approximate linearity at low doses and an approximately cubic relationship within the higher range of doses that was studied. For liver tumors induced by NDMA, the Weibull b value was approximately proportional to the sixth power of dose rate + 0.1 mg/kg/day [males, 37 (d + 0.1)6; females, 51 (d + 0.1)6], again with some variation between liver cell types, and again implying approximate linearity at low doses. These algebraic formulae should, of course, be trusted only in the range of doses where they were derived, and particularly not above it.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic nitrosamine ingestion in 1040 rodents: the effect of the choice of nitrosamine, the species studied, and the age of starting exposure.

In parallel with a larger experiment on 4080 rats fed 16 different concentrations of N-nitrosodiethylamine (NDEA) or N-nitrosodimethylamine (NDMA) from 6 weeks of age, a variety of smaller experiments on a total of 1040 rodents were undertaken and are the subject of the present report. Three separate subjects were addressed. Studies of 16 different concentrations of N-nitrosopyrrolidine and N-nitrosopiperidine given from age 6 weeks onwards to small groups of rats yielded dose-response relationships for the effects of N-nitrosopyrrolidine on liver tumors and for those of N-nitrosopiperidine on tumors of the liver and upper gastrointestinal tract that resembled those seen for NDMA and NDEA, respectively, except that N-nitrosopyrrolidine and N-nitrosopiperidine were less potent [the respective dose rates needed to halve the proportion of tumorless survivors after 2 years of treatment being approximately 0.4 (males) and 0.6 (females) mg/kg adult body weight/day for each agent]. Alternatively, it was estimated that the risks to rats from lifelong exposure to 1 microgram/kg adult body weight/day of each agent might be about 0.1%, and that the risks to rats from lower doses would be proportionately less. Studies of 16 different concentrations of NDEA on small groups of female mice and female hamsters yielded the types of dose response that would be expected for upper gastrointestinal tumors, liver cell tumors, and Kupffer cell tumors in mice (no other types of liver tumor being produced, in contrast with previous reports) and for tracheal and liver cell tumors in hamsters (no clear effect on upper gastrointestinal tumors being apparent in hamsters). The dose rates needed to halve the proportion of tumorless survivors after 2 years of treatment were approximately 0.3 mg/kg adult body weight/day, i.e., 5 times that for the same agent in rats. In part, however, this may be because treatment started at an older age in these species. Studies were undertaken of the effects on esophageal and liver tumorigenesis of starting the treatment of rats with NDEA at 3 or at 20 weeks of age instead of at 6 weeks of age (as in the main experiment). Earlier treatment resulted in slightly greater dosage rates, if dosage was measured in mg/kg/day, and hence in a correspondingly more rapid yield of esophageal tumors, but the effect was not large. By contrast, an earlier start to treatment resulted, after a fixed duration of treatment, in animals having a 3-fold higher incidence rate of liver tumors, while a later start resulted in a 2-fold decrease.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term toxicity study of Ponceau 4R in rats using animals exposed in utero.

Groups of 66 (treated) or 114 (control) rats of each sex were fed diets providing 0 (control), 50, 500 or 1250 mg Ponceau 4R/kg body weight/day for 60 days. The animals were then mated and allowed to rear their litters. At weaning, pups were selected for the long-term study to give treated groups of 54 (of each sex) and a control group of 96 (of each sex), with offspring always receiving the same treatment as their parents. Treatment continued until approximately 20% of animals survived, resulting in a duration of 114 wk for males and 118 wk for females. Body weight, food and water intake and clinical conditions were monitored regularly throughout the study. Blood and urine from 20 rats/sex/group from the high-dose and control groups were examined at months 3, 6, 12, 18 and 24. At the end of the study each animal was autopsied, selected organs were weighed, and a full range of tissues was preserved. High-dose animals showed a lower body-weight gain without any reduction in food intake. Water intake was higher than that of the controls in the medium- and high-dose groups and this was related to caecal enlargement and softening of faeces. No adverse changes were seen in the investigations of blood or urine apart from a higher incidence of females with higher levels of protein in urine at the 1250 mg/kg/day dose. No other findings of significance were seen and survival and tumour incidence were similar in all groups. A no-untoward-effect level was established at 500 mg Ponceau 4R/kg/day.

Long-term toxicity study of Green S in mice.

Groups of 105 (control) or 65 (treated) female CD-1 mice were mated one to one with equal numbers of males after both sexes had received diets containing 0 (control), 0.033, 0.33 or 0.66% Green S for 9 wk. The number of animals pregnant, the number of young born and the number surviving were similar in all groups. One male and one female from each litter were used to provide groups of 85 (control) or 50 (treated) mice of each sex for the long-term study. Treatment with Green S continued throughout pregnancy and rearing. Body weight and condition were regularly monitored for each animal throughout the study. Blood was examined from groups of 20 mice from the control and highest treatment groups at wk 14, 28 and 51 and from all survivors at the end of the study. A post-mortem examination was carried out on all animals in the long-term study and a full range of tissues was preserved and examined by light microscopy. Organ weights were recorded at the autopsy of all mice reaching the end of the study. No effects that could be attributed to treatment were seen in any of the observations. The no-untoward-effect level of Green S fed to mice for 2 yr is concluded to be 0.66% of the diet, equivalent to intakes of approximately 530 and 660 mg/kg body weight/day in males and females, respectively.

Nitrosamine carcinogenesis in 5120 rodents: chronic administration of sixteen different concentrations of NDEA, NDMA, NPYR and NPIP in the water of 4440 inbred rats, with parallel studies on NDEA alone of the effect of age of starting (3, 6 or 20 weeks) and of species (rats, mice or hamsters).

A Weibull analysis is presented of the dose and time relationships for the effects on 4 080 inbred rats of chronic ingestion in the drinking water of 16 different doses of N-nitroso-dimethylamine (NDMA) and of N-nitrosodiethylamine (NDEA). The sites chiefly affected were the liver (by both agents) and the oesophagus (by NDEA only). Since the experiment continued into extreme old age, effects became clearly measurable even at a dose of only 0.01 mg/kg per day, which is an order of magnitude lower than previously achieved. (After only two years of treatment, however, the 'TD50' doses needed to halve the proportion of tumourless survivors would have been about 0.06 mg/kg per day of NDEA and about 0.12 mg/kg per day of NDMA.) The general pattern of response was that the log probability of remaining tumourless was given by the product of two terms, the first (the 'Weibull b-value') depending on the dose-rate but not on the duration of exposure, and the second depending not on dose at all but on (approximately the seventh power of) duration. For oesophageal tumours, the 'Weibull b-value' was approximately proportional to the cube of the dose-rate of NDEA (males 21 d3, females 11 d3, where d = dose-rate in mg/kg adult body weight/day, and the background incidence was unmeasurably low. For liver tumours induced by NDEA, the b-value was approximately proportional to the fourth power of dose-rate + 0.04 mg/kg per day (males, 19 (d + 0.04)4; females, 32 (d + 0.04)4), although the relationships were slightly different for the different subsites of liver tumour. This one formula implies both approximate linearity at low doses and an approximately cubic relationship within the higher range of doses that was studied. For liver tumours induced by NDMA, the Weibull b-value was approximately proportional to the sixth power of dose rate + 0.1 mg/kg per day (males, 37 (d + 0.1)6; females, 51 (d + 0.1)6), again with variation between liver subsites, and again implying approximate linearity at low doses. The difference between the latter two relationships may represent differences in the induction of particular DNA repair enzymes by NDMA and NDEA, or in the effects of those enzymes on methylated and on ethylated DNA. These formulae should, of course, be trusted only in the range of doses from which they were derived, and particularly not for those above it.(ABSTRACT TRUNCATED AT 400 WORDS)