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)

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.

Studies on the effects of orally administered Di-(2-ethylhexyl) phthalate in the ferret.

A target-organ study of the effects of the phthalate ester di-(2-ethylhexyl) phthalate (DEHP) has been conducted in mature male albino ferrets. DEHP treatment caused a loss of body weight when administered as a 1% (w/w) diet for 14 months. Additionally marked liver enlargement with associated morphological and biochemical changes was observed. These changes consisted of liver cell enlargement, lysosomal changes, dilatation of the endoplasmic reticulum and the depression of a number of marker enzyme activities. The only other tissue observed to be affected by DEHP treatment was the testes where histological evidence of tissue damage was observed in some animals. Studies on the metabolism of [14C]DEHP in the ferret indicated that the diester was metabolised to derivatives of mono-(2-ethylhexyl) phthalate which were excreted in the urine both unconjugated and as glucuronides. The results obtained have been compared with previous studies in the rat and it is concluded that DEHP is hepatotoxic in both species.

The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 1. Selection and Distribution of the Test Chemicals.

An international validation study on in vitro tests for skin corrosivity was conducted during 1996 and 1997 under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The main objectives of the study were to assess the performances of selected in vitro tests in discriminating between: (a) corrosives (C) and non-corrosives (NC), for selected groups of chemicals (e.g. organic acids, phenols) and/or for all chemicals (single chemical entities only); and (b) known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. Each test was evaluated for reliability and relevance by using a test set of 60 coded chemicals. In this paper, the test chemicals used in the validation study are identified; they include organic acids (6C/5NC), organic bases (7C/3NC), neutral organics (9NC), phenols (2C/3NC), inorganic acids (6C/1NC), inorganic bases (2C/2NC), inorganic salts (1C/2NC), electrophiles (3C/5NC) and soaps/surfactants (3NC). The in vivo classifications and important physicochemical properties (e.g. logP, pKa) of the test chemicals are given. The main criterion for including chemicals in the test set was that their corrosivity classifications were based on unequivocal animal data. Where available, structure-activity information was also used to support the corrosivity classifications. Despite the small numbers of chemicals in some of the categories, it was felt that the test set chosen represented the best possible for evaluating the performances of the in vitro tests for predicting skin corrosivity, given the limited availability of unequivocal animal data. The prediction of skin corrosivity from pH data was also investigated for those chemicals with extreme pH values (i.e. pH2 or 11.5). Nine of the 12 strongly acidic or alkaline chemicals in the test set, which were predicted to be C on the basis of their pH values, had also been found to be C in vivo.

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.

The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II (Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test.

To date, no standardized international guideline for the testing of chemicals for phototoxic potential has been accepted for regulatory purposes. In 1991, the European Commission (EC), represented initially by the Directorate General XI and later by ECVAM (the European Centre for the Validation of Alternative Methods) and COLIPA (the European Cosmetic, Toiletry and Perfumery Association), agreed to establish a joint EU/COLIPA programme on the development and validation of in vitro phototoxicity tests. The first phase (phase I, 1992-93) was designed as a prevalidation study, to identify in vitro test procedures and test protocols for a formal validation trial under blind conditions. In the second phase (phase II, 1994-95), the formal validation study, the most promising in vitro phototoxicity tests were validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial. The 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) was performed as a core test in nine laboratories, since it provided the best results in phase I of the study. The purpose of phase II was to confirm the reliability and relevance of the in vitro tests for predicting phototoxic effects and for identifying phototoxic chemicals. In phase II the phototoxic potential of test chemicals in the 3T3 NRU PT test was either assessed by determining the phototoxicity factor (PIF) by using a cut-off value of 5 as in phase I of the study, or by determining the mean photo effect (MPE) by using a cut-off value of 0.1, as recently proposed by Holzhütter (1997). Results obtained with both approaches in the 3T3 NRU PT test in phase II were reproducible in the nine laboratories, and the correlation between in vitro and in vivo data was very high. Therefore, ECVAM and COLIPA conclude from this formal validation trial under blind conditions that the 3T3 NRU PT test is a scientifically validated in vitro test which is ready to be considered for regulatory purposes for assessing the phototoxic potential of chemicals. A draft OECD Guideline for "In Vitro Phototoxicity Testing", incorporating the standard protocol of the 3T3 NRU PT test, will be submitted to the OECD test guidelines programme in due course.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

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.

A three-generation reproduction study of Ponceau 4R in the rat.

Ponceau 4R was fed to three generations of rats, at dietary concentrations to provide 0, 50, 500 or 1250 mg/kg body weight/day. In each generation treated groups consisted of 36 rats of each sex while 60 females served as controls. Apart from the F0 generation, which started treatment as weanlings, treatment was continued throughout the study, providing in utero exposure of all offspring. The F0 generation was bred twice, on the first occasion to provide animals for the next generation and for a long-term study, and a second time to provide data on in utero and post-partum development. In each generation approximately one third of the females from each group were killed before parturition to provide data on in utero development. The foetuses from these animals were examined for skeletal abnormalities. Remaining animals were allowed to litter and the offspring were monitored for 21 days after birth for survival and development. All animals were killed and subjected to a post-mortem examination which, for a proportion of each group in each generation, included recording of organ weights. Although a few adult rats died during the study these deaths were not associated with treatment. Fur of the treated animals was coloured pink, and faeces and caecal contents of animals from the two highest dose groups were yellow, the faeces also being softer than those of the controls. Treatment had no observed effect on clinical observations, body weight or food and water intake at any stage of the study. Animals fed the two highest doses for prolonged periods had enlarged caeca, but this effect was not seen in weanling animals on the same treatment. Neither the caecal enlargement nor the liver weights seen in the F2 and F3 offspring were considered to be an adverse effect of treatment. No treatment-related effects were seen in the uterine contents of females at any generation, but the skeletons of treated foetuses showed a slightly more advanced development than those of the controls. Postnatal development of offspring was not affected by treatment at any stage of the study. Tissues from the F3 animals were examined by light microscopy and revealed no treatment-related effects. It is concluded that the no-adverse-effect level for Ponceau 4R is 1250 mg/kg body weight/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.

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.

A study of the toxicity of five mineral hydrocarbon waxes and oils in the F344 rat, with histological examination and tissue-specific chemical characterisation of accumulated hydrocarbon material.

Five food-grade mineral hydrocarbon (MHC) materials; a low melting point wax (LMPW), a synthetic wax (C80W) and three white oils (N15H, N70H and P70H) were administered orally to female Fischer-344 rats for 28 and 90 days at a dose level of 2% in the diet. Tissues were examined at autopsy for any treatment-related histopathological changes. The histology of target organs was the same as found in previous studies on LMPW and mineral oils and similar effects were also observed from feeding C80W. Chemical analysis showed no detectable levels of MHCs in urine and no discernible differences in the MHC profile in faeces extracts compared to diets. The presence of MHCs in most tissues was not always associated with observable histological changes. The notable observations were MHC material was detected in all tissues of rats fed with diets containing LMPW and C80W. The levels found ranged from 0.04 to 1.52% by weight for the LMPW and from 0.01 to 0.75% for the C80W. MHC material was detected in all samples of small intestine, heart and kidney for all groups. Only the livers from rats administered with LMPW and C80W were analysed, which were found to contain MHC material. Preferential accumulation of MHCs was in the alkane range approximately C(20)-C(35). The findings indicate that the size and the structure of individual components play a role both in determining their propensity to accumulate in different tissues and in the severity of any response that they elicit once they have accumulated. The implication of these findings are discussed in the context of specifications for 'food-grade' mineral hydrocarbons such as used as food additives. The data presented here suggests that the current specifications are not prescriptively adequate in controlling the amount of MHC material between C(25) and C(35) that can accumulate.

Three-generation toxicity study of rats ingesting Brown HT in the diet.

Brown HT was fed to rats of both sexes over three generations at dietary concentrations designed to provide daily intakes of 0, 50, 250 and 500 mg Brown HT/kg body weight/day. During the study a number of females died or failed to nurse their litters. This was so severe following the first mating of F1 adults that the animals were remated to provide the next generation. None of these effects were related to treatment. Body weight and food and water intakes were not adversely affected by treatment. No effects of treatment were seen on reproductive performance or foetal and pup development, apart from slight evidence of a treatment-related retarded ossification of the third sternebrae. Organ weights at autopsy showed two changes, one of which was increased kidney weights which, although not present in every generation, seemed to be related to treatment. The other, increased caecum weights, occurred in adult high-dose females of early generations, but not in males or later generations of the study. Apart from brown coloration of tissues, macroscopic and microscopic examination revealed no treatment-related changes. It was concluded that the no-untoward-effect level in the present study was 250 mg Brown HT/kg/day.

A 28-day feeding study with methyl isoeugenol in rats.

Methyl isoeugenol was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 30, 100 and 300 mg/kg body weight/day. A further group of 16 male and 16 female rats was given the rodent diet as a control. The administration of methyl isoeugenol in the diet did not adversely affect the growth or general health of the animals or their food intakes. Although high dose animals of both sexes had increased lymphocyte and total white blood cell counts, these are not considered, in isolation, to be an adverse effect of treatment. None of the minor variations observed in the serum chemical analyses or urine analyses is considered to be indicative of a treatment-related toxic effect. An increase in liver weight, adjusted for body weight, was seen in male and female rats receiving 300 mg methyl isoeugenol/kg body weight. Few histopathological abnormalities were observed. Although the incidence of kidney and Harderian gland lesions was higher for high dose animals compared with the controls, the lesions are of a type that occurs spontaneously and are thus not considered to be attributable to treatment with methyl isoeugenol. While the increased liver weight and white blood cell counts of rats given 300 mg methyl isoeugenol/kg body weight may represent effects of treatment, it is not considered that there is any reason to regard these as adverse effects.

A 28-day feeding study with ethyl acetoacetate in rats.

Ethyl acetoacetate encapsulated in gum arabic was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 100, 300 and 1000 mg/kg body weight/day. A further group of 16 male and 16 female rats was given rodent diet containing gum arabic as a control. The administration of ethyl acetoacetate in the diet did not adversely affect the growth or general health of the animals or their food intakes. None of the minor variations observed in the haematology, serum chemical analyses or urine analyses are considered to be indicative of a treatment-related toxic effect. Caecal enlargement was seen in male rats treated with the top dose of ethyl acetoacetate, but this was accompanied by a normal histopathology. Few histopathological abnormalities were observed. Proteinaceous casts were found in the bladder of approximately half the male rats given 1000 mg ethyl acetoacetate/kg, and nephrocalcinosis was a common occurrence in female rats in this dose group. Renal function was unimpaired in treated male and female rats, and the histopathological findings are common in the strain of rats chosen for this study. Although the caecal enlargement and the changes in kidney and bladder of rats given 1000 mg ethyl acetoacetate/kg are noted, it is considered that ethyl acetoacetate did not produce treatment-related adverse effects in rats during this study.

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)

Ninety-day feeding study in Fischer-344 rats of highly refined petroleum-derived food-grade white oils and waxes.

Subchronic 90-day feeding studies were conducted in male and female Fischer-344 (F-344) rats on highly refined white mineral oils and waxes representative of those used for food applications. The goal was to help clarify the mixed results found in other toxicity studies with laboratory animals. Seven white oils and 5 waxes were fed at dietary doses of 20,000, 2,000, 200, and 20 ppm and compared with control groups on untreated diet; toxicity was assessed at 90 days and also after a reversal period of 28 days and/or 85 days. Higher molecular-sized hydrocarbons (microcrystalline waxes and the higher viscosity oils) were without biological effects. Paraffin waxes and low- to midviscosity oils produced biological effects that were inversely related to molecular weight, viscosity, and melting point; oil type and processing did not appear to be determinants. Biological effects were more pronounced in females than in males. Effects occurred mainly in the liver and mesenteric lymph nodes and included increased organ weights, microscopic inflammatory changes, and evidence for the presence of saturated mineral hydrocarbons in affected tissues. Inflammation of the cardiac mitral valve was also observed at high doses in rats treated with paraffin waxes. Further studies are required to elucidate the mechanism for the responses observed and the relevance of these inflammatory responses in the F-344 rat to other species, including humans.