The effects of continued versus discontinued administration of 2-acetylaminofluorene on the morphology and behavior of hepatocellular and urinary bladder tumors in mice.

2-Acetylaminofluorene (2-AAF) was administered in the feed (60, 75, 100 or 150 ppm) to 3,314 BALB/c female mice for periods of either 9, 12, 15 or 24 mon. All mice were killed at 24 mon to compare the effects of continued versus discontinued administration of 2-AAF on morphologic characteristics of hepatocellular and urinary bladder neoplasms. Hepatocellular neoplasms included ratio of hepatocellular adenomas to carcinomas, degree of differentiation and incidence of pulmonary metastases. Characteristics evaluated for the bladder tumors included ratio of local to diffuse carcinomas, ratio of invasiveness to noninvasiveness of the carcinomas, ratio of types of bladder carcinoma and incidence of pulmonary metastases. Differences in the ratio of adenomas to carcinomas, degree of differentiation and incidence of pulmonary metastases were not different between the continued and discontinued group. Although the incidence of bladder carcinomas was higher in the mice on the continued 2-AAF diet, other morphologic and biologic characteristics were similar.

Response to the society of toxicology task force re-examination of the ED01 study.

This communication has re-examined and justified certain of the NCTR's analyses and recommendations from the ED01 Study, which were either misunderstood or misinterpreted by the SOT Task Force. In addition, we have shown that some of the Task Force's own analyses and interpretations are subject to review on scientific grounds. The Task Force's rejection of the linear extrapolation method recommended by the NCTR was stated because of a suspected force-fitting of a linear model to data, an approach that is not part of the NCTR procedure. In suspecting a protective effect of 2-AAF against bladder tumors, the Task Force used an inappropriate model that overpredicted the background bladder tumor rate in control mice. Contrary to the Task Force's belief, a failure to account adequately for time to tumor response was more characteristic of analyses performed by the Task Force rather than those performed by the NCTR. The Task Force's questioning of the multistage model for risk assessment was based on its use of inappropriate, crude tumor data rather than upon NCTR's use of the multistage model with time-adjusted tumor data. The Hartley-Sielken model did not fit the ED01 tumor data as well as the Task Force had presumed. In a risk extrapolation comparison by the Task Force, a coarse time partition of the ED01 data that had been questioned by the Task Force actually produced more stable results than a finer partition proposed by the Task Force. Another problem in the Task Force report concerns the change of protocol. Instead of resulting in a loss of strength as anticipated by the Task Force, the change of protocol during the ED01 Study resulted in an increase in information as alluded to by the Task Force. If the Task Force's proposal for restricting the length of feeding studies had been followed in the ED01 Study, most of the dose related tumor information would not have been obtained. Also, the Task Force's belief that low doses of 2-AAF had some effect on the prevention of early death was not supported by a statistical analysis. In summary, the review conducted by the SOT Task Force contained some misleading representations of the data from the ED01 Study and statements that showed a misunderstanding of NCTR's analyses of the study.

Influence of cause-of-death assignment on time-to-tumor analyses in animal carcinogenesis studies.

The importance of cause-of-death determination in an animal carcinogenesis study with respect to estimation of time-to-tumor distributions of internally occurring (occult) tumors is discussed. A nontechnical description of time-to-tumor estimation is presented. The information obtained from time-to-tumor estimation when cause-of-death designation was used is illustrated for liver tumors in female mice of the inbred strain BALB/cStCrlfC3Hf/Nctr from the ED01 study with N-2-fluorenylacetamide done at the National Center for Toxicological Research. A time-to-tumor analysis of reticulum cell sarcoma data from the same study has provided insight into some difficulties involved in routine case-by-case determination of cause of death. A more flexible system for assigning of cause of death to dead animals and cause of morbidity to moribund animals is described as a way to improve cause-of-death assignment.

Correlations between gross and microscopic lesions in carcinogenic studies in mice.

Microscopic diagnoses of a number of spontaneous and induced neoplasms in mice were correlated with the gross findings of the ED01 and a number of other carcinogenic studies conducted at NCTR to determine the value of detailed histopathologic examinations in bioassay testing. The results indicated that for organs such as thymus, lung, adrenal, Harderian gland and urinary bladder 50% or more of the neoplastic lesions would be missed if at least one histological section were not examined from each organ. For organs such as the liver and mammary gland, a single tissue section did not greatly improve the ability to detect neoplastic lesions beyond that afforded by a thorough necropsy examination.

Biologic and morphologic characteristics of urinary bladder neoplasms induced in BALB/c female mice with 2-acetylaminofluorene.

2-Acetylaminofluorene (2-AAF) induced transitional cell carcinomas, transitional cell carcinomas with squamous metaplasia, squamous cell carcinomas and undifferentiated carcinomas in the urinary bladders of BALB/c female mice. The transitional cell carcinoma was the most common, but was less aggressive and infiltrative than the other three types. The carcinomas were most likely to occur in the vertex of the bladder. A positive correlation existed between dose and length of administration of 2-AAF and the incidence, type, depth of invasion, and the location of the bladder carcinoma.

Effects of dose and time in a long-term, low-dose carcinogenic study.

Large numbers of female BALB/c StCrlfC3Hf/Nctr mice were exposed for up to 33 months to low doses of 2-acetylaminofluorene (2-AAF) under controlled conditions. The study design consisted of sacrifice intervals, life span, and discontinued dosing groups. Two separate and distinct endpoints, that is urinary bladder neoplasms and liver neoplasms, resulted in 2 different types of dose response relationships. Although bladder neoplasms exhibited a minimum effect level (or a nonlinear response) for specific conditions, the total results were consistent with a "no threshold concept." The late appearing liver neoplasms displayed a nearly linear type response that extrapolated directly to zero dose. Time of exposure was shown to be an important factor in that, as animals were sacrificed at 18, 24, and 33 months, a positive response was noted at the next lower dose as time was extended. Discontinuing dosing and sacrificing at 18 and 24 months also demonstrated the effects of exposure to the carcinogen 2-AAF. Induction of bladder neoplasms was shown to occur early in the study, but was dependent upon the continuous presence of 2-AAF. The liver neoplasms appeared very late in the study but were shown to be induced at a very early point in the exposures and did not require the continuous presence of the carcinogen in order to develop. A standard 18 month bioassay study, if conducted under the same conditions, would have classified this chemical as a weak acting carcinogen. These studies demonstrate the importance of the time factor in safety evaluation or risk assessment in carcinogenesis.

Effects of continuous and discontinued exposure to 2-AAF on urinary bladder hyperplasia and neoplasia.

The effect of discontinued feeding of 2-acetylaminofluorine (2-AAF) was compared with the effect of continuous 2-AAF feeding on the urinary bladder of BALB/c female mice. Dietary concentrations of 2-AAF examined in this regard were 60, 75, 100, and 150 ppm. These diets were fed for 9, 12, or 15 months and then replaced with control feed, and the animals were killed at 18 or 24 months. Observations on these animals were contrasted with observations made at the same intervals on animals continuously fed these concentrations of 2-AAF throughout the study. Discontinuing 2-AAF exposure decreased prevalence of bladder tumors below that found in continuously exposed mice and resulted in a decrease in severity and prevalence of hyperplasia. Mild hyperplasia was retained in a portion of each population and the prevalence of this hyperplasia was dependent on both the period of exposure and the dose of 2-AAF. Mild persistent hyperplasia was correlated closely with neoplasia and may be a necessary stage in neoplastic development. Moderate or severe hyperplasia apparently regress when 2-AAF feeding is discontinued and do not appear to be essential steps in the neoplastic process.

Dose and time responses models for the incidence of bladder and liver neoplasms in mice fed 2-acetylaminofluorene continuously.

The incidence of bladder and liver neoplasms developed in mice continuously fed 2-acetylaminofluorene (2-AAF) in a multidisciplined study conducted at the National Center for Toxicological Research (NCTR) is represented using a probit model. The estimated incidences are made from time intervals using both sacrificed and dead and moribund mice. A probit log dose model is fit for each type of neoplasm at each time interval where the incidence permits. A probit log time model is fit for each of the neoplastic types for each dose level where the incidence permits. Finally, the response of liver neoplasms to 2-AAF is represented in the dose time plane using a probit plane model. 2-AAF appears to be an early acting bladder carcinogen and a late acting liver carcinogen. The bladders seem to be much more uniform in their response to the carcinogen than the livers and the uniformity of neither response appears to be age dependent. Although the time to appearence of both liver and bladder neoplasms is dose related, the increment in incidence of liver neoplasms with respect to time is not dose related while that of the bladder neoplasms is dose related. The data for bladder neoplasms do not contradict the "no threshold" theory of carcinogenesis while the liver data strongly support it.

Analysis of life-shortening effects in female BALB/C mice fed 2-acetylaminofluorene.

A total of 4320 female BALB/cStCrlfC3H/Nctr mice were continuously fed 2-acetylaminofluorene (2-AAF) in the diet at various concentrations in order to obtain a quantitative assessment of dose-related life-shortening effects. Statistical procedures which adjust for extraneous deaths were used to analyze the survival data. In addition to the demonstration of a reduced survival time associated with bladder and liver tumors induced by 2-AAF, a further toxic effect was observed at 150 ppm. Since the experiment was replicated six times, variability in the survival times of animals in different replicates was studied. Although all replicates were conducted under specific pathogen free / defined flora (SPF/DF) conditions, a significant change in survival time was demonstrated across replicates.

Estimation of distributions of time to appearance of tumor and time to death from tumor after appearance in mice fed 2-acetylaminofluorene.

A recently-developed statistical technique is employed to estimate parameters of the distributions of time to appearance of tumors and time to death from tumors after their appearance using both interval sacrifice and survival information. Estimates are obtained for three types of tumors at each of eight dose levels of 2-AAF, namely, 0, 30, 35, 60, 75, 100 and 150 ppm, which were administered to a total of 20880 female BALB/cStCrlfC3Hf/Nctr mice. For bladder neoplasms, the estimated median times to tumor ranged from 19.8 months at 150ppm to 1876.6 months at 0 ppm, while for liver neoplasms the extremes were 25.2 months at 150 ppm and 37.7 months at 0 ppm. Reticulum cell sarcomas occurred on the average at 24.7 months in all dose groups except 150 ppm, where the average time to appearance was 22.9 months. Although a pronounced dose-response for time-to-tumor was expressed for both bladder and liver neoplasms, the time required for death from either neoplasm following its appearance was not dose-related. The estimated median times to death after appearance of tumors were as follows: bladder neoplasm, 6.8 months; liver neoplasm, 5.7 months; reticulum cell sarcoma, 3.0 months.

An unbalanced experimental design for dose response studies.

Sixty sets of real data for 15 different pesticides from both sexes of Balb/C mice in two different experimental designs were generated at NCTR. The quantal responses for the dose groups in this data ranged from 1% to 90%. It was shown that the data could be represented equally well by a probit or logit transformation. It was further shown that the investment in terms of 7 times as many animals would greatly increase the confidence in estimating the parameters of the model and in predicting the dose at the low end of the dose response. Most important, it was shown that the estimation of a safe dose for a specified risk was greatly influenced by the choice of experimental design and method of extrapolation. It might be worth the investment in better experimental design to both the consumer and to the chemical industry if higher safe doses could be established which would allow the chemical to better accomplish its purpose and yet improve the assurance of the safety of the consumer.

Concomitant aortic valve replacement and myocardial revascularization.

Twenty-six consecutive patients underwent combined aortic valve replacement and myocardial revascularization at the Emory University Affiliated Hospitals between May, 1973 and March, 1976. Acute myocardial infarction resulted in two operative deaths (8%). There have been four late deaths, all Class IV preoperative. The age range was 37 to 79 years with an average age of 60. Preoperatively all patients were Class IV or late Class III. Twenty-three patients had symptoms of angina pectoris; congestive heart failure was evident in 56%. Postoperatively, 70% are now Class 1 or II. Single coronary bypass was performed in 16 patients, double in 6, and triple in three. Double bypass plus mitral valve replacement was required in two with aneurysmectomy in one. The rate of intraoperative infarction was 27% for the series but only 7% in the last year. The methods of intraoperative myocardial preservation and the technical approach for the operative procedures were variable. Results with each method are correlated, and currently preferred techniques are presented and discussed. Best results were obtained in patients who presented early in their symptomatic course with isolated proximal coronary lesions and good renoff vessels. Excellent results could be achieved despite advanced age of patients, requirement for multiple bypass grafts, and correction of other associated cardiac lesions. Poorest results were obtained when long-standing ventricular failure was combined with poor vessels distal to coronary stenoses.

Estimation of the LD1 and extrapolation of the LD0.1 for five organophosphate pesticides.

The oral LD50,s for organophosphate pesticides have been determined in CD-1 strain male and female mice. The values in mg/kg are: Trichlorfon, 800 and 800; Naled, 409 and 330; Dichlorvos, 139 and 133, GC6506, 23.4 and 17.8; Fospirate, 225 and 263 respectively. Toxicity was greater in males with Fospirate and greater in females with Naled and GC6506. The predicted LD1's and the extrapolated LD0.1's have been determined for the 5 organophosphates from an unbalanced design, loaded heavily toward the lower end of the dose-response curve. It has been shown that the slopes of the curves obtained with 50, 100 and 660 animals are parallel for all compound except Fospirate in the 660 mouse experiments. This is probably related to excessive female deaths in the upper segment of the dose-response curve. Sex dependent lethality was observed with Trichlorfon, Dichlorvos and Fospirate with the males being more susceptible than the females except in the case of Fospirate where there was a reversal at the LD50 with greater susceptibility in the females. The conditions for obtaining accurate results in such experiments have been established. The implications of human exposure to low levels of the environmental pollutants have been discussed.

Estimation of the LD1 and extrapolation of the LD0.1 for five organothiophosphate pesticides.

The oral LD50's for five organothiophosphate pesticides have been determined in CD-1 strain male and female mice. The values in mg/kg are: parathion, 15.0 and 14.3; methyl parathion, 14.5 and 19.5; guthion, 7.15 and 6.35; imidan, 25.2 and 23.1; sumithion, 1,045 and 1,220 respectively. Toxicity was greater parathion but there was no difference in the males. The relationship between chemical structure and toxicity has been discussed. The addition of a methyl group in the meta position of the nitrophenyl group (sumithion) decreased toxicity 72 times compared to methyl parathion. The predicted LD1 and extrapolated LD0.1's have been determined for the five organothiophosphates and the conditions required for accurate results have been established. It has been shown that the slopes of the curves (males vs. females) obtained with 50, 100 and 660 animals are parallel for all compounds health implications of exposure to low levels of environmental pesticides have been discussed.