Derek B. Janszen

Haber's rule: a special case in a family of curves relating concentration and duration of exposure to a fixed level of response for a given endpoint.

The concept that the product of the concentration (C) of a substance and the length of time (t) it is administered produces a fixed level of effect for a given endpoint has been ascribed to Fritz Haber, who was a German scientist in the early 1900s. He contended that the acute lethality of war gases could be assessed by the amount of the gas in a cubic meter of air (i.e. the concentration) multiplied by the time in min that the animal had to breathe the air before death ensued (i.e. C x t=k). While Haber recognized that C x t=k was applicable only under certain conditions, many toxicologists have used his rule to analyze experimental data whether or not their chemicals, biological endpoints, and exposure scenarios were suitable candidates for the rule. The fact that the relationship between C and t is linear on a log-log scale and could easily be solved by hand, led to early acceptance among toxicologists, particularly in the field of entomology. In 1940, a statistician named Bliss provided an elegant treatment on the relationships among exposure time, concentration, and the toxicity of insecticides. He proposed solutions for when the log-log plot of C and t was composed of two or more rectilinear segments, for when the log-log plot was curvilinear, and for when the slope of the dosage-mortality curve was a function of C. Despite the fact that Habers rule can underestimate or overestimate effects (and consequently risks), it has been used in various settings by regulatory bodies. Examples are presented from the literature of data sets that follow Habers rule as well as those that do not. Habers rule is put into perspective by showing that it is simply a special case in a family of power law curves relating concentration and duration of exposure to a fixed level of response for a given endpoint. Also shown is how this power law family can be used to examine the three-dimensional surface relating C, t, and varying levels of response. The time has come to move beyond the limited view of C and t relationships inferred by Habers rule to the use of the broader family of curves of which this rule is a special case.

Mortality of iron foundry workers. IV: Analysis of a subcohort exposed to formaldehyde

In the final phase of the mortality study of workers at an automotive iron foundry, a subset (N = 3929) of the original cohort of 8147 men, consisting of those exposed to formaldehyde during the period from January 1960 through May 1987, was analyzed. In addition to the external US population, an internal population (N = 2032), consisting of men who had worked in the same foundry during the same time period but not in formaldehyde-exposed jobs, was also used as a referent. Follow-up continued through December 31, 1989. Smoking status was ascertained for 65.4% of the exposed and for 55.1% of the unexposed cohorts. Detailed work histories and evaluation of occupational exposures by an industrial hygienist enabled us to categorize cumulative formaldehyde and silica exposures. Standardized mortality ratios were used to compare the mortality experience of the exposed cohort with the US population and, because of concerns about the healthy worker effect, with an occupational referent population. Relative risks for race, formaldehyde exposure status, smoking status, and silica exposure level were estimated by fitting a Poisson regression model to four causes of death: cancers of the buccal cavity and pharynx, lung cancer, diseases of the respiratory system, and emphysema. No association between formaldehyde exposure and deaths from malignant or nonmalignant diseases of the respiratory system was found. Cigarette smoking and silica exposure were found to be significantly associated with deaths attributed to lung cancer and disease of the respiratory system.

Benchmark dose Risk Assessment for formaldehyde using airflow modeling and a single-compartment, DNA-protein cross-link dosimetry model to estimate human equivalent doses

Formaldehyde induced squamous-cell carcinomas in the nasal passages of F344 rats in two inhalation bioassays at exposure levels of 6 ppm and above. Increases in rates of cell proliferation were measured by T. M. Monticello and colleagues at exposure levels of 0.7 ppm and above in the same tissues from which tumors arose. A risk assessment for formaldehyde was conducted at the CIIT Centers for Health Research, in collaboration with investigators from Toxicological Excellence in Risk Assessment (TERA) and the U.S. Environmental Protection Agency (U.S. EPA) in 1999. Two methods for dose-response assessment were used: a full biologically based modeling approach and a statistically oriented analysis by benchmark dose (BMD) method. This article presents the later approach, the purpose of which is to combine BMD and pharmacokinetic modeling to estimate human cancer risks from formaldehyde exposure. BMD analysis was used to identify points of departure (exposure levels) for low-dose extrapolation in rats for both tumor and the cell proliferation endpoints. The benchmark concentrations for induced cell proliferation were lower than for tumors. These concentrations were extrapolated to humans using two mechanistic models. One model used computational fluid dynamics (CFD) alone to determine rates of delivery of inhaled formaldehyde to the nasal lining. The second model combined the CFD method with a pharmacokinetic model to predict tissue dose with formaldehyde-induced DNA-protein cross-links (DPX) as a dose metric. Both extrapolation methods gave similar results, and the predicted cancer risk in humans at low exposure levels was found to be similar to that from a risk assessment conducted by the U.S. EPA in 1991. Use of the mechanistically based extrapolation models lends greater certainty to these risk estimates than previous approaches and also identifies the uncertainty in the measured dose-response relationship for cell proliferation at low exposure levels, the dose-response relationship for DPX in monkeys, and the choice between linear and nonlinear methods of extrapolation as key remaining sources of uncertainty.

Nasal Epithelium as a Sentinel for Airborne Environmental Pollution

A wide range of chemicals, particulate matter, and gaseous air pollutants are present in urban atmospheres and may pose a significant health risk for human populations. Nasal passages are the first site of contact of the respiratory tract with the environment and offer significant protection to the lower respiratory tract by conditioning the inspired air. This activity, which includes removal of certain pollutants, places the nose at risk of pathological changes, including cancer. Mexico City residents are exposed to a complex mixture of air pollutants. Based on predicted nasal air flow characteristics, four nasal biopsy sites were selected for study in adult male volunteers from a control low polluted town (n = 12) and southwest metropolitan Mexico City permanent residents (n = 54). Clinical data with emphasis on nasal symptoms and histopathological changes including basal and goblet cell hyperplasia, squamous metaplasia, epithelial dysplasia, and neovascularization were evaluated. Immunohistochemical staining was used to assess accumulation of p53 protein. Control individuals had no respiratory symptoms and their biopsies were unremarkable. Mexico City residents complained of epistaxis, rhinorrea, nasal crusting, dryness, and nasal obstruction. Their biopsies showed patchy shortening of cilia, deciliated areas, basal cell hyperplasia, and squamous metaplasia. Dysplastic lesions were predominantly located on antral squamous epithelium and in squamous metaplastic epithelium of the posterior inferior turbinates and they exhibited p53 nuclear accumulation. Individuals with > 10 h of daily outdoor exposure for 5 years or more had the highest rate of dysplasia. Subjects with epistaxis were more likely to have dysplasias and neovascularization. Results of this study suggest: (a) Nasal lesions in Mexico City residents are likely the result of many potentially toxic and/or carcinogenic pollutants, including ozone, aldehydes, particulate matter, and unmeasured pollutants; (b) the alteration of the nasal mucociliary defense mechanisms and the effects of reactive and/or water-soluble materials and particulates could be playing a major role in the nasal pathology; (c) the accumulation of p53 protein in dysplastic nasal lesions in the context of prolonged exposure to air pollutants raises the possibility that p53 mutations are already present and are providing the squamous cells with a selective advantage for clonal expansion; and (d) the nasal passages provide a valuable sentinel tissue for the detection of toxic air pollutants.

Phase-Specific Developmental Toxicity in Mice Following Maternal Methanol Inhalation

Methanol is toxic to embryos of mice and rats when inhaled by dams at high concentrations. The present studies examined methanol-induced developmental toxicity following inhalation exposure (6 hr/day) of pregnant CD-1 mice to 5000, 10,000 or 15,000 ppm either throughout organogenesis (GD 6-15), during the period of neural tube development and closure (GD 7-9), or during a time of potential neural tube reopening (GD 9-11). Transient neurologic signs and reduced body weights were observed in up to 20% of dams exposed to 15,000 ppm. Examination of near-term fetuses revealed embryotoxicity (increased resorptions, reduced fetal weights, and/or fetal malformations) at 10,000 and 15,000 ppm, while 3-day exposures at 5000 ppm yielded no observable adverse effects. Terata included neural and ocular defects, cleft palate, hydronephrosis, deformed tails, and limb (paw and digit) anomalies. Neural tube defects and ocular lesions occurred after methanol inhalation between GD 7-9, while limb anomalies were induced only during GD 9-11; cleft palate and hydronephrosis were observed after exposure during either period. These findings were consistent with prior reports that maternal methanol inhalation at high levels induces developmental toxicity in a concentration-dependent manner. Furthermore, our data indicate that the spectrum of teratogenic effects depended upon both the timing (i.e., stage of embryonic development) and the number of methanol exposures.

Dose‐, route‐, and sex‐dependent urinary excretion of phenol metabolites in B6C3F1 mice

Phenol is the major oxidized metabolite of benzene, a known human leukemogen and ubiquitous environmental pollutant. Unlike benzene, phenol does not induce tumors in mice following oral exposure; benzene also exhibits sex-related differences in genotoxicity to bone marrow cells that are not observed following phenol administration. We studied the urinary excretion of phenol metabolites in mice as a means to further investigate the metabolic basis for differences in benzene- and phenol-induced toxicity. Male and female B6C3F1 mice (n = 3/group) were exposed to 15, 40, 100, or 225 mumol [14C]phenol/kg by i.v. tail vein injection (6 microCi/mouse). First-pass intestinal metabolism of phenol was evaluated by comparison of urinary excretion of phenol metabolites following i.v. administration with additional groups of male mice that received the same dose levels by oral gavage. Mice were placed in glass metabolism cages, and urine was collected over dry ice for 48 h. Urinary metabolites were separated by high-pressure liquid chromatography (HPLC) and quantified by liquid scintillation spectrometry. Urinary excretion of conjugated metabolites of phenol was dose-dependent in both male and female mice administered phenol by i.v. injection or gavage. The major urinary metabolites of phenol were phenol sulfate (PS), phenol glucuronide (PG), and hydroquinone glucuronide (HQG). Sulfation was the dominant pathway at all dose levels, but decreased as a percent of the excreted dose with a concomitant increase in glucuronidation as the dose level increased. Male mice consistently excreted a higher proportion of phenol as the oxidized conjugated metabolite, HQG, compared to female mice, suggesting that male mice oxidize phenol to hydroquinone more rapidly than female mice. Increased oxidation of phenol to hydroquinone by male mice compared to female mice is consistent with both the greater sensitivity of male mice to the genotoxic effects of benzene and the greater potency of hydroquinone compared to phenol as a genotoxicant. Intestinal conjugation of phenol prior to absorption was significant only at low doses and thus alone does not provide an explanation for the lack of carcinogenicity of phenol in bioassays conducted at much higher dose levels.

Effect of different sampling designs on outcome of endocrine disruptor studies.

In this article, we demonstrate how sampling strategy can influence the outcome of endocrine disruptor studies. In a study of the weak xenoestrogen bisphenol A (BPA), possible treatment-related effects on ventral prostate (VP) fresh weight were found in rat offspring at 6 months of age when only one or two male pups were randomly selected from each litter. In subsequent BPA and di-n-butyl phthalate studies, large intralitter variability of this specific end point was apparent when the VP weights from entire litter complements were examined. We modeled the effects of sampling 1, 2, or 3 pups from each litter using the full-litter complement data. When one pup was randomly selected, a substantial percentage of incorrect conclusions about the presence or absence of treatment effects occurred. These statistical modeling analyses raise significant concern about the selection of one pup per litter for highly variable end points.

Neurotoxicological Evaluation of Ethyl Tertiary‐Butyl Ether Following Subchronic (90‐day) Inhalation in the Fischer 344 Rat

The purpose of this study was to evaluate whether repeated 6‐h exposure (65 exposures over a 14‐week period) of male and female Fischer‐344 rats (n = 12 rats/sex/concentration) to ethyl tertiary‐butyl ether (ETBE) atmospheres at 500, 1750, or 5000 ppm would result in neurotoxicity. Neurotoxicity was assessed by a blinded functional observational battery (FOB), motor activity, and terminal neuropathology. Motor activity was assessed 4 days prior to ETBE exposure and following 20, 42, and 65 days of exposure. The FOB was assessed 4 days prior to ETBE exposure and following 1, 6, 10, 20, 42, and 65 days of exposure. Transient ataxia, a sign of narcosis, was noted in male rats immediately following the 6‐h exposure to 5000 ppm ETBE. Statistically significant treatment effects on motor activity were not observed. Minor changes in grip strength and hindlimb splay were observed; however, none demonstrated a dose–response relationship or a consistent pattern of neurological dysfunction. No gross or microscopic abnormalities were observed in the central, peripheral, or autonomic nervous systems of rats exposed to 5000 ppm ETBE. No statistically significant differences in brain weight or size were observed in ETBE‐exposed rats. A statistically significant increase in body weight was observed in female rats exposed to 5000 ppm following 42 and 65 exposure days. Although ataxia was a common feature of acute ETBE neurotoxicity in rats following high‐level exposure, adverse neurological effects are not expected in the general public at the anticipated exposure levels associated with automotive refueling.

Evaluation of the metabolism and hepatotoxicity of styrene in F344 rats, B6C3F1 mice, and CD-1 mice following single and repeated inhalation exposures

Styrene is used for the manufacture of plastics and polymers. The metabolism and hepatotoxicity (mice only) of styrene was compared in male B6C3F1 mice, CD-1 mice, and F344 rats to evaluate biochemical mechanisms of toxicity. Rats and mice were exposed to 250 ppm styrene for 6 h/day for 1 to 5 days, and liver (mice only) and blood were collected following each day of exposure. Mortality and increased serum alanine aminotransferase (ALT) activity were observed in mice but not in rats. Hepatotoxicity in B6C3F1 mice was characterized by severe centrilobular congestion after one exposure followed by acute centrilobular necrosis. Hepatotoxicity was delayed by 1 day in CD-1 mice, and the increase in ALT and degree of necrosis was less than observed for B6C3F1 mice. Following exposure to unlabeled styrene for 0, 2, or 4 days, rats and mice were exposed to [7-14C]-styrene (60 microCi/mmol) for 6 h. Urine, feces, and expired air were collected for up to 48 h. Most styrene-derived radioactivity was excreted in urine. The time-course of urinary excretion indicates that rats and CD-1 mice eliminated radioactivity at a faster rate than B6C3F1 mice following a single 250 ppm exposure, consistent with a greater extent of liver injury for B6C3F1 mice. The elimination rate following 3 or 5 days of exposure was similar for rats and both mouse strains. Following three exposures, the total radioactivity eliminated in excreta was elevated over that measured for one exposure for both mouse strains. An increased excretion of metabolites on multiple exposure is consistent with the absence of ongoing acute necrosis following 4 to 5 daily exposures. These data indicate that an induction in styrene metabolism occurs after multiple exposures, resulting in an increased uptake and/or clearance for styrene.

Pleural Lesions in Syrian Golden Hamsters and Fischer-344 Rats Following Intrapleural Instillation of Man-made Ceramic or Glass Fibers*

The mesothelium is a target of the toxic and carcinogenic effects of certain natural mineral and man-made fibers. Long-term inhalation of a ceramic fiber (RCF-1) results in a high incidence of pleural mesotheliomas in Syrian golden hamsters but not in identically exposed Fischer-344 rats. The present study compared the histopathology of the early pleural response in rats and hamsters instilled with artificial fibers. Groups of Syrian golden hamsters and Fischer-344 rats were instilled with ceramic (RCF-1) or glass (MMVF-10) fibers directly into the pleural space. Each species received approximately equal numbers of long, thin fibers per g body weight. Fiber-induced lesions were compared 7 and 28 days postinstillation. Both hamsters and rats developed qualitatively similar dose-dependent inflammatory lesions that were not fiber-type specific. Both species developed fibrosis in conjunction with inflammation in the visceral pleura, but a striking interspecies difference was noted in the pattern of mesothelial cell response. Hamsters developed greater surface mesothelial cell proliferation and had focal aggregates of mesothelial cells embedded deep within regions of visceral pleural fibrosis. It is hypothesized from the present study that the marked fiber-induced proliferative mesothelial cell response of the hamster visceral pleura may explain the high number of pleural mesotheliomas found in long-term fiber studies in this species.