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Chemicals possessing persistence (P) and high mobility (M) can present a hazard to drinking water resources by traversing natural barriers like riverbanks and artificial barriers found in water treatment plants. If the chemical is also toxic (T), i.e. classifiable as a PMT, the agent might be of particular concern as a potential drinking water contaminant. During routine water sampling, detection and quantitation of polar substances with high mobility can be problematic. The German Environment Agency (UBA) is considering the use of the Log Koc value as a proxy for mobility (M). Log Koc is related to Log P by the equation Log Koc = 0.69 Log P + 0.22. In this study, we demonstrate that chemicals with log P values at or very close to 2.0, 3.0 or 4.0 (and their concomitant log Koc values) can vary significantly in their chemical structures, molecular weights, molar volumes, and calculated molar refractivity (CMR), which is related to the mean polarizability of a molecule. The large degree of potential diversity in chemical structure and molecular parameters related to chemical behavior at a particular log P or log Koc value suggests that log Koc might not contain enough information to function as a standalone surrogate for the mobility (M) of a chemical, i.e. as related to its ability to move from a drinking water resource through the water plant purification process.

Exposure to chemicals produced by natural processes is ubiquitous. First, in addition to the products of normal metabolism produced in humans of normal body weight, adipose tissue produces a large number of chemicals, including estrogen, testosterone from the produced estrogen, thyroid-stimulating hormone, leptin and approximately 500 other molecules termed adipokines, and a large number of inflammatory mediators. Second, the gut biome contains approximately the same number of bacteria as cells found in the entire body and produces a large number of small molecules. Third, the overwhelming majority (99.9%) of pesticide exposure occurs during ingestion of natural plant pesticides from eating vegetables. Fourth, consumption of cooked muscles meats leads to significant exposure to mutagenic and carcinogenic heterocyclic amines, polycyclic aromatic amines, and nitropyrenes. Fifth, many common beverages, for example, beer, coffee, and tea contain organic chemicals that display mutagenic activity. As compared with man-made production levels, from 1945 to 2015, an estimated 5000-fold more organic compounds were produced by a variety of natural processes, including common wood-degrading and forest litter-degrading fungi, microorganisms in temperate and boreal forest soils, bacteria in marine sponges, marine macro-algae, volcanoes, and forest fires. Exposure to these naturally produced organic compounds occurs via inhalation of ambient air, ingestion of food and water, and contact with soil, freshwater, and seawater. Contact with several thousand different endogenous or exogenous chemicals per day is unavoidable. This understanding might assist in better allocating resources toward controlling exposures to agents of highest concern as determined by current concepts of chronic disease causation.

The bacterial reverse mutation assay, that is, the Ames test, measures mutations that reverse the inactivation of a gene involved in the synthesis of either histidine in Salmonella bacteria or tryptophan in Escherichia coli. The classic dose–response curve of an Ames assay plots number of reverse mutations (“revertants”) on the y-axis versus dose of the test chemical on the x-axis. Frequently, the dose–response curve resembles a parabola with a linear initial slope resulting from the accumulation of mutations, which transitions to a downward curvature resulting from cell killing (cytotoxicity) at increasingly higher doses of the test chemical. For regulatory purposes, a positive Ames test is usually considered as induction of twice the number of reverse mutations above background levels. For research purposes, the potency of the mutagenic response can be calculated from measuring the initial slope of the mutagenic response. This initial slope can be calculated in a manner that disentangles the downward pull on the initial slope value provided by the initiation of cytotoxicity. For a dose–response curve resembling a parabola, both the initial positive slope representing mutagenicity and the secondary negative slope representing cytotoxicity can be calculated from the same dose–response curve. The Ames test is the most commonly conducted genotoxicity assay. When a series of molecular congeners are assayed in the Ames test for mutagenicity, additional consideration of the cytotoxicity can provide important structure–activity relationship information.

In 2018, the National Institutes of Health estimated that 1,735,350 new cancer cases were diagnosed in the United States and 609,640 patients died from various forms of cancer. The vast majority of those cancer deaths occurred from cancers derived from epithelial cells, that is, carcinomas. The most lethal cancers in American women based upon estimated deaths occurred in the lung and bronchus (352,209), breast (205,675), colon and rectum (123,617), pancreas (96,571), and ovary (71,141). The largest cancer killers in men occurred in the lung and bronchus (427,587), prostate (140,086), colon and rectum (135,542), pancreas (100,599), and liver (80,526). Histologically different carcinomas are initiated, clonally expanded, and progressed in association with particular changes in genes and also epigenetic alterations. Currently employed genotoxicity testing protocols emphasize testing for the initiating (mutational) potential of the test agent. While 2-year chronic rodent cancer bioassays test for the entire spectrum of carcinogenic transformation and development, the high doses used in these assays induce cytotoxicity leading to increased cellular proliferation rates and high false-positive rates of tumor induction in non-genotoxic chemicals. The low cancer induction from high radiation exposures experienced by atomic bomb survivors in Hiroshima and Nagasaki, Japan, and the epidemiological evidence showing that cigarette smoking duration and not intensity is associated with lung cancer risk both support a more important role for tumor promotion rather than initiation in the clinical presentation of human carcinomas. Cancer hazard assessment testing protocols and weight-of-the-evidence analysis of agent-specific cancer risk should be better aligned with the pathogenesis of human carcinoma.

Chemical-induced pulmonary carcinogenesis in humans is usually a multi-decade process. The average age at diagnosis of lung cancer in cigarette smokers, and mesothelioma in asbestos workers, is approximately 70. The carcinogenic process consists of genetic changes to normal cells usually sequenced as initiation (mutations), promotion (clonal expansion of initiated cells), and progression (carcinoma in situ to invasive carcinoma to metastatic carcinoma). Angiogenesis, the sprouting of new vessels from preexisting capillaries, plays an important role in the progression of small avascular tumors to vascularized invasive carcinomas and metastatic carcinomas. While the overall carcinogenic process is multi-decade, the transition from avascular to vascularized carcinoma is believed to take place over the course of a much more limited time span. Tumor progression is a late-stage event in carcinogenesis. About 70% of human cancers including lung cancer express hypoxia-inducible factor (HIF)-1 in the absence of cobalt exposure. Cobalt compounds administered to transformed cell lines and primary cultures of human endothelial cells, smooth muscle cells, and mesenchymal stem cells can elicit overexpression of HIF-1. Cobalt-induced expression of HIF-1 would not be expected to interact with either the avascular initiation or promotion phases of carcinoma development. Humans exposed long-term to cobalt leaching from implants do not have an elevated cancer risk, and neither do goats ingesting up to 667 times the recommended daily human cobalt dietary requirement throughout their 15- to 18-year life span. In vitro hypoxia-mimetic characteristics of cobalt compounds do not correlate with the absence of increased risk following long-term systemic exposure to cobalt in humans.

The American Conference of Governmental and Industrial Hygienists (ACGIH ® ) states “…the TLV ® -CS Committee preferably relies on published, peer-reviewed literature available in the public domain.” Many studies used to document threshold limit values (TLVs) are not peer reviewed, with many unpublished. The academic studies published in the peer-reviewed literature upon which ACGIH relies to determine TLVs rarely report data not already statistically transformed and thus incalculable. From 2008 to 2018, ACGIH established or reviewed TLVs for 145 different substances or groups of substances. Studies underlying development of these 145 TLVs demonstrate limited reporting of data sufficient to facilitate independent statistical analysis. None of the 145 TLVs showed sufficient data to independently substantiate ACGIH’s evaluations. In the majority of cases, sentinel studies upon which calculation of the TLV was based cannot be determined. Occupational Safety and Health Administration (OSHA) recommends workplaces rely on ACGIH TLVs and National Institute for Occupational Safety and Health recommended exposure limits rather than older OSHA permissible exposure limit values to optimize worker safety. Inaccurate or poorly substantiated determination of TLVs can adversely affect both human health and commerce, as net US sales of the 145 substances represent an estimated USD$189 billion, about one-fourth of the USD$800 billion chemical market in the United States. ACGIH should provide the studies relied upon, individual data values supporting the TLVs, and statistical calculation methods underlying the development of a TLV. The current system of poorly supported and underreported TLVs has the potential to propagate errors into the standard setting process of other regulatory organizations both nationally and internationally, as the ACGIH TLVs are used by many foreign regulatory bodies. Increased transparency by ACGIH is consistent with recent reforms implemented by US EPA.

The maximum tolerated dose (MTD) provides the highest probability of a positive result in a toxicology bioassay. The assumption underlying the MTD in animal bioassays is that adverse effects at very high doses are qualitatively the same as those occurring at low doses. In contrast with the MTD, the optimal top dose in a toxicology animal study is the highest dose that does not produce a pathological end point that presents no risk at lower doses, for example, the dose below which cytotoxicity induces tumors in the absence of genotoxicity or other carcinogenic mechanisms. Normal concentrations or biological activity levels of many substances necessary for normal physiological function induce pathology when found at high levels. For example, the demonstration that ingestion of abnormally high levels of certain dietary fats can cause or exacerbate atherosclerosis in relevant animal models like rhesus macaques does not demonstrate that normal levels of these fats should be considered as toxic. Excessive estrogenic stimulation is associated with breast, ovarian, and endometrial cancers. This does not imply that normal age-appropriate levels of estrogen are toxic. Normal wound healing is associated with transforming growth factors beta 1 and 2. Excessive stimulation of fibroblasts by these growth factors results in hypertrophic scarring and keloid formation. An understanding of the mode of action of a test substance can facilitate the selection of dose levels much higher than those expected to be experienced by humans, but not beyond a dose level at which pathology is an experimental artefact of the high-dose level.

The immobility of plants exerted evolutionary selection pressures resulting in the production of thousands of chemical substances thought to function as pesticides against predation by insects and animals. More than 10,000 plant-derived compounds have been isolated with the existence of about 100,000 such compounds postulated. In 1990, Ames et al. reported that 99.99% by weight of the pesticides ingested in a normal human diet are derived from natural plant-based sources. This surprising result raised the question as to whether these natural plant pesticides were toxic to humans. These authors examined a relatively small subset of natural pesticides and determined that their tumorigenicity in rodent cancer bioassays was similar to synthetic pesticides. In this analysis, we used standard United States Environmental Protection Agency programs to estimate the toxicity (T.E.S.T. 4.2) and persistence (EPI Suite 4.1) of a series of high-volume synthetic and natural pesticides. On average, synthetic pesticides were more persistent in the environment than were natural pesticides. This result is consistent with cost, time, and logistical constraints under which farmers apply a limited number of applications of pesticides during a crop cycle. Synthetic and natural pesticides are predicted to possess toxicities including mutagenicity and developmental toxicity. Synthetic pesticides are less often mutagenic.

The National Toxicology Program has conducted 594, 2-year studies exposing various strains of rats and mice via different routes of exposure. In the current study, we analyze the results from 108 chemicals tested in 106, 2-year studies conducted by exposing F334/N rats and B6C3F1 mice via gavage. An additional 18, 2-year gavage studies have been conducted in Osborne–Mendel rats and B6C3F1 mice on 19 different chemicals. We analyze the results from 23 chemicals tested in 21, 2-year studies conducted by exposing F334/N rats and B6C3F1 mice via drinking water; 18 chemicals tested in 18, 2-year studies conducted by exposing F334/N rats and B6C3F1 mice via dermal application; and 11 chemicals tested in 11, 2-year studies conducted by exposing F334/N rats and B6C3F1 mice via intraperitoneal injection. The results from these 174 studies are analyzed and discussed separately. The neoplasticity of each chemical was analyzed for tumor incidence by species–sex category, tumor site concordance across species, and tumor site concordance across sex within species. When available the Ames Salmonella mutagenicity assay results, and any results from a test for genotoxicity other than the Ames test, were correlated with the neoplasticity results. Tumor site concordance across sex within species is generally higher than tumor site concordance across species. In addition, the high degree of variability of Ames test results suggests that historical Ames test data are less reliable than recent results conducted under good laboratory practices and employing Organization for Economic Cooperation and Development protocols relevant to the physicochemical characteristics of the test chemical.

The degree of correlation between tumors predicted by OncoLogic™ (Oncologic) and the actual formation of tumors as observed in the National Toxicology Program (NTP) 2-year rodent studies is lower for “justification reports” that incorporate historical data than for “data reports” that do not. The correlation between the ordinal ranking of the observed carcinogenicity of parent NTP chemicals and the predicted “level of carcinogenicity concern” from the justification reports obtained from Oncologic is poor ( r = 0.56). Similarly, the correlation between the ordinal ranking of the carcinogenicity of metabolites from parent NTP chemicals and the predicted “level of carcinogenicity concern” from the justification report obtained from Oncologic is also poor ( r = 0.43). In contrast, the correlation between the ordinal ranking of the observed carcinogenicity of parent NTP chemicals and the predicted level of carcinogenicity concern from the data reports obtained from Oncologic is comparatively better ( r = 0.75). The correlation between the ordinal ranking of the carcinogenicity of metabolites from parent NTP chemicals and the predicted “level of carcinogenicity concern” from the data reports generated from Oncologic is also comparatively good ( r = 0.68). The level of correlation between the ordinal tumorigenicity ranks of parent chemicals and between the ordinal tumorigenicity ranks of chemicals reported to induce liver tumors in the National Center for Toxicological Research liver cancer database was also investigated. There was a higher degree of correlation seen for Oncologic “data reports” as compared with Oncologic “justification reports.” Incorporation of additional information via “justification reports” weakens the predictive power of Oncologic.

Since its inception in 1976, the National Toxicology Program (NTP) has conducted 594 2-year studies on rats and mice by a number of routes of administration including inhalation, feed, drinking water, dermal, and intraperitoneal injection. Of these studies, the results on 470 chemicals were of adequate technical quality to be incorporated into final technical reports. In this study, the 470 chemicals were categorized from 1 to 48 by the level of “clear” neoplastic evidence in male and female rats, and in male and female mice, and given an ordinal rank from 1 to 135 following additional considerations regarding tumor site concordance and tumor multiplicity. The resultant tumorigenicity category score and ordinal rank score were examined for associations with results in the Ames Salmonella mutagenicity assay; presence or absence of structural alerts of carcinogenicity; and three Hansch Quantitative Structure-Activity Relationship (QSAR) parameters, namely, calculated base 10 logarithm of the octanol–water partition coefficient (ClogP), calculated molar refractivity (CMR), and McGowan molecular volume (MgVol). Smaller molecular volumes were found to be associated with higher levels of tumorigenicity. Whereas lower rather than higher levels of lipophilicity were found to be associated with higher levels of tumorigenicity. Positive Ames test results were positively correlated with overall tumorigenicity and with possession of structural alerts. Since larger organic molecules have more chemical reaction centers, it was not surprising that higher ClogP values were positively correlated with the number of structural alerts. The results from this study demonstrate the ability to devise rational rules for relative tumorigenicity that correlate, in biologically plausible ways, with known parameters of toxicity.

This study presents an analysis of species, sex concordance in tumorigenicity among National Toxicology Program (NTP) chemicals tested for carcinogenicity by feed. It includes mutagenicity and other genetic toxicology test results, as well as Log P values. A total of 213 chemicals were tested in 212 2-year studies by exposing F334/N rats and B6C3F1 mice in their feed. Twenty-two of the 213 chemicals tested were clearly neoplastic in male and female rats and mice. Thirteen of these 22 chemicals were positive in at least one Ames test representing a prediction accuracy of 59.1%. Seventy-six of 213 chemicals tested were not neoplastic in either rodent species. Of the 22 ubiquitously neoplastic chemicals, 14 were concordant for developing tumors at the same anatomical site both across species and across sex within species. Of the 22 ubiquitously neoplastic chemicals, none were concordant for developing tumors at the same anatomical site across species, but not across sex within species. The neoplastic response to chemicals administered in feed displays more concordance within either male or female rats, or male and female mice, than between male rats and male mice, or female rats and female mice. In the NTP feed studies, negative Ames test results are more predictive of neoplastic responses than positive Ames test results. Log P values for the chemicals that produced tumors in male and female rats and mice were compared to chemicals that did not produce tumors. A statistical comparison between these Log P values showed no relationship between Log P values and neoplasticity.

The term oxidative stress was introduced by the National Toxicology Program (NTP) in 1985. It is now invoked as a contributing mechanism to the induction of tumors in chronic rodent cancer bioassays following administration of a chemical agent, especially via inhalation. The term oxidative stress is confusing as there are two different types of oxidative stress. Direct oxidative stress derives from chemical damage to mitochondrial energy-producing structures with concomitantly altered mitochondrial function. Indirect oxidative stress is frequently quantitatively greater than direct oxidative stress in terms of release of activated oxygen or nitrogen species secreted by one or more of 12 potential in situ or recruited inflammatory cell types. Evidence supporting a role for direct oxidative stress in chemical carcinogenesis comes from cell culture studies wherein the cells have been removed from their natural environment and denuded of defense systems, for example, removal of particles by cilia, macrophage engulfment of particles, and suspension and dilution of chemicals or particles in pulmonary surfactant. In contrast, extensive direct evidence from bronchoalveolar lung washes conducted on humans shows elevations in pulmonary macrophage and neutrophil counts, release of intravascular proteins into the extracellular space, and activation of pro-inflammatory cytokines that correlate with observed pulmonary pathology. Higher rates of lung cancer in smokers with chronic obstructive pulmonary disease (COPD) than in smokers at the same cigarette consumption level but without COPD demonstrate a positive contribution by pulmonary inflammation to pulmonary carcinogenesis. Indirect oxidative stress from pulmonary inflammation should be considered before allocating a primary carcinogenic role for direct oxidative stress.

International Agency for Research on Cancer (IARC) classifies agents as to their potential carcinogenicity in humans. Monographs 1–123 categorize 120 agents as group 1 (carcinogenic to humans); 82 agents as group 2A (probably carcinogenic to humans); 311 agents as group 2B (possibly carcinogenic to humans); 499 agents as group 3 (not classifiable as to its carcinogenicity to humans); but only a single agent, caprolactam, a precursor for the manufacture of Nylon 6, fibers, and plastic as group 4 (probably not carcinogenic to humans). The evidence regarding carcinogenic potential to humans for a randomly selected sample of 100 group 3 agents was studied to determine whether the paucity of agents classified as group 4 represented the state of the scientific evidence or a reluctance on the part of IARC to classify a chemical into the lowest risk category. Of the 100 agents evaluated, a significant minority estimated at 24% did not possess structural elements of carcinogenicity, mutagenic activity in the Ames Salmonella assay or activity in a cell transformation assay. The results of this analysis suggest that the lack of agents classified as group 4 represents a reluctance on the part of IARC to place agents into the lowest category of risk. Failure to appropriately place agents into group 4 unnecessarily exhausts valuable staff resources as these low risk compounds require periodic updating of the evidentiary basis of their classification as a group 3 agent. It would be advantageous for both IARC and public health agencies if a clearer differentiation of group 3 and group 4 agents was employed.

A viable approach for the analysis of the enantiomeric distributions of volatile components in essential oils based on automatic injection solid-phase microextraction-chiral-gas chromatography-mass selective detection (AutoSPME-chiral-GC-MSD) is demonstrated. From a selection of polar and non-polar fibers of varying thickness, the relatively non-polar thin-film polydimethylsiloxane fiber is found to be the best fiber for the analysis. Fiber exposure to the headspace above sub-microliter quantities of the oils for approximately 6 s is all that is required to obtain both the required sensitivity and resolution to produce excellent reproducibilities (generally ≤ 5.0%). The findings using this approach are in good agreement with previous findings based on chiral-GC-flame ionization detection analysis of solutions of essential oils. These results clearly establish AutoSPME as a simple, rapid, accurate, and precise method of sample preparation for the qualitative and quantitative analysis of enantiomeric distributions within essential oils. This work will be expanded to include studies directed at determining the purity of additional essential oils as well as experiments directed at determining the production location of essential oils.

Menthol occurs naturally in oils of the Mentha species in the (1 R, 3R, 4S)-(–) form (l-menthol), whereas synthetic menthol is available either in the same form or as a racemic mixture (d- and l-menthol). Quantitative analysis of the presence of the (1S, 3S, 4R)-(+)- form (d-menthol) is achieved by using gas chromatographic analysis on a chiral capillary column with selective ion monitoring detection. Detection of the presence of as little as 0.01 % (d-menthol in the total menthol concentration is possible with relative standard deviation values averaging around 7%. Minimal sample preparation with short sample analysis times of 30 min provide for a rapid sample turn around. This method should be applicable to the speciation of menthol in a wide variety of menthol-containing products, including cigarettes.

Based on the characteristics of a wide range of natural products, a more comprehensive understanding of the role of the Maillard reaction has been developed which describes a molecular basis for the production of a common set of flavourful, volatile aromatic compounds from the reactions between amino acids and sugars at elevated temperature (pressure cooking). By employing a variety of natural products, a clearer knowledge of the common mechanisms for the production of many important flavourants and odourants has been established. Regardless of the starting natural product the same qualitative profile of volatile materials is produced upon ‘pressure cooking’ an aqueous extract of the natural product. The notable differences in aroma arise not from significant differences in the qualitative nature of the materials but from variations in the quantitative distribution of the volatile materials produced.

Percutaneous penetration enhancers (PPEs) are chemicals used to enhance the transdermal delivery of drugs. Fifty-eight of the approximately 150 PPEs used for the transdermal delivery of drugs have been reported in cigarette mainstream smoke (MS). MS is a complex aerosol of minute liquid droplets (termed the particulate phase) suspended within a mixture of gases (CO(2), CO, NO(x), etc.) and semi-volatile compounds. The gases and many of the semi-volatiles are termed the vapor phase. Twenty-nine of the 58 PPEs have been identified in MS vapor phase, 15 in the particulate phase and 14 in both the vapor and particulate phases. There is a tendency for MS PPEs to be hydrophobic, with 40 of the 58 compounds (69%) being either hydrophobic or strongly hydrophobic, and only 24% being hydrophilic. Many of the 4800 known constituents of MS are hydrophilic and would not be expected to readily cross cell membranes or penetrate tissue when delivered as single compounds. The in vivo effect on biological activity of the juxtaposition within the cigarette smoke aerosol of the large number of hydrophilic constituents with the 58 PPEs is currently unknown. As an initial step in understanding this potential complex interaction, the 58 PPEs in MS have been identified and a number of molecular parameters related to the ability to penetrate tissue have been calculated, including MS concentration, measured and calculated base ten logarithm of the octanol-water partition coefficient (Mlog P and Clog P), molecular volume (MgVol) and calculated molar refractivity (CMR).

Cigarette smoke is a complex aerosol of minute liquid droplets (termed the particulate phase) suspended within a mixture of gases (CO(2), CO, NO(x), etc.) and semi-volatile compounds. The International Agency for Research on Cancer (IARC) has classified a number of the chemical constituents reported in cigarette mainstream smoke (MS) as carcinogens. Previously, we published a series of historical reviews reporting that 11 IARC Group 1 (known human), nine Group 2A (probable human) and 48 Group 2B (possible human) carcinogens have been observed in MS. Here, we expand the list of IARC classified carcinogens from 68 to 81 compounds (11 Group 1, 14 Group 2A and 56 Group 2B) reported in MS. A number of the IARC compounds reported in MS are found in the vapor phase including three Group 1, eight Group 2A and 18 Group 2B constituents. Several IARC MS compounds are found in both the vapor and particulate phases including two in Group 1, one in Group 2A and one in Group 2B. Forty-eight IARC MS carcinogens are found in the particulate phase only. Lipophilicity, as determined by the base 10 logarithm of the calculated octanol-water partition coefficient and denoted as Clog P, is reported for each of the 71 non-metallic MS IARC carcinogens. Clog P correlates with a number of biological activities including in vitro mutagenicity and carcinogenicity in rodents, and in the absence of any additional toxicological or epidemiological data, a high log P compound is more likely to be carcinogenic than a low log P compound.

Tobacco smoke aerosols with fewer mutagens in the particulate fraction may present reduced risk to the smoker. The objective of this study was to test the hypothesis that the temperature at which tobacco is pyrolyzed or combusted can affect the mutagenicity of the particulate fraction of the smoke aerosol. Tobacco smoke aerosol was generated under precisely controlled temperature conditions from 250 to 550 degrees C by heating compressed tobacco tablets in air. The tobacco aerosols generated had a cigarette smoke-like appearance and aroma. The tobacco smoke aerosol was passed through a Cambridge filter pad to collect the particulate fraction, termed the smoke condensate. Although condensates of tobacco smoke and whole cigarette mainstream smoke share many of the same chemical components, there are physical and chemical differences between the two complex mixtures. The condensates from smoke aerosols prepared at different temperatures were assayed in the Ames Salmonella microsome test with metabolic activation by rat liver S9 using tester strains TA98 and TA100. Tobacco smoke condensates were not detectably mutagenic in strain TA98 when the tobacco smoke aerosol was generated at temperatures below 400 degrees C. Above 400 degrees C, condensates were mutagenic in strain TA98. Similarly, condensates prepared from tobacco smoke aerosols generated at temperatures below 475 degrees C were not detectably mutagenic in strain TA100. In contrast, tobacco tablets heated to temperatures of 475 degrees C or greater generated smoke aerosol that was detectably mutagenic as measured in TA100. Therefore, heating and pyrolyzing tobacco at temperatures below those found in tobacco burning cigarettes reduces the mutagenicity of the smoke condensate. Highly mutagenic heterocyclic amines derived from the pyrolysis of tobacco leaf protein may be important contributors to the high temperature production of tobacco smoke Ames Salmonella mutagens. The relevance of these findings regarding cancer risk in humans is difficult to assess because of the lack of a direct correlation between mutagenicity in the Ames Salmonella test and carcinogenicity.