Affiliation: Professor, Johns Hopkins Bloomberg School of Public Health
Country: United States
Phone: 1 (410) 706-1736
Fax: 1 (410) 706-8013
Past position(s): Professor, Senior Scientist, Environmental Defense Fund Washington, DC
Education: AB, 1967, Vassar College; PhD, 1972, Johns Hopkins
Areas of interest: Environmental toxicology; molecular epidemiology
Neurotoxicity and reproductive toxicity are important areas for risk assessment, since the nervous and reproductive systems are highly sensitive to xenobiotic effects. Many agents have been identified as toxic to these systems in humans (Barlow and Sullivan 1982; OTA 1990). Many pesticides are deliberately designed to disrupt reproduction and neurological function in target organisms, such as insects, through interference with hormonal biochemistry and neurotransmission.
It is difficult to identify substances potentially toxic to these systems for three interrelated reasons: first, these are among the most complex biological systems in humans, and animal models of reproductive and neurological function are generally acknowledged to be inadequate for representing such critical events as cognition or early embryofoetal development; second, there are no simple tests for identifying potential reproductive or neurological toxicants; and third, these systems contain multiple cell types and organs, such that no single set of mechanisms of toxicity can be used to infer dose-response relationships or predict structure-activity relationships (SAR). Moreover, it is known that the sensitivity of both the nervous and reproductive systems varies with age, and that exposures at critical periods may have much more severe effects than at other times.
Neurotoxicity Risk Assessment
Neurotoxicity is an important public health problem. As shown in table 1, there have been several episodes of human neurotoxicity involving thousands of workers and other populations exposed through industrial releases, contaminated food, water and other vectors. Occupational exposures to neurotoxins such as lead, mercury, organophosphate insecticides and chlorinated solvents are widespread throughout the world (OTA 1990; Johnson 1978).
Table 1. Selected major neurotoxicity incidents
|400 BC||Rome||Lead||Hippocrates recognizes lead toxicity in the mining industry.|
|1930s||United States (Southeast)||TOCP||Compound often added to lubricating oils contaminates “Ginger Jake,” an alcoholic beverage; more than 5,000 paralyzed, 20,000 to 100,000 affected.|
|1930s||Europe||Apiol (with TOCP)||Abortion-inducing drug containing TOCP causes 60 cases of neuropathy.|
|1932||United States (California)||Thallium||Barley laced with thallium sulphate, used as rodenticide, is stolen and used to make tortillas; 13 family members hospitalized with neurological symptoms; 6 deaths.|
|1937||South Africa||TOCP||60 South Africans develop paralysis after using contaminated cooking oil.|
|1946||—||Tetraethyl lead||More than 25 individuals suffer neurological effects after cleaning gasoline tanks.|
|1950s||Japan (Minimata)||Mercury||Hundreds ingest fish and shellfish contaminated with mercury from chemical plant; 121 poisoned, 46 deaths, many infants with serious nervous system damage.|
|1950s||France||Organotin||Contamination of Stallinon with triethyltin results in more than 100 deaths.|
|1950s||Morocco||Manganese||150 ore miners suffer chronic manganese intoxication involving severe neurobehavioural problems.|
|1950s-1970s||United States||AETT||Component of fragrances found to be neurotoxic; withdrawn from market in 1978; human health effects unknown.|
|1956||—||Endrin||49 persons become ill after eating bakery foods prepared from flour contaminated with the insecticide endrin; convulsions result in some instances.|
|1956||Turkey||HCB||Hexachlorobenzene, a seed grain fungicide, leads to poisoning of 3,000 to 4,000; 10 per cent mortality rate.|
|1956-1977||Japan||Clioquinol||Drug used to treat travellers’ diarrhoea found to cause neuropathy; as many as 10,000 affected over two decades.|
|1959||Morocco||TOCP||Cooking oil contaminated with lubricating oil affects some 10,000 individuals.|
|1960||Iraq||Mercury||Mercury used as fungicide to treat seed grain used in bread; more than 1,000 people affected.|
|1964||Japan||Mercury||Methylmercury affects 646 people.|
|1968||Japan||PCBs||Polychlorinated biphenyls leaked into rice oil; 1,665 people affected.|
|1969||Japan||n-Hexane||93 cases of neuropathy occur following exposure to n-hexane, used to make vinyl sandals.|
|1971||United States||Hexachlorophene||After years of bathing infants in 3 per cent hexachlorophene, the disinfectant is found to be toxic to the nervous system and other systems.|
|1971||Iraq||Mercury||Mercury used as fungicide to treat seed grain is used in bread; more than 5,000 severe poisonings, 450 hospital deaths, effects on many infants exposedprenatally not documented.|
|1973||United States (Ohio)||MIBK||Fabric production plant employees exposed to solvent; more than 80 workers suffer neuropathy, 180 have less severe effects.|
|1974-1975||United States (Hopewell, VA)||Chlordecone (Kepone)||Chemical plant employees exposed to insecticide; more than 20 suffer severe neurologicalproblems, more than 40 have less severe problems.|
|1976||United States (Texas)||Leptophos (Phosvel)||At least 9 employees suffer severe neurological problems following exposure to insecticide during manufacturing process.|
|1977||United States (California)||Dichloropropene (Telone II)||24 individuals hospitalized after exposure to pesticide Telone following traffic accident.|
|1979-1980||United States (Lancaster, TX)||BHMH (Lucel-7)||Seven employees at plastic bathtub manufacturing plant experience serious neurologicalproblems following exposure to BHMH.|
|1980s||United States||MPTP||Impurity in synthesis of illicit drug found to cause symptoms identical to those of Parkinson’s disease.|
|1981||Spain||Contaminated toxic oil||20,000 persons poisoned by toxic substance in oil, resulting in more than 500 deaths; many suffer severe neuropathy.|
|1985||United States and Canada||Aldicarb||More than 1,000 individuals in California and other Western States and British Columbia experience neuromuscular and cardiac problems following ingestion of melons contaminated with the pesticide aldicarb.|
|1987||Canada||Domoic acid||Ingestion of mussels contaminated with domoic acid causes 129 illnesses and 2 deaths; symptoms include memory loss, disorientation and seizures.|
Source: OTA 1990.
Chemicals may affect the nervous system through actions at any of several cellular targets or biochemical processes within the central or peripheral nervous system. Toxic effects on other organs may also affect the nervous system, as in the example of hepatic encephalopathy. The manifestations of neurotoxicity include effects on learning (including memory, cognition and intellectual performance), somatosensory processes (including sensation and proprioreception), motor function (including balance, gait and fine movement control), affect (including personality status and emotionality) and autonomic function (nervous control of endocrine function and internal organ systems). The toxic effects of chemicals upon the nervous system often vary in sensitivity and expression with age: during development, the central nervous system may be especially susceptible to toxic insult because of the extended process of cellular differentiation, migration, and cell-to-cell contact that takes place in humans (OTA 1990). Moreover, cytotoxic damage to the nervous system may be irreversible because neurons are not replaced after embryogenesis. While the central nervous system (CNS) is somewhat protected from contact with absorbed compounds through a system of tightly joined cells (the blood-brain barrier, composed of capillary endothelial cells that line the vasculature of the brain), toxic chemicals can gain access to the CNS by three mechanisms: solvents and lipophilic compounds can pass through cell membranes; some compounds can attach to endogenous transporter proteins that serve to supply nutrients and biomolecules to the CNS; small proteins if inhaled can be directly taken up by the olfactory nerve and transported to the brain.
US regulatory authorities
Statutory authority for regulating substances for neurotoxicity is assigned to four agencies in the United States: the Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), the Occupational Safety and Health Administration (OSHA), and the Consumer Product Safety Commission (CPSC). While OSHA generally regulates occupational exposures to neurotoxic (and other) chemicals, the EPA has authority to regulate occupational and nonoccupational exposures to pesticides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). EPA also regulates new chemicals prior to manufacture and marketing, which obligates the agency to consider both occupational and nonoccupational risks.
Agents that adversely affect the physiology, biochemistry, or structural integrity of the nervous system or nervous system function expressed behaviourally are defined as neurotoxic hazards (EPA 1993). The determination of inherent neurotoxicity is a difficult process, owing to the complexity of the nervous system and the multiple expressions of neurotoxicity. Some effects may be delayed in appearance, such as the delayed neurotoxicity of certain organophosphate insecticides. Caution and judgement are required in determining neurotoxic hazard, including consideration of the conditions of exposure, dose, duration and timing.
Hazard identification is usually based upon toxicological studies of intact organisms, in which behavioural, cognitive, motor and somatosensory function is assessed with a range of investigative tools including biochemistry, electrophysiology and morphology (Tilson and Cabe 1978; Spencer and Schaumberg 1980). The importance of careful observation of whole organism behaviour cannot be overemphasized. Hazard identification also requires evaluation of toxicity at different developmental stages, including early life (intrauterine and early neonatal) and senescence. In humans, the identification of neurotoxicity involves clinical evaluation using methods of neurological assessment of motor function, speech fluency, reflexes, sensory function, electrophysiology, neuropsychological testing, and in some cases advanced techniques of brain imaging and quantitative electroencephalography. WHO has developed and validated a neurobehavioural core test battery (NCTB), which contains probes of motor function, hand-eye coordination, reaction time, immediate memory, attention and mood. This battery has been validated internationally by a coordinated process (Johnson 1978).
Hazard identification using animals also depends upon careful observational methods. The US EPA has developed a functional observational battery as a first-tier test designed to detect and quantify major overt neurotoxic effects (Moser 1990). This approach is also incorporated in the OECD subchronic and chronic toxicity testing methods. A typical battery includes the following measures: posture; gait; mobility; general arousal and reactivity; presence or absence of tremor, convulsions, lacrimation, piloerection, salivation, excess urination or defecation, stereotypy, circling, or other bizarre behaviours. Elicited behaviours include response to handling, tail pinch, or clicks; balance, righting reflex, and hind limb grip strength. Some representative tests and agents identified with these tests are shown in table 2.
Table 2. Examples of specialized tests to measure neurotoxicity
|Weakness||Grip strength; swimming endurance; suspension from rod; discriminative motor function; hind limb splay||n-Hexane, Methylbutylketone, Carbaryl|
|Incoordination||Rotorod, gait measurements||3-Acetylpyridine, Ethanol|
|Tremor||Rating scale, spectral analysis||Chlordecone, Type I Pyrethroids, DDT|
|Myoclonia, spasms||Rating scale, spectral analysis||DDT, Type II Pyrethroids|
|Auditory||Discriminant conditioning, reflex modification||Toluene, Trimethyltin|
|Visual toxicity||Discriminant conditioning||Methyl mercury|
|Somatosensory toxicity||Discriminant conditioning||Acrylamide|
|Pain sensitivity||Discriminant conditioning (btration); functional observational battery||Parathion|
|Olfactory toxicity||Discriminant conditioning||3-Methylindole methylbromide|
|Habituation||Startle reflex||Diisopropylfluorophosphate (DFP)|
|Classical conditioning||Nictitating membrane, conditioned flavour aversion, passive avoidance, olfactory conditioning||Aluminium, Carbaryl, Trimethyltin, IDPN, Trimethyltin (neonatal)|
|Operant or instrumental conditioning||One-way avoidance, Two-way avoidance, Y-maze avoidance, Biol watermaze, Morris water maze, Radial arm maze, Delayed matching to sample, Repeated acquisition, Visual discrimination learning||Chlordecone, Lead (neonatal), Hypervitaminosis A, Styrene, DFP, Trimethyltin, DFP. Carbaryl, Lead|
Source: EPA 1993.
These tests may be followed by more complex assessments usually reserved for mechanistic studies rather than hazard identification. In vitro methods for neurotoxicity hazard identification are limited since they do not provide indications of effects on complex function, such as learning, but they may be very useful in defining target sites of toxicity and improving the precision of target site dose-response studies (see WHO 1986 and EPA 1993 for comprehensive discussions of principles and methods for identifying potential neurotoxicants).
The relationship between toxicity and dose may be based upon human data when available or upon animal tests, as described above. In the United States, an uncertainty or safety factor approach is generally used for neurotoxicants. This process involves determining a “no observed adverse effect level” (NOAEL) or “lowest observed adverse effect level” (LOAEL) and then dividing this number by uncertainty or safety factors (usually multiples of 10) to allow for such considerations as incompleteness of data, potentially higher sensitivity of humans and variability of human response due to age or other host factors. The resultant number is termed the reference dose (RfD) or reference concentration (RfC). The effect occurring at the lowest dose in the most sensitive animal species and gender is generally used to determine the LOAEL or NOAEL. Conversion of animal dose to human exposure is done by standard methods of cross-species dosimetry, taking into account differences in lifespan and exposure duration.
The use of the uncertainty factor approach assumes that there is a threshold, or dose below which no adverse effect is induced. Thresholds for specific neurotoxicants may be difficult to determine experimentally; they are based upon assumptions as to mechanism of action which may or may not hold for all neurotoxicants (Silbergeld 1990).
At this stage, information is evaluated on sources, routes, doses and durations of exposure to the neurotoxicant for human populations, subpopulations or even individuals. This information may be derived from monitoring of environmental media or human sampling, or from estimates based upon standard scenarios (such as workplace conditions and job descriptions) or models of environmental fate and dispersion (see EPA 1992 for general guidelines on exposure assessment methods). In some limited cases, biological markers may be used to validate exposure inferences and estimates; however, there are relatively few usable biomarkers of neurotoxicants.
The combination of hazard identification, dose-response and exposure assessment is used to develop the risk characterization. This process involves assumptions as to the extrapolation of high to low doses, extrapolation from animals to humans, and the appropriateness of threshold assumptions and use of uncertainty factors.
Reproductive Toxicology—Risk Assessment Methods
Reproductive hazards may affect multiple functional endpoints and cellular targets within humans, with consequences for the health of the affected individual and future generations. Reproductive hazards may affect the development of the reproductive system in males or females, reproductive behaviours, hormonal function, the hypothalamus and pituitary, gonads and germ cells, fertility, pregnancy and the duration of reproductive function (OTA 1985). In addition, mutagenic chemicals may also affect reproductive function by damaging the integrity of germ cells (Dixon 1985).
The nature and extent of adverse effects of chemical exposures upon reproductive function in human populations is largely unknown. Relatively little surveillance information is available on such endpoints as fertility of men or women, age of menopause in women, or sperm counts in men. However, both men and women are employed in industries where exposures to reproductive hazards may occur (OTA 1985).
This section does not recapitulate those elements common to both neurotoxicant and reproductive toxicant risk assessment, but focuses upon issues specific to reproductive toxicant risk assessment. As with neurotoxicants, authority to regulate chemicals for reproductive toxicity is placed by statute in the EPA, OSHA, the FDA and the CPSC. Of these agencies, only the EPA has a stated set of guidelines for reproductive toxicity risk assessment. In addition, the state of California has developed methods for reproductive toxicity risk assessment in response to a state law, Proposition 65 (Pease et al. 1991).
Reproductive toxicants, like neurotoxicants, may act by affecting any of a number of target organs or molecular sites of action. Their assessment has additional complexity because of the need to evaluate three distinct organisms separately and together—the male, the female and the offspring (Mattison and Thomford 1989). While an important endpoint of reproductive function is the generation of a healthy child, reproductive biology also plays a role in the health of developing and mature organisms regardless of their involvement in procreation. For instance, loss of ovulatory function through natural depletion or surgical removal of oocytes has substantial effects upon the health of women, involving changes in blood pressure, lipid metabolism and bone physiology. Changes in hormone biochemistry may affect susceptibility to cancer.
The identification of a reproductive hazard may be made on the basis of human or animal data. In general, data from humans are relatively sparse, owing to the need for careful surveillance to detect alterations in reproductive function, such as sperm count or quality, ovulatory frequency and cycle length, or age at puberty. Detecting reproductive hazards through collection of information on fertility rates or data on pregnancy outcome may be confounded by the intentional suppression of fertility exercised by many couples through family-planning measures. Careful monitoring of selected populations indicates that rates of reproductive failure (miscarriage) may be very high, when biomarkers of early pregnancy are assessed (Sweeney et al. 1988).
Testing protocols using experimental animals are widely used to identify reproductive toxicants. In most of these designs, as developed in the United States by the FDA and the EPA and internationally by the OECD test guidelines program, the effects of suspect agents are detected in terms of fertility after male and/or female exposure; observation of sexual behaviours related to mating; and histopathological examination of gonads and accessory sex glands, such as mammary glands (EPA 1994). Often reproductive toxicity studies involve continuous dosing of animals for one or more generations in order to detect effects on the integrated reproductive process as well as to study effects on specific organs of reproduction. Multigenerational studies are recommended because they permit detection of effects that may be induced by exposure during the development of the reproductive system in utero. A special test protocol, the Reproductive Assessment by Continuous Breeding (RACB), has been developed in the United States by the National Toxicology Program. This test provides data on changes in the temporal spacing of pregnancies (reflecting ovulatory function), as well as number and size of litters over the entire test period. When extended to the lifetime of the female, it can yield information on early reproductive failure. Sperm measures can be added to the RACB to detect changes in male reproductive function. A special test to detect pre- or postimplantation loss is the dominant lethal test, designed to detect mutagenic effects in male spermatogenesis.
In vitro tests have also been developed as screens for reproductive (and developmental) toxicity (Heindel and Chapin 1993). These tests are generally used to supplement in vivo test results by providing more information on target site and mechanism of observed effects.
Table 3 shows the three types of endpoints in reproductive toxicity assessment—couple-mediated, female-specific and male-specific. Couple-mediated endpoints include those detectable in multigenerational and single-organism studies. They generally include assessment of offspring as well. It should be noted that fertility measurement in rodents is generally insensitive, as compared to such measurement in humans, and that adverse effects on reproductive function may well occur at lower doses than those that significantly affect fertility (EPA 1994). Male-specific endpoints can include dominant lethality tests as well as histopathological evaluation of organs and sperm, measurement of hormones, and markers of sexual development. Sperm function can also be assessed by in vitro fertilization methods to detect germ cell properties of penetration and capacitation; these tests are valuable because they are directly comparable to in vitro assessments conducted in human fertility clinics, but they do not by themselves provide dose-response information. Female-specific endpoints include, in addition to organ histopathology and hormone measurements, assessment of the sequelae of reproduction, including lactation and offspring growth.
Table 3. Endpoints in reproductive toxicology
|Multigenerational studies||Other reproductive endpoints|
|Mating rate, time to mating (time to pregnancy1)
Litter size (total and live)
Number of live and dead offspring (foetal death rate1)
External malformations and variations1
Internal malformations and variations1
Postnatal structural and functional development1
Visual examination and histopathology
|Testes, epididymides, seminal vesicles, prostate, pituitary
Testes, epididymides, seminal vesicles, prostate, pituitary
Sperm number (count) and quality (morphology, motility)
Luteinizing hormone, follicle stimulating hormone, testosterone, oestrogen, prolactin
Testis descent1, preputial separation, sperm production1, ano-genital distance, normality of external genitalia1
Visual examination and histopathology
Oestrous (menstrual1) cycle normality
Ovary, uterus, vagina, pituitary
Ovary, uterus, vagina, pituitary, oviduct, mammary gland
Vaginal smear cytology
LH, FSH, oestrogen, progesterone, prolactin
Normality of external genitalia1, vaginal opening, vaginal smear cytology, onset of oestrus behaviour (menstruation1)
Vaginal smear cytology, ovarian histology
1 Endpoints that can be obtained relatively noninvasively with humans.
Source: EPA 1994.
In the United States, the hazard identification concludes with a qualitative evaluation of toxicity data by which chemicals are judged to have either sufficient or insufficient evidence of hazard (EPA 1994). “Sufficient” evidence includes epidemiological data providing convincing evidence of a causal relationship (or lack thereof), based upon case-control or cohort studies, or well-supported case series. Sufficient animal data may be coupled with limited human data to support a finding of a reproductive hazard: to be sufficient, the experimental studies are generally required to utilize EPA’s two-generation test guidelines, and must include a minimum of data demonstrating an adverse reproductive effect in an appropriate, well-conducted study in one test species. Limited human data may or may not be available; it is not necessary for the purposes of hazard identification. To rule out a potential reproductive hazard, the animal data must include an adequate array of endpoints from more than one study showing no adverse reproductive effect at doses minimally toxic to the animal (EPA 1994).
As with the evaluation of neurotoxicants, the demonstration of dose-related effects is an important part of risk assessment for reproductive toxicants. Two particular difficulties in dose-response analyses arise due to complicated toxicokinetics during pregnancy, and the importance of distinguishing specific reproductive toxicity from general toxicity to the organism. Debilitated animals, or animals with substantial nonspecific toxicity (such as weight loss) may fail to ovulate or mate. Maternal toxicity can affect the viability of pregnancy or support for lactation. These effects, while evidence of toxicity, are not specific to reproduction (Kimmel et al. 1986). Assessing dose response for a specific endpoint, such as fertility, must be done in the context of an overall assessment of reproduction and development. Dose-response relationships for different effects may differ significantly, but interfere with detection. For instance, agents that reduce litter size may result in no effects upon litter weight because of reduced competition for intrauterine nutrition.
An important component of exposure assessment for reproductive risk assessment relates to information on the timing and duration of exposures. Cumulative exposure measures may be insufficiently precise, depending upon the biological process that is affected. It is known that exposures at different developmental stages in males and females can result in different outcomes in both humans and experimental animals (Gray et al. 1988). The temporal nature of spermatogenesis and ovulation also affects outcome. Effects on spermatogenesis may be reversible if exposures cease; however, oocyte toxicity is not reversible since females have a fixed set of germ cells to draw upon for ovulation (Mattison and Thomford 1989).
As with neurotoxicants, the existence of a threshold is usually assumed for reproductive toxicants. However, the actions of mutagenic compounds on germ cells may be considered an exception to this general assumption. For other endpoints, an RfD or RfC is calculated as with neurotoxicants by determination of the NOAEL or LOAEL and application of appropriate uncertainty factors. The effect used for determining the NOAEL or LOAEL is the most sensitive adverse reproductive endpoint from the most appropriate or most sensitive mammalian species (EPA 1994). Uncertainty factors include consideration of interspecies and intraspecies variation, ability to define a true NOAEL, and sensitivity of the endpoint detected.
Risk characterizations should also be focused upon specific subpopulations at risk, possibly specifying males and females, pregnancy status, and age. Especially sensitive individuals, such as lactating women, women with reduced oocyte numbers or men with reduced sperm counts, and prepubertal adolescents may also be considered.
Toxicology plays a major role in the development of regulations and other occupational health policies. In order to prevent occupational injury and illness, decisions are increasingly based upon information obtainable prior to or in the absence of the types of human exposures that would yield definitive information on risk such as epidemiology studies. In addition, toxicological studies, as described in this chapter, can provide precise information on dose and response under the controlled conditions of laboratory research; this information is often difficult to obtain in the uncontrolled setting of occupational exposures. However, this information must be carefully evaluated in order to estimate the likelihood of adverse effects in humans, the nature of these adverse effects, and the quantitative relationship between exposures and effects.
Considerable attention has been given in many countries, since the 1980s, to developing objective methods for utilizing toxicological information in regulatory decision-making. Formal methods, frequently referred to as risk assessment, have been proposed and utilized in these countries by both governmental and non-governmental entities. Risk assessment has been varyingly defined; fundamentally it is an evaluative process that incorporates toxicology, epidemiology and exposure information to identify and estimate the probability of adverse effects associated with exposures to hazardous substances or conditions. Risk assessment may be qualitative in nature, indicating the nature of an adverse effect and a general estimate of likelihood, or it may be quantitative, with estimates of numbers of affected persons at specific levels of exposure. In many regulatory systems, risk assessment is undertaken in four stages: hazard identification, the description of the nature of the toxic effect; dose-response evaluation, a semi-quantitative or quantitative analysis of the relationship between exposure (or dose) and severity or likelihood of toxic effect; exposure assessment, the evaluation of information on the range of exposures likely to occur for populations in general or for subgroups within populations; risk characterization, the compilation of all the above information into an expression of the magnitude of risk expected to occur under specified exposure conditions (see NRC 1983 for a statement of these principles).
In this section, three approaches to risk assessment are presented as illustrative. It is impossible to provide a comprehensive compendium of risk assessment methods used throughout the world, and these selections should not be taken as prescriptive. It should be noted that there are trends towards harmonization of risk assessment methods, partly in response to provisions in the recent GATT accords. Two processes of international harmonization of risk assessment methods are currently underway, through the International Programme on Chemical Safety (IPCS) and the Organization for Economic Cooperation and Development (OECD). These organizations also maintain current information on national approaches to risk assessment.
Structure activity relationships (SAR) analysis is the utilization of information on the molecular structure of chemicals to predict important characteristics related to persistence, distribution, uptake and absorption, and toxicity. SAR is an alternative method of identifying potential hazardous chemicals, which holds promise of assisting industries and governments in prioritizing substances for further evaluation or for early-stage decision making for new chemicals. Toxicology is an increasingly expensive and resource-intensive undertaking. Increased concerns over the potential for chemicals to cause adverse effects in exposed human populations have prompted regulatory and health agencies to expand the range and sensitivity of tests to detect toxicological hazards. At the same time, the real and perceived burdens of regulation upon industry have provoked concerns for the practicality of toxicity testing methods and data analysis. At present, the determination of chemical carcinogenicity depends upon lifetime testing of at least two species, both sexes, at several doses, with careful histopathological analysis of multiple organs, as well as detection of preneoplastic changes in cells and target organs. In the United States, the cancer bioassay is estimated to cost in excess of $3 million (1995 dollars).
Even with unlimited financial resources, the burden of testing the approximately 70,000 existing chemicals produced in the world today would exceed the available resources of trained toxicologists. Centuries would be required to complete even a first tier evaluation of these chemicals (NRC 1984). In many countries ethical concerns over the use of animals in toxicity testing have increased, bringing additional pressures upon the uses of standard methods of toxicity testing. SAR has been widely used in the pharmaceutical industry to identify molecules with potential for beneficial use in treatment (Hansch and Zhang 1993). In environmental and occupational health policy, SAR is used to predict the dispersion of compounds in the physical-chemical environment and to screen new chemicals for further evaluation of potential toxicity. Under the US Toxic Substances Control Act (TSCA), the EPA has used since 1979 an SAR approach as a “first screen” of new chemicals in the premanufacture notification (PMN) process; Australia uses a similar approach as part of its new chemicals notification (NICNAS) procedure. In the US SAR analysis is an important basis for determining that there is a reasonable basis to conclude that manufacture, processing, distribution, use or disposal of the substance will present an unreasonable risk of injury to human health or the environment, as required by Section 5(f) of TSCA. On the basis of this finding, EPA can then require actual tests of the substance under Section 6 of TSCA.
Rationale for SAR
The scientific rationale for SAR is based upon the assumption that the molecular structure of a chemical will predict important aspects of its behaviour in physical-chemical and biological systems (Hansch and Leo 1979).
The SAR review process includes identification of the chemical structure, including empirical formulations as well as the pure compound; identification of structurally analogous substances; searching databases and literature for information on structural analogs; and analysis of toxicity and other data on structural analogs. In some rare cases, information on the structure of the compound alone can be sufficient to support some SAR analysis, based upon well-understood mechanisms of toxicity. Several databases on SAR have been compiled, as well as computer-based methods for molecular structure prediction.
With this information, the following endpoints can be estimated with SAR:
It should be noted that SAR methods do not exist for such important health endpoints as carcinogenicity, developmental toxicity, reproductive toxicity, neurotoxicity, immunotoxicity or other target organ effects. This is due to three factors: the lack of a large database upon which to test SAR hypotheses, lack of knowledge of structural determinants of toxic action, and the multiplicity of target cells and mechanisms that are involved in these endpoints (see “The United States approach to risk assessment of reproductive toxicants and neurotoxic agents”). Some limited attempts to utilize SAR for predicting pharmacokinetics using information on partition coefficients and solubility (Johanson and Naslund 1988). More extensive quantitative SAR has been done to predict P450-dependent metabolism of a range of compounds and binding of dioxin- and PCB-like molecules to the cytosolic “dioxin” receptor (Hansch and Zhang 1993).
SAR has been shown to have varying predictability for some of the endpoints listed above, as shown in table 1. This table presents data from two comparisons of predicted activity with actual results obtained by empirical measurement or toxicity testing. SAR as conducted by US EPA experts performed more poorly for predicting physical-chemical properties than for predicting biological activity, including biodegradation. For toxicity endpoints, SAR performed best for predicting mutagenicity. Ashby and Tennant (1991) in a more extended study also found good predictability of short-term genotoxicity in their analysis of NTP chemicals. These findings are not surprising, given current understanding of molecular mechanisms of genotoxicity (see “Genetic toxicology”) and the role of electrophilicity in DNA binding. In contrast, SAR tended to underpredict systemic and subchronic toxicity in mammals and to overpredict acute toxicity to aquatic organisms.
Table 1. Comparison of SAR and test data: OECD/NTP analyses
|Endpoint||Agreement (%)||Disagreement (%)||Number|
|Acute mammalian toxicity (LD50 )||80||201||142|
|Carcinogenicity3 : Two year bioassay||72–954||—||301|
Source: Data from OECD, personal communication C. Auer ,US EPA. Only those endpoints for which comparable SAR predictions and actual test data were available were used in this analysis. NTP data are from Ashby and Tennant 1991.
1 Of concern was the failure by SAR to predict acute toxicity in 12% of the chemicals tested.
2 OECD data, based on Ames test concordance with SAR
3 NTP data, based on genetox assays compared to SAR predictions for several classes of “structurally alerting chemicals”.
4 Concordance varies with class; highest concordance was with aromatic amino/nitro compounds; lowest with “miscellaneous” structures.
For other toxic endpoints, as noted above, SAR has less demonstrable utility. Mammalian toxicity predictions are complicated by the lack of SAR for toxicokinetics of complex molecules. Nevertheless, some attempts have been made to propose SAR principles for complex mammalian toxicity endpoints (for instance, see Bernstein (1984) for an SAR analysis of potential male reproductive toxicants). In most cases, the database is too small to permit rigorous testing of structure-based predictions.
At this point it may be concluded that SAR may be useful mainly for prioritizing the investment of toxicity testing resources or for raising early concerns about potential hazard. Only in the case of mutagenicity is it likely that SAR analysis by itself can be utilized with reliability to inform other decisions. For no endpoint is it likely that SAR can provide the type of quantitative information required for risk assessment purposes as discussed elsewhere in this chapter and Encyclopaedia.
The study and characterization of chemicals and other agents for toxic properties is often undertaken on the basis of specific organs and organ systems. In this chapter, two targets have been selected for in-depth discussion: the immune system and the gene. These examples were chosen to represent a complex target organ system and a molecular target within cells. For more comprehensive discussion of the toxicology of target organs, the reader is referred to standard toxicology texts such as Casarett and Doull, and Hayes. The International Programme on Chemical Safety (IPCS) has also published several criteria documents on target organ toxicology, by organ system.
Target organ toxicology studies are usually undertaken on the basis of information indicating the potential for specific toxic effects of a substance, either from epidemiological data or from general acute or chronic toxicity studies, or on the basis of special concerns to protect certain organ functions, such as reproduction or foetal development. In some cases, specific target organ toxicity tests are expressly mandated by statutory authorities, such as neurotoxicity testing under the US pesticides law (see “The United States approach to risk assessment of reproductive toxicants and neurotoxic agents,” and mutagenicity testing under the Japanese Chemical Substance Control Law (see “Principles of hazard identification: The Japanese approach”).
As discussed in “Target organ and critical effects,” the identification of a critical organ is based upon the detection of the organ or organ system which first responds adversely or to the lowest doses or exposures. This information is then used to design specific toxicology investigations or more defined toxicity tests that are designed to elicit more sensitive indications of intoxication in the target organ. Target organ toxicology studies may also be used to determine mechanisms of action, of use in risk assessment (see “The United States approach to risk assessment of reproductive toxicants and neurotoxic agents”).
Methods of Target Organ Toxicity Studies
Target organs may be studied by exposure of intact organisms and detailed analysis of function and histopathology in the target organ, or by in vitro exposure of cells, tissue slices, or whole organs maintained for short or long term periods in culture (see “Mechanisms of toxicology: Introduction and concepts”). In some cases, tissues from human subjects may also be available for target organ toxicity studies, and these may provide opportunities to validate assumptions of cross-species extrapolation. However, it must be kept in mind that such studies do not provide information on relative toxicokinetics.
In general, target organ toxicity studies share the following common characteristics: detailed histopathological examination of the target organ, including post mortem examination, tissue weight, and examination of fixed tissues; biochemical studies of critical pathways in the target organ, such as important enzyme systems; functional studies of the ability of the organ and cellular constituents to perform expected metabolic and other functions; and analysis of biomarkers of exposure and early effects in target organ cells.
Detailed knowledge of target organ physiology, biochemistry and molecular biology may be incorporated in target organ studies. For instance, because the synthesis and secretion of small-molecular-weight proteins is an important aspect of renal function, nephrotoxicity studies often include special attention to these parameters (IPCS 1991). Because cell-to-cell communication is a fundamental process of nervous system function, target organ studies in neurotoxicity may include detailed neurochemical and biophysical measurements of neurotransmitter synthesis, uptake, storage, release and receptor binding, as well as electrophysiological measurement of changes in membrane potential associated with these events.
A high degree of emphasis is being placed upon the development of in vitro methods for target organ toxicity, to replace or reduce the use of whole animals. Substantial advances in these methods have been achieved for reproductive toxicants (Heindel and Chapin 1993).
In summary, target organ toxicity studies are generally undertaken as a higher order test for determining toxicity. The selection of specific target organs for further evaluation depends upon the results of screening level tests, such as the acute or subchronic tests used by OECD and the European Union; some target organs and organ systems may be a priori candidates for special investigation because of concerns to prevent certain types of adverse health effects.
Toxicology is the study of poisons, or, more comprehensively, the identification and quantification of adverse outcomes associated with exposures to physical agents, chemical substances and other conditions. As such, toxicology draws upon most of the basic biological sciences, medical disciplines, epidemiology and some areas of chemistry and physics for information, research designs and methods. Toxicology ranges from basic research investigations on the mechanism of action of toxic agents through the development and interpretation of standard tests characterizing the toxic properties of agents. Toxicology provides important information for both medicine and epidemiology in understanding aetiology and in providing information as to the plausibility of observed associations between exposures, including occupations, and disease. Toxicology can be divided into standard disciplines, such as clinical, forensic, investigative and regulatory toxicology; toxicology can be considered by target organ system or process, such as immunotoxicology or genetic toxicology; toxicology can be presented in functional terms, such as research, testing and risk assessment.
It is a challenge to propose a comprehensive presentation of toxicology in this Encyclopaedia. This chapter does not present a compendium of information on toxicology or adverse effects of specific agents. This latter information is better obtained from databases that are continually updated, as described in the last section of this chapter. Moreover, the chapter does not attempt to set toxicology within specific subdisciplines, such as forensic toxicology. It is the premise of the chapter that the information provided is relevant to all types of toxicological endeavours and to the use of toxicology in various medical specialities and fields. In this chapter, topics are based primarily upon a practical orientation and integration with the intent and purpose of the Encyclopaedia as a whole. Topics are also selected for ease of cross-reference within the Encyclopaedia.
In modern society, toxicology has become an important element in environmental and occupational health. This is because many organizations, governmental and non-governmental, utilize information from toxicology to evaluate and regulate hazards in the workplace and nonoccupational environment. As part of prevention strategies, toxicology is invaluable, since it is the source of information on potential hazards in the absence of widespread human exposures. Toxicological methods are also widely used by industry in product development, to provide information useful in the design of specific molecules or product formulations.
The chapter begins with five articles on general principles of toxicology, which are important to the consideration of most topics in the field. The first general principles relate to understanding relationships between external exposure and internal dose. In modern terminology, “exposure” refers to the concentrations or amount of a substance presented to individuals or populations—amounts found in specific volumes of air or water, or in masses of soil. “Dose” refers to the concentration or amount of a substance inside an exposed person or organism. In occupational health, standards and guidelines are often set in terms of exposure, or allowable limits on concentrations in specific situations, such as in air in the workplace. These exposure limits are predicated upon assumptions or information on the relationships between exposure and dose; however, often information on internal dose is unavailable. Thus, in many studies of occupational health, associations can be drawn only between exposure and response or effect. In a few instances, standards have been set based on dose (e.g., permissible levels of lead in blood or mercury in urine). While these measures are more directly correlated with toxicity, it is still necessary to back-calculate exposure levels associated with these levels for purposes of controlling risks.
The next article concerns the factors and events that determine the relationships between exposure, dose and response. The first factors relate to uptake, absorption and distribution—the processes that determine the actual transport of substances into the body from the external environment across portals of entry such as skin, lung and gut. These processes are at the interface between humans and their environments. The second factors, of metabolism, relate to understanding how the body handles absorbed substances. Some substances are transformed by cellular processes of metabolism, which can either increase or decrease their biological activity.
The concepts of target organ and critical effect have been developed to aid in the interpretation of toxicological data. Depending upon dose, duration and route of exposure, as well as host factors such as age, many toxic agents can induce a number of effects within organs and organisms. An important role of toxicology is to identify the important effect or sets of effects in order to prevent irreversible or debilitating disease. One important part of this task is the identification of the organ first or most affected by a toxic agent; this organ is defined as the “target organ”. Within the target organ, it is important to identify the important event or events that signals intoxication, or damage, in order to ascertain that the organ has been affected beyond the range of normal variation. This is known as the “critical effect”; it may represent the first event in a progression of pathophysiological stages (such as the excretion of small-molecular-weight proteins as a critical effect in nephrotoxicity), or it may represent the first and potentially irreversible effect in a disease process (such as formation of a DNA adduct in carcinogenesis). These concepts are important in occupational health because they define the types of toxicity and clinical disease associated with specific exposures, and in most cases reduction of exposure has as a goal the prevention of critical effects in target organs, rather than every effect in every or any organ.
The next two articles concern important host factors that affect many types of responses to many types of toxic agents. These are: genetic determinants, or inherited susceptibility/resistance factors; and age, sex and other factors such as diet or co-existence of infectious disease. These factors can also affect exposure and dose, through modifying uptake, absorption, distribution and metabolism. Because working populations around the world vary with respect to many of these factors, it is critical for occupational health specialists and policy-makers to understand the way in which these factors may contribute to variabilities in response among populations and individuals within populations. In societies with heterogeneous populations, these considerations are particularly important. The variability of human populations must be considered in evaluating the risks of occupational exposures and in reaching rational conclusions from the study of nonhuman organisms in toxicological research or testing.
The section then provides two general overviews on toxicology at the mechanistic level. Mechanistically, modern toxicologists consider that all toxic effects manifest their first actions at the cellular level; thus, cellular responses represent the earliest indications of the body’s encounters with a toxic agent. It is further assumed that these responses represent a spectrum of events, from injury through death. Cell injury refers to specific processes utilized by cells, the smallest unit of biological organization within organs, to respond to challenge. These responses involve changes in the function of processes within the cell, including the membrane and its ability to take up, release or exclude substances; the directed synthesis of proteins from amino acids; and the turnover of cell components. These responses may be common to all injured cells, or they may be specific to certain types of cells within certain organ systems. Cell death is the destruction of cells within an organ system, as a consequence of irreversible or uncompensated cell injury. Toxic agents may cause cell death acutely because of certain actions such as poisoning oxygen transfer, or cell death may be the consequence of chronic intoxication. Cell death can be followed by replacement in some but not all organ systems, but in some conditions cell proliferation induced by cell death may be considered a toxic response. Even in the absence of cell death, repeated cell injury may induce stress within organs that compromises their function and affects their progeny.
The chapter is then divided into more specific topics, which are grouped into the following categories: mechanism, test methods, regulation and risk assessment. The mechanism articles mostly focus on target systems rather than organs. This reflects the practice of modern toxicology and medicine, which studies organ systems rather than isolated organs. Thus, for example, the discussion of genetic toxicology is not focused upon the toxic effects of agents within a specific organ but rather on genetic material as a target for toxic action. Likewise, the article on immunotoxicology discusses the various organs and cells of the immune system as targets for toxic agents. The methods articles are designed to be highly operational; they describe current methods in use in many countries for hazard identification, that is, the development of information related to biological properties of agents.
The chapter continues with five articles on the application of toxicology in regulation and policy-making, from hazard identification to risk assessment. The current practice in several countries, as well as IARC, is presented. These articles should enable the reader to understand how information derived from toxicology tests is integrated with basic and mechanistic inferences to derive quantitative information used in setting exposure levels and other approaches to controlling hazards in the workplace and general environment.
A summary of available toxicology databases, to which the readers of this encyclopaedia can refer for detailed information on specific toxic agents and exposures, can be found in Volume III (see “Toxicology databases” in the chapter Safe handling of chemicals, which provides information on many of these databases, their information sources, methods of evaluation and interpretation, and means of access). These databases, together with the Encyclopaedia, provide the occupational health specialist, the worker and the employer with the ability to obtain and use up-to-date in- formation on toxicology and the evaluation of toxic agents by national and international bodies.
This chapter focuses upon those aspects of toxicology relevant to occupational safety and health. For that reason, clinical toxic-ology and forensic toxicology are not specifically addressed as subdisciplines of the field. Many of the same principles and approaches described here are used in these subdisciplines as well as in environmental health. They are also applicable to evaluating the impacts of toxic agents on nonhuman populations, a major concern of environmental policies in many countries. A committed attempt has been made to enlist the perspectives and experiences of experts and practitioners from all sectors and from many countries; however, the reader may note a certain bias towards academic scientists in the developed world. Although the editor and contributors believe that the principles and practice of toxic-ology are international, the problems of cultural bias and narrowness of experience may well be evident in this chapter. The chapter editor hopes that readers of this Encyclopaedia will assist in ensuring the broadest perspective possible as this important reference continues to be updated and expanded.