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The United States Approach to Risk Assessment of Reproductive Toxicants and Neurotoxic Agents

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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

Year(s) Location Substance Comments
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.

Hazard identification

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

Function Procedure Representative agents
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
Learning, memory
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).

Dose-response assessment

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).

Exposure assessment

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.

Risk characterization

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.

Hazard identification

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

  Couple-mediated endpoints
Multigenerational studies Other reproductive endpoints
Mating rate, time to mating (time to pregnancy1)
Pregnancy rate1
Delivery rate1
Gestation length1
Litter size (total and live)
Number of live and dead offspring (foetal death rate1)
Offspring gender1
Birth weight1
Postnatal weights1
Offspring survival1
External malformations and variations1
Offspring reproduction1
Ovulation rate

Fertilization rate
Preimplantation loss
Implantation number
Postimplantation loss1
Internal malformations and variations1
Postnatal structural and functional development1
  Male-specific endpoints
Organ weights

Visual examination and histopathology

Sperm evaluation1

Hormone levels1

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
  Female-specific endpoints
Body weight
Organ weights
Visual examination and histopathology

Oestrous (menstrual1) cycle normality
Hormone levels1

Senescence (menopause1)

Ovary, uterus, vagina, pituitary
Ovary, uterus, vagina, pituitary, oviduct, mammary gland
Vaginal smear cytology
LH, FSH, oestrogen, progesterone, prolactin
Offspring growth
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).

Dose-response assessment

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.

Exposure assessment

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).

Risk characterization

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.



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