Occupational exposure limits for 30 organophosphate pesticides based on inhibition of red blood cell acetylcholinesterase.

Toxicity and other relevant data for 30 organophosphate pesticides were evaluated to suggest inhalation occupational exposure limits (OELs), and to support development of a risk assessment strategy for organophosphates in general. Specifically, the value of relative potency analysis and the predictability of inhalation OELs by acute toxicity measures and by repeated oral exposure NOELs was assessed. Suggested OELs are based on the prevention of red blood cell (RBC) acetylcholinesterase (AChE) inhibition and are derived using a weight-of-evidence risk assessment approach. Suggested OEL values range from 0.002 to 2 mg/m(3), and in most cases, are less than current permissible exposure levels (PELs) or threshold limit values(R) (TLVs(R)). The available data indicate that experimental data for most organophosphates evaluated are limited; most organophosphates are equally potent RBC AChE inhibitors in different mammalian species; NOELs from repeated exposure studies of variable duration are usually equivalent; and, no particular grouping based on organophosphate structure is consistently more potent than another. Further, relative potency analyses have limited usefulness in the risk assessment of organophosphates. The data also indicated that equivalent relative potency relationships do not exist across either exposure duration (acute vs. repeated) or exposure route (oral vs. inhalation). Consideration of all variable duration and exposure route studies are therefore usually desirable in the development of an OEL, especially when data are limited. Also, neither acute measures of toxicity nor repeated oral exposure NOELs are predictive of weight-of-evidence based inhalation OELs. These deviations from what is expected based on the common mechanism of action for organophosphates across exposure duration and route - AChE inhibition - is likely due to the lack of synchrony between the timing of target tissue effective dose and the experimental observation of equivalent response. Thus, comprehensive interpretation of all toxicity data in the context of available toxicokinetic, toxicodynamic and exposure information for each individual organophosphate in a weight-of-evidence based risk assessment is desirable when deriving inhalation OELs.

Derivation of an occupational exposure limit (OEL) for methylene chloride based on acute CNS effects and relative potency analysis.

The Occupational Safety and Health Administration (OSHA) methylene chloride Permissible Exposure Level (PEL) or 25 ppm is quantitatively derived from mouse tumor results observed in a high-exposure National Toxicology Program bioassay. Because this approach depends on controversial interspecies and low-dose extrapolations, the PEL itself has stimulated heated debate. Here, an alternative safety assessment for methylene chloride is presented. It is based on an acute human lowest-observed-adverse-effect level (LOAEL) of 200 ppm for subtle central nervous system (CNS) depression. Steep, parallel exposure-response curves for anesthetic and subanesthetic CNS effects associated with compounds mechanistically and structurally related to methylene chloride are shown to support a safety factor of two to account for inter-individual variability in response. LOAEL/no-observed-adverse-effect ratios for subtle CNS effects associated with structurally related solvents are shown to support a safety factor range of two to four to account for uncertainty in identifying a subthreshold exposure level. Anesthetic relative potencies and anesthetic/subanesthetic effect level ratios are shown to be constant for the compounds evaluated, demonstrating that subanesthetic relative potencies are also constant. Relative potencies among similarly derived occupational exposure limits (OELs) for solvents structurally related to methylene chloride are therefore used to validate the derived methylene chloride OEL range of 25-50 ppm. Because this safety assessment is based on human (rather than rodent) data and empirical (rather than theoretical) exposure-response relationships and is supported by relative potency analysis, it is a defensible alternative to to the OSHA risk assessment and should positively contribute to the debate regarding the appropriate basis and value for a methylene chloride PEL.

Evaluation of alternative methods for establishing safe levels of occupational exposure to vinyl halides.

The facts that reduction of occupational vinyl chloride exposures to levels within or below the 0.5-5 ppm range has so far been successful in eliminating vinyl chloride-induced liver angiosarcoma and that humans appear to be less sensitive to the carcinogenic effect of vinyl chloride than rats offered an opportunity to verify or dispute risk assessment extrapolation models used, and proposed, by the U.S. EPA. Safe occupational vinyl chloride exposures were defined as levels associated with an incidence of one angiosarcoma in 100,000 exposed workers, determined from rat bioassay data using default no-threshold (linearized multistage model and benchmark dose approach with linear extrapolation) and threshold (NOEL/LOEL and benchmark dose uncertainty factor approaches) models, and then compared against the likely protective range of 0.5-5 ppm. Safe levels derived using either no-threshold model are equivalent and are two to three orders of magnitude below the 0.5-5 ppm range. Safe levels derived using either threshold model, when applying uncertainty factors which reflect equal or less sensitivity in humans compared to rats, fall within the 0.5-5 ppm range. Similar results were obtained for vinyl bromide and vinyl fluoride. These results undermine the U.S. EPA default assumption of no-threshold for vinyl halides as well as for other DNA-reactive carcinogens while simultaneously supporting the notion that a practical threshold exists. They further suggest that when threshold models are appropriate, the default assumption of greater sensitivity in humans compared to rats should be carefully evaluated.

Reduction of azo dyes during in vitro percutaneous absorption.

The azoreduction of phenylazo-2-naphthol (Sudan I), 5-(phenylazo)-6-hydroxynaphthalene-2-sulfonic acid [aniline subsidiary color of FD&C Yellow No. 6 (ANSC)], and phenylazophenol [Solvent Yellow 7 (SY7)] in skin during percutaneous absorption was measured and the contributions of cytosolic and microsomal reductions were characterized. By using a series of azo dyes with a common U-14C-labeled phenylazo moiety, percutaneous absorption and metabolism were measured in vitro in flow-through diffusion cells with Sencar mouse, hairless guinea pig, and human skin. Azoreductase assays using subcellular fractions from skin of all species were used to examine intracellular rates and distribution for the series of dyes. The absorption of the lipophilic dyes Sudan I and SY7 from a finite surface dose of 5 micrograms/cm2 was extensive in all species. In mouse, 32.8 +/- 2.8% of the applied Sudan I dose and 64.1 +/- 3.3% of the applied SY7 dose were absorbed in 24 hr. In the hairless guinea pig, 57.6 +/- 5.9% of the applied Sudan I dose and 67.8 +/- 4.6% of the applied SY7 dose were absorbed in 24 hr. Human skin was least permeable, with 26.4 +/- 6.7% of the applied Sudan I dose and 36.1 +/- 4.5% of the applied SY7 dose absorbed in 24 hr. Sudan I and SY7 were extensively reduced in skin of all species during percutaneous absorption (29.5 and 26.5%, respectively, of the absorbed dose in human skin and greater than 50% of the applied dose in other species). ANSC was the least absorbed, with 5% or less penetrating. SY7 was preferentially reduced in the skin cytosol of all species, whereas Sudan I was equally reduced in the skin cytosol and microsomal fractions. The site of azoreduction in the cell may affect the metabolic fate of the liberated arylamine. The extensive azoreduction observed during percutaneous absorption may modulate the toxicities of these compounds and must be considered when effective doses are determined for quantitative risk assessments from dermal exposures.

Metabolism of xenobiotics during percutaneous penetration: role of absorption rate and cutaneous enzyme activity.

The role of absorption rate and enzyme activity on cutaneous metabolism of topically applied xenobiotics was assessed by determining the simultaneous percutaneous penetration/metabolism of benzo[a]pyrene (B[a]P) and 7-ethoxycoumarin (7-EC) in intact, metabolically viable skin of Sencar mice, hairless guinea pigs, and humans. In addition, specific activities of aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin deethylase (ECDE) were determined in cutaneous microsomal fractions. Both compounds were readily absorbed but only minimally metabolized. Sencar mouse and hairless guinea pig skin absorbed 55-60% of the applied B[a]P dose and metabolized only 6 and 3%, respectively, of that absorbed. Human skin absorbed 31% of the applied dose and B[a]P metabolism was not detectable. All three species absorbed 60-80% of the applied 7-EC dose. Sencar mouse and hairless guinea pig skin metabolized 1.3 and 1.2% of the absorbed dose, respectively. and human skin metabolized only 0.05%. When 7-EC absorption was increased to the maximum possible rate, its metabolism by Sencar mouse and hairless guinea pig skin was also substantially increased. In human skin, a much smaller increase in 7-EC absorption rate was possible and no increase in 7-EC metabolism occurred. Thus relatively slower absorption of 7-EC and B[a]P by human skin may limit cutaneous metabolism of these penetrating compounds. Specific activities of AHH and ECDE were significantly lower in human skin than in Sencar mouse and hairless guinea pig skin, suggesting that low enzyme activity contributes as well to a low rate of metabolism by human skin compared to other species. Thus absorption rate and cutaneous enzyme activity are interrelated determinants of the extent of cutaneous metabolism of B[a]P and 7-EC occurring during their percutaneous penetration, and slow absorption and low enzyme activity limit cutaneous metabolism of B[a]P and 7-EC in human skin in particular.

Extent of cutaneous metabolism during percutaneous absorption of xenobiotics.

In vitro percutaneous absorption studies generally do not determine whether biotransformation occurs during passage of a substance through the skin. Since it has recently been demonstrated that several chemicals are metabolized during skin permeation, we investigated the metabolism of five additional compounds (14C-labeled) after application to fuzzy rat skin: caffeine, p,p'-DDT, butylated hydroxytoluene (BHT), salicylic acid, and acetyl ethyl tetramethyltetralin (AETT). The viability of skin was maintained with a tissue culture medium. Radioactivity of each substrate and any metabolites in skin and receptor fluid was measured so that the absorption and metabolism of water-insoluble compounds would be accurately determined. Percutaneous absorption ranged from a low of 13% of the applied dose for BHT to a high of 49% for DDT. BHT was metabolized in skin to 4-hydroxy-BHT and an unknown metabolite. Of the absorbed radioisotope, 6.6% was isolated in biotransformed products found mainly in the receptor fluid. AETT was also metabolized during absorption, with 1.9% of the absorbed radioisotope found in two unknown peaks. Caffeine, DDT, and salicylic acid were not metabolized during skin permeation. Skin and liver microsomal metabolism was measured for all compounds except DDT. Metabolism in skin was observed only for the compounds also biotransformed in the diffusion cell; BHT and AETT were metabolized at 113 and 2.5 pmol/min/mg protein, respectively. In this study, as in others, skin metabolism was substantially less than the corresponding metabolism in liver. Therefore, a low rate of liver metabolism such as that found for caffeine, salicylic acid, and DDT might often be predictive of the absence of measurable metabolism during skin permeation. It seems likely that for many compounds, the biotransformations in skin will be small in terms of the percentage of absorbed material that is metabolized. Nevertheless, with potent compounds, even small quantities of a metabolite can be important and for pharmacokinetic studies, viability of skin must be maintained.

Disruption of neostriatal development in rats following perinatal exposure to mild, but chronic carbon monoxide.

The vulnerability of the developing neostriatum to mild, but chronic hypoxia was evaluated in weanling rats exposed only in utero or from conception through postnatal day 10 to 0, 75, 150, and 300 ppm carbon monoxide (CO). The exposure conditions produced maternal carboxyhemoglobin (HbCO) levels of about 11, 19, and 27 percent. HbCO levels of 5 percent are maintained by human cigarette smokers while comparable levels in non-smokers average less than 1%. Significant elevations in DNA and the neurotransmitter, dopamine (DA), were observed in the striatum of 21-day-old rats following the combined pre- and neonatal CO exposure. These neurochemical changes were observed 11 days after CO exposure was terminated and, therefore, cannot be interpreted as acute responses to reduced oxygen. These data indicate that the immature neostriatum is altered by even mild hypoxic insults presented during the time of neuronal proliferation and synaptogenesis.

Postnatal alterations in cerebellar GABA content, GABA uptake and morphology following exposure to carbon monoxide early in development.

Rats were exposed to either 0, 75, 150 or 300 ppm carbon monoxide (CO) from the day of conception until 10 days after birth. Exposure to CO resulted in significant deficits in cerebellar weight and deficits in total cerebellar content of gamma-aminobutyric acid (GABA) among rats at 10 days and 21 days after birth. Total cerebellar high-affinity 3H-GABA uptake was decreased among rats exposed to 300 ppm at 21 days of age, while total high-affinity 3H-glutamate uptake was unaffected. Moreover, cerebella of rats exposed to 300 ppm had fewer fissures, although major fissures were of normal depth, when examined histologically at 21 days of age. These results identify developmental processes involving GABAergic neuronal maturation and foliation of the cerebellum as vulnerable to early CO exposure.

Prenatal carbon monoxide exposure differentially affects postnatal weight and monoamine concentration of rat brain regions.

Regional weight, protein concentration, and monoamine concentration were determined in the pons/medulla, neocortex, hippocampus, and cerebellum of 21- and 42-day-old offspring of rats exposed to either air or 75, 150, or 300 ppm carbon monoxide (CO) throughout pregnancy. Norepinephrine and serotonin concentrations decreased linearly with increasing CO exposure concentrations in the pons/medulla of CO-exposed offspring at 21 days of age, but not by 42 days of age. Norepinephrine concentration increased linearly with increasing CO exposure concentrations in neocortex and tended to increase in hippocampus of CO-exposed offspring at 42 days of age, but not at 21 days of age. Regional weights and protein concentrations of the neocortex, hippocampus, or pons/medulla were not affected by CO exposure at either age. Cerebellar weights of CO-exposed offspring, however, decreased linearly with increasing CO exposure concentrations at both 21 and 42 days after birth. No significant effect of CO exposure on cerebellar monoamine concentrations was observed. The results suggest that prenatal CO exposure disrupts the development of noradrenergic and serotonergic neuronal systems, and the development of the cerebellum.

Alteration in the postnatal ontogeny of cerebellar norepinephrine content following chronic prenatal carbon monoxide.

The postnatal ontogeny of norepinephrine content in the cortex and cerebellum was determined in rats exposed prenatally to a chronic low level of carbon monoxide (150 parts per million). In the cerebellum, norepinephrine concentration and total norepinephrine content among carbon monoxide-exposed rats were consistently elevated over that of control rats from the second through the sixth postnatal weeks. In the cortex, norepinephrine concentration and total norepinephrine content among carbon monoxide-exposed rats did not differ from that of control rats over the same period. These results identify the cerebellum as a region whose postnatal development is altered by prenatal exposure to low levels of carbon monoxide-induced hypoxia.

Diethyldithiocarbamate depresses the acoustic startle response in rats.

The relationship between norepinephrine (NE) content in cortex and spinal cord and acoustic startle amplitude was investigated in two experiments. Administration of diethyldithiocarbamate (DDC) depressed startle amplitude at the same time and dose that it most severely depleted NE content. These results support the conclusion that NE facilitates the normal elaboration of the acoustic startle reflex and also support evidence that NE activity in the spinal cord may be of particular importance in the maintenance of normal startle amplitude.