Assessment of pyrethroid-induced paraesthesias: comparison of animal model and human data.

The quantification of upper respiratory tract (URT) sensory irritation is considered to be important in rodent inhalation studies, since it may be also used as an endpoint mimicking trigeminal paraesthesias observed in humans. URT sensory irritation is known to be associated with rodent-specific secondary physiological effects such as depression of body temperature and changes in heart rate. In acutely exposed rats, these endpoints have been addressed by telemetrical measurements. The analysis of the ventilation pattern during acute inhalation studies of rats exposed to the alpha-cyano-pyrethroid cyfluthrin demonstrates that concentration-dependent URT sensory irritation was associated with a hypothermic response. The no-effect levels (NO(A)EL) based on the URT sensory irritation endpoint following acute inhalation exposure for 1 h and following a repeated 4-week or 13-week inhalation exposure for 6 h/day on 5 days week were virtually identical (approximately 0.1 mg/m3 air). An additional objective was to examine whether human volunteers experience comparable signs when acutely exposed for 1 h to airborne concentrations slightly above or in the range of the NO(A)EL. In human volunteers there were no clinically significant or pyrethroid related abnormalities in vital signs, ECG's or in any clinical laboratory tests after either exposure, although transient effects related to URT (sensory) irritation were reported. In conclusion, an initial actual exposure concentration of approximately 0.1 mg cyfluthrin/m3 air appears to be in the range of the sensory irritant threshold concentration for both rats and humans. Thus, with regard to physiological afferent portal-of-entry effects, the interspecies response was consistent.

Adequacy of required regulatory hazard testing for the detection of potential hormonal activity of crop protection chemicals.

The capacity of some synthetic chemicals, the so-called "endocrine-disrupting chemical," to alter hormonal activity, as well as the adequacy of the testing of chemicals to evaluate this capacity, has been called into question. Among the chemicals indicted have been certain crop protection agents or pesticides. Crop protection chemicals rank among the most closely regulated and thoroughly tested chemicals in use in both the human health and environmental hazard areas. However, it has been proposed that in vitro and in vivo screening tests be used to identify potential endocrine-active chemicals and to supplement or replace required regulatory bioassays. In vitro tests, such as receptor binding, examine a single chemical event, do not measure toxicity, post-receptor-mediated biological response, or the absorption, distribution, metabolism, and elimination of a chemical. Therefore, data derived solely from such a limited testing technique should not be used as a basis for selection of chemicals for making regulatory decisions. In vivo screening tests, such as the uterotrophic assay, which promise to provide a rapid answer to a targeted question, do not capture the complexity of the biological response. As in the case with in vitro tests, results from a single in vivo test, such as a change in uterine weight, should not be used as a basis for regulatory decision making. Further, it has been suggested that such a screening battery should be put into place for ecotoxicity testing. Yet it is well recognized that endocrine-active chemicals that affect fish and wildlife in their natural habitat have been shown to cause similar adverse effects in laboratory test animals. Therefore, these screening tests do not add value to the current regulatory test battery. Evidence is presented that demonstrates that the regulatory safety assessment paradigm has a low likelihood of missing potential endocrine-active chemicals and has served society well.

Learning and memory of rats after long-term administration of low doses of parathion.

A set of four learning and memory tests (Morris Maze I for reference memory, Morris Maze II for working memory, one-way active avoidance, and passive avoidance) were employed to address the questions whether parathion impaired cognitive functions after low, long-term exposure and could cause persistent changes in cognition. Motor activity and general behavior were investigated in a functional observational battery. Parathion was administered in rat food in low doses which caused no clinical symptoms and no or borderline brain acetylcholinesterase inhibition. Parathion doses of 0.5, 2, or 8 ppm in rat food produced the averaged uptake of 24, 100, or 400 microg/kg body weight per group per day in male rats and 36, 152, or 550 microg/kg per day in female rats in week 13. Learning tests were performed in weeks 1 to 4 and 10 to 14, as well as 30 to 34 weeks after the end of treatment, when the male and female rats were about 13 months old. Low doses of parathion given daily for 13 weeks had no cumulative or adverse effects on learning and memory, either during treatment or after the extended treatment-free period, in any of the tests. A significant improvement of learning compared to control observed in the Morris Water Maze I during the first week of treatment (males dose group 0.5 ppm) shows that parathion can improved cognitive functions in rats. Results of the study indicate that adverse effects changing learning and memory in animals may occur only at higher doses of organophosphates, at which the peripheral and brain acetylcholinesterases are inhibited to a greater extent than those in the present study.

[Toxicologic evaluation of pyrethroids in indoor air: demonstrated with the example of cyfluthrin and permethrin]. / Toxikologische Bewertung von Pyrethroiden in Innenräumen: Aufgezeigt am Beispiel Cyfluthrin und Permethrin.

Pyrethroids have varying activities depending on vehicle or route of administration (oral, dermal, inhalational). Specific features like the sensory irritation potential of the alpha-cyano-pyrethroids on the respiratory tract can only be quantified adequately by inhalation testing. Thus equitoxic dosages can vary between inhalative and oral application, especially for alpha-cyano-pyrethrolds. The no-effect values for chronic exposures derived for permethrin (type I pyrethroid) and cyfluthrin (type II pyrethroid) show clearly, that each pyrethroid has to be considered as an individual substance toxicologically, and that any extrapolation from the oral to the inhalative route should only be done after a thorough assessment of the specific toxicological profile. The study of simulated pest control measures on carpets pretreated with permethrin showed, that no significant enrichment of permethrin in total dust could be seen from a carpet additionally treated with pyrethroids. The missing correlation between absolute (mg pyrethroid/m3 air) and relative (mg pyrethroid/kg dust) concentrations in air-borne dust as well as the low degree of translocation of pyrethroids from carpets (only about 0.044% x m(-2) x h(-1) of the cyfluthrin applied to the carpet can be regarded as possibly respirable) prove, that analyses of pyrethroids in household sedimented dust ("vacuum cleaner bag analyses") without knowing the absolute surface concentration and respective air concentrations are of little value for risk assessment. The data allow the conclusion, that a scientific assessment of health risks is only possible based on absolute concentrations of pyrethroids in indoor air.

Electroretinographic assessment of early retinopathy in rats.

Amoscanate, a substance which damages photoreceptors, was administered orally to Wistar rats in doses of 10, 40, and 125 mg/kg body weight once daily for 3 or 10 days. At both times electroretinographic, ophthalmological, and histopathological examinations of the retina were carried out to compare the sensitivity of conventional methods and to test electroretinography (ERG) for suitability for use in toxicity studies. Time-dependent and dose-dependent effects were found by electroretinography and light microscopy. However, signs of retinal changes appeared earlier and more distinctly in the electroretinogram. Ophthalmological fundus examination in albino rats yielded no characteristic correlate. In conclusion, electroretinography constitutes a valuable supplement to histopathology and is suitable for use in toxicity studies.

Analysis of alternative methods for determining ocular irritation.

According to classification and labelling requirements, chemicals, dyes, agrochemicals and pharmaceutical formulations have to be evaluated for their potential to induce eye irritancy or corrosion. An attempt was made to analyse the predictive power of the bovine eye-chicken egg chorioallantoic membrane (BE-CAM) assay in comparison with results obtained using the conventional Draize method. In summary, results showed limited correlation between reactions in vitro and responses of eyes in vivo. In a pilot study, ultrasonic pachymetry showed high sensitivity and fairly good correlation between corneal thickness and clinical observations in eyes.

Difference between species in response to a 3,5-dichloropyridyloxyphenoxy compound: induction of cytochrome P-450 and/or peroxisome proliferation.

In toxicological studies, the test compound FOE 3440 A, a [(3,5-dichloro-2-pyridyl)oxy]phenoxypropanoate with herbicidal properties, produced a severe increase in weight and an intensive induction of monoxygenases activity in the mouse, but not in the rat. Comparative subacute studies were performed with oral administration of 0, 5, 20 and, in some instances, 80 mg kg-1 body weight to mice, rats, hamsters, dogs and rhesus monkeys. Liver enzyme activities were measured. The evaluation of the enzyme activity results showed an unusually severe dose-related induction of the monooxygenases [7-ethoxycoumarin-O-deethylase (EOD), 7-ethoxyresorufin-O-deethylase (EOR) and aldrin epoxidase (ALD)] in the mouse and a much weaker reaction in the other species tested. This exceptional position of the mouse was also demonstrated in vitro by a cytochrome P-450 interaction (inhibition of ALD). The primary metabolite of FOE 3440 A produced a distinct inhibition of the ALD in mice liver microsomes. There were no interactions for the other species. Tests for this cytochrome P-450 interaction using microsomes from three different human livers gave no indications of an inhibition in any case. The 'phenoxypropanoic acid' moiety of FOE 3440 A is structurally similar to the pharmaceutical clofibrate, a familiar model substance for peroxisome proliferation. In order to answer the question of whether peroxisome proliferation is the second mechanism for affecting liver, the carnitine acetyl transferase activity (CA-T), a marker enzyme for peroxisome proliferation, was determined in all liver samples from the comparative species studies. The most striking result of the measurement of CA-T activity was the very large increase in the male rat in the low dose group of 5 mg kg-1. Lesser increases in the CA-T activity were measured in the female rat, the mouse, and also in the 20 mg kg-1 group of the hamster. By comparison, the changes of the activity in dog and monkey were very small. Comparative studies in mouse and hamster using a model substance described in the literature (1,4-bis[2-(3,5-dichloropyridyloxy]-benzene (TCPOBOP] indicated that the '3,5-dichloropyridyloxy' moiety of FOE 3440 A is responsible for the induction of the monooxygenases in the mouse and the 'phenoxypropanoic acid' moiety for the peroxisome proliferation in rodents.

Methodological aspects of the determination of the acute inhalation toxicity of spray-can ingredients.

Spray-can ingredients, if liberated in confined spaces, are potential health hazards for man. Thus, appropriate inhalation toxicity studies have to be performed in accordance with internationally recognized guidelines, e.g. the US Environmental Protection Agency: Federal Insecticide, Fungicide and Rodenticide Act (FIFRA no. 81-3) or OECD no. 403. One of the essential requirements of such guidelines is that test animals (preferably rats) be exposed to a steady-state concentration in a dynamic inhalation chamber for at least 4 hours. This is not easy to achieve with vapours released from a pressurized spray-can. The method described here makes it possible to expose experimental animals in an inhalation chamber to a steady-state concentration of intermittently released spray jets of constant doses per jet. Animal experiments and theoretical considerations (computer simulations) have shown that the method presented allows an up-to-date determination of the acute inhalation toxicity of spray-can ingredients.

Effects of inhaled cholinesterase inhibitors on bronchial tonus and on plasma and erythrocyte acetylcholine esterase activity in rats.

Young adult male and female Wistar rats were inhalationally exposed head-only for 1 or 4 h to different anticholinesterase aerosols. The compounds tested were dichlorvos, fenamiphos, methamidophos, parathion, a pyrimidine thiophosphate and the carbamate propoxur. These compounds are direct or indirect inhibitors of cholinesterase activity. Immediately after termination of exposure to the compounds, the rats were anesthetized with barbiturate and subjected to pulmonary function tests. An acetylcholine provocation test was performed to correlate the effect of the cholinesterase inhibition and lung resistance. The results basically revealed that by inhalation exposure bronchoconstriction in the absence of acetylcholine provocation did not occur at toxicologically significant doses of the pesticides. An increase in lung resistance was observed only after provocation. However, measurements of plasma cholinesterase activity proved to be more sensitive than the provocation test. With regard to their diagnostic value, the results of the reported study may be summarized as follows (beginning with the most sensitive parameter): plasma cholinesterase activity depression greater than or equal to acetylcholine-induced bronchoconstriction greater than or equal to cholinergic symptoms greater than erythrocyte cholinesterase activity depression greater than pulmonary resistance without acetylcholine provocation.

The effect of cyclohexylamine on the embryo following oral administration to mice and rats.

The possible embryotoxic effects of cyclohexylamine hydrochloride doses (expressed as free base) of 10, 30 and 100 mg/kg of body weight per day administered orally in water to mice and rats from the 6th to 15th day post-coitum were investigated. Treatment with daily doses of up to 100 mg/kg of body weight to mice and of up to 30 mg/kg to rats had no adverse effect on the mothers and the embryos. In the rat the dose of 100 mg/kg led to decreased weight gain in the mothers during the treatment period. In parallel, a non-specific retardation of the embryo was found, since the weight of the foetus and of the placenta were significantly reduced. Cyclohexylamine had neither a teratogenic effect nor a primary toxic effect on the embryo in either of the animal species. The tolerated dose without any harm for the development of the embryo was 100 mg/kg/day in mice and 30 mg/kg/day in rats.

Mutagenicity studies with 2,4,5-T on bacteria and mammalian germ cells.

The herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) was evaluated for potential mutagenicity by a Salmonella/mammalian-microsome test, a dominant lethal test on female rats, and by a cytogenetic assay on spermatogonia of Chinese hamster. In the Salmonella/mammalian-microsome test on four Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, and TA 98), doses of up to and including 2500 micrograms/plate did not cause any mutagenic effects. In a dominant lethal test on female rats, 8-week dietary administration of 2,4,5-T at doses of up to and including 10 mg/kg/day did not cause any increase in preimplantation loss or the rate of dead implants, and did not have any effect on the fertilization quota. Cytogenetic analysis of the spermatogonia of male Chinese hamsters orally dosed five times at 24-hr intervals with 2,4,5-T at levels of up to and including 100 mg/kg did not provide any indication of 2,4,5-T having chromosome-damaging effects. Therefore, none of the three test systems provided any indication of 2,4,5-T having a mutagenic effect.

Chronic toxicity of metrifonate.

From chronic toxicity studies with metrifonate (identical with trichlorfon) on different animal species, the following conclusion can be reached: 1. There is no conclusive evidence that trichlorfon has a carcinogenic effect. 2. There is no indication that trichlorfon has a significant cumulative toxic effect. 3. There is no confirmation that trichlorfon has a specific effect on liver, blood and haematopoietic organs. 4. The toxicological profile of trichlorfon in the chronic toxicity study consists, on the one hand, in a depression of cholinesterase activity whereby the different animal species differed in their sensitivity. In rats, cholinesterase activity was depressed in serum at dietary levels of 500 p.p.m. and above and also in erythrocytes at a dietary concentration of 1000 p.p.m. In dogs, cholinesterase activity was depressed in serum and erythrocytes at a dietary level of only 200 p.p.m. The Rhesus monkey seemingly is very sensitive because erythrocyte cholinesterase activity is possibly reduced at a daily dose of 0.2 mg/kg per os. On the other hand, induction of an inhibition of spermatogenesis in rats and dogs at a high dose level is discussed, an effect which seemingly is morphologically detectable but nonetheless seems not to affect function as animal experiments have demonstrated.

Mutagenicity studies with praziquantel, a new anthelmintic drug, in mammalian systems.

Praziquantel, a new anthelmintic drug with antischistosomal and anticestodal properties, was tested in comparison with a placebo control and a 'positive control' with cyclophosphamide in mammalian test system in vivo for potential mutagenic effects. The test systems used and the tested doses of Praziquantel were: (1) Dominant lethal test on male NMRI mice, 12 mating periods of 4 days each, 1 X 1200 mg/kg BW by mouth; (2) Dominant lethal test on female NMRI mice, treatment during pre-estrus, 1 X 1200 mg/kg BW by mouth; (3) Micronucleus test on male and female NMRI mice, two doses with a 24-h interval and preparation of the femoral marrow 6 h after the second dose, 2 X 300 mg/kg and 2 X 600 mg/kg BW by mouth; (4) Spermatogonial test on the Chinese hamster, two doses with a 24-h interval and preparation of the spermatogonia 48 h after the second dose, 2 X 600 mg/kg BW by mouth. The 1200 mg/kg BW dose corresponded to approximately 1/2 of the LD50 after oral application in the mouse and about 40 times the therapeutic dose (1 X 30 mg/kg BW). The cyclophosphamide doses in the test systems were 1 X 200 mg/kg or 2 X 200 mg/kg BW by mouth. No indication was found of any mutagenic potency of Praziquantel. This agrees with the results of point-mutation tests done by other authors.

Evaluation of the mutagenic potential of cyclohexylamine on spermatogonia of the Chinese hamster.

In a cytogenetic study on the spermatogonia of Chinese hamster, cyclohexylamine (neutral sulphate) was evaluated for mutagenic effects in comparison with an untreated control group and a group treated with the mutagenic compound cyclophosphamide, by assessing spermatogonial metaphases of treated Chinese hamster for chromosomal structural changes. Each test group comprised 8 male hamsters selected at random. Approximately 100 metaphases from each animal were assessed. The doses were 5 X 150 mg cyclohexylamine sulphate (approx. 5 X 102 mg base) per kg body-weight orally, and 5 X 100 mg cyclophosphamide per kg body-weight orally. The individual doses were administered at intervals of 24 h. Preparations were made 24 h after the final treatment, essentially by the method of Hoo and Bowles [10]. Gaps, breaks, fragments, deletions and translocations were assessed as structural changes; frequencies were determined of the metaphases (a) with aberration(s) including gaps, (b)with aberration(s) less gaps and (c)with translocation(s). Aberrations occurred in the untreated negative control group (1.24% incl. gaps, 0.25% without gaps). Translocations were not seen in the untreated group. In the cyclochexylamine group, the frequencies of the aberrant metaphases were sometimes less than in the control group (0.87% including gaps, 0.37% without gaps). Statistically, the results were not significantly different from the control data. No translocations were seen after administration of cyclohexylamine. The positive cyclophosphamide control group clearly differed from the untreated control and from the cyclohexylamine group in the parameters (a) to (c); mainly, the results were highly significantly different from those obtained in the untreated control group. The frequencies of the aberrant metaphases were 3.41% including gaps and 1.99% without gaps. The frequency of the translocations was 0.71% (5 out of 704). Cyclohexylamine sulphate, administered 5 times at 150 mg/kg body-weight orally, had no mutagenic effect, whereas cyclophosphamide, adminstered 5 times at 100 mg/kg body-weight orally, had a chromosome-damaging effect on Chinese hamster spermatogonia.