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.

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.

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.

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.

Experiences with the dominant lethal test in female mice: effects of alkylating agents and artificial sweeteners on pre-ovulatory oocyte stages.

Pre-ovulatory oocytes are especially sensitive to mutagenic influences. Since post-dictyotene oocytes are not subject to selection and elimination before fertilization, they may reveal mutagenic effects directly and unrestrictedly. Presuming that a chemical is administered at pro-estrus to female mice one can conclude that the substance or its active metabolite has the chance to reach the gamete during the sensitive pre-fertilization stages. We proved the usefulness of the test system by investigating the effects of alkylating agents. A second step was to investigate other substances. The following treatments induced dominant lethal effects: methyl methanesulfonate 100 mg/kg i.m., cyclophosphamide 200 mg/kg per os, triaziquone 0.25 mg/kg i.p. In contrast, the following agents were ineffective and can be classified as not mutagenic in this method: sodium cyclamate 10 000 mg/kg per os, saccharine sodium, 10 000 mg/kg per os, cyclohexamine sulfate 150 mg/kg per os, ethanol 5 ml/kg per os.

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.

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.

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.

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.

Studies of embryo toxicity in rats and rabbits.

Embryo toxicity studies were carried out to investigate the effects of different doses of fluorescent whitening agents (FWAs) after oral administration to pregnant rats from the 6th to the 15th day of pregnancy and to rabbits from the 6th to the 18the day of pregnancy. In own studies two FWAs of the bis(triazinylamino or triazoly)stilbenedisulfonic acid type showed no embryo-toxic or teratogenic effect on wither species at daily doses up to 1000 mg/kg. Also the toxic dose to the mothers, 3000 mg/kg, given to rabbits in case of one FWA was without these effects. In the studies of KEPLINGER, et al. (Toxicol. Appl. Parmacol. 27: 494-506, 1974) a triazolylstilbenemonosulfonic acid derivative, two bis(triazinylamino)stilbenedisulfonic acid derivatives and a bis(sulfostyryl)diphenyl derivative had no terato-enic effect on rabbits when administered orally in doses of up to 30 mg/kg/day.

Testing mutagenic properties with the dominant lethal test on the male mouse.

Mytagecity studies were carried out with fluorescent whitening agents (FWAs) using the dominant lethal test on male mice. Own tests with five FWAs, and those of KEPLINGER, et al. (Toxicol. Appl. Pharmacol.27: 494-506, 1974), with four FWAs, are described. In our tests, acute oral administration of five FWAs at a dose of 5000 mg/kg body weight gave no evidence of a mutagenic effect during 8 weeks' mating. The FWAs used were three bis(triazinylamino)stilbenedisulfonic acid derivatives and a 1,3-diphenyl-2-pyrazoline derivative. The results of the tests carried out by KEPLINGER, et al. showed that intraperitoneal injection of the four FWAs produced no mutagenic effect during six weeks' mating; the whiteners used were a triazolylstilbenemonosulfonic acid derivative (50 mg/kg), two bis(triazinylamino)stilbenedisulfonic acid derivatives (50 mg/kg) and a bis(sulfostyryl)biphenyl derivative (10 mg/kg).

Method for testing mutagenic effects of chemicals on spermatogonia of the Chinese hamster: results obtained with cyclophosphamide, saccharin, and cyclamate.

The action of different cyclophosphamide doses on spermatogonia of the Chinese hamster was examined. Two oral treatments at an interval of 24 h were carried out and spermatogonia were prepared for examination 24 or 48 h after the second dose. Accordingly the effects of 5 oral cyclophosphamide doses given on five consecutive days were tested on spermatogonia and preparations were made 24 or 72 h after the last treatment. The results so obtained form the basis of reference for findings following oral administration of saccharin sodium, sodium cyclamate, or trimethylphosphate. Male Chinese hamsters, 6-8 per group, were used, from each of which about 100 metaphases were evaluated. Preparation was carried out essentially according to Hoo and Bowles [Mutation Res. 13, 85-88 (1971)]. gaps, breaks, fragments, deletions and translocations were rated as structural aberrations. For every dose and every time of preparation the incidence of metaphases with aberrations, with or without gaps, and with translocations were assessed. The different experiments led to the following conclusions: 1. By analysis of spermatogonial metaphases of treated Chinese hamsters chemically induced chromosome aberrations can be proved with certainty. 2. Incidence of metaphases with translocations is a sensitive measure which is distinctly superior to the summary determination of all aberrations. In this way it was possible to show a mutagenic influence of 2 times 8 mg/kg cyclophosphamide p.o. 3. Following two cyclophosphamide doses administered at an interval of 24 h it was found that preparations of spermatogonia 48 h after the second dose was better suited for the evaluation than that at 24 h, for aberrations were more frequent with the same treatment. After five cyclophosphamide treatments at 24-h intervals, aberrations were somewhat more frequent 24 h after the last dose than at 72 h; in any case the values exceeded significantly the results of untreated controls. 4. A conclusive numeric chromosome analysis is not possible with the spermatogonia test, since a relatively high percentage of non-diploid cells is apparently of methodological origin. 5. Tests with 2 times 500 or 5 times 1000 mg/kg trimethylphosphate orally showed an increase in chromosome aberrations compared with controls indicating mutagenic effects in both cases; with 5 times 1000 mg/kg p.o., however, the figures were low as a result of marked mitotic inhibition. 6. The results of the spermatogonia test on Chinese hamsters revealed no mutagenic effects of saccharin sodium 2 times 5000 mg/kg orally, and of sodium cyclamate 5 times 2000 mg/kg orally. This is based on comparisons of the results both with untreated controls and positive controls treated with trimethylphosphate or cyclophosphamide.

[Influence of several weeks' treatment of male and female mice with saccharin, cyclamate or cyclohexylamine sulfate on fertility and dominant lethal effects (author's transl)]. / Einfluss einer mehrwöchigen Behandlung männlicher und weiblicher Mäuse mit Saccharin, Cyclamat oder Cyclohexylaminsulfat auf Fertilität und Dominant-Letal-Effekte

The purpose of this investigation was to find out whether long-term treatment of male and female mice with saccharin sodium, sodium cyclamate or cyclohexylamine sulfate, would reduce fertility or induce dominant lethal mutations. Before mating, saccharin sodium or sodium cyclamate were added to the food in a concentration of 1%, while cyclohexylamine sulfate was added in a concentration of 0.11% for at least 10 weeks. This treatment corresponded, in the case of saccharin sodium and sodium cyclamate, to an active substance intake of approx. 2000 mg/kg per day and for cyclohexylamine sulfate to an intake of approx. 200 mg/kg per day (corresponding to approx. 136 mg cyclohexylamine per kilogram per day). These doses affected neither the females nor the males in respect of appearance, behaviour, and weight gain. The doses were also compatible with the normal fertility of the animals. Furthermore, in all cases the treatment did not cause a biologically important increase of pre-implantative and post-implantative losses. The dominant lethal tests did not indicate a mutagenic action of saccharin sodium or sodium cyclamate (1% in the food) and of cyclohexylamine sulfate (0.11% in the food) after 10 weeks' treatment of male and female mice. These results, obtained after long-term treatment, corresponded generally to the findin

PIP: An attempt to determine whether long-term treatment of male and female mice with saccharin sodium, sodium cyclamate or cyclohexylamine sulfate, would reduce fertility or induce dominant lethal mutations is reported. Before mating, saccharin sodium or sodium cyclamate were added to the food in a concentration of 1%, while cyclohexylamine sulfate was added in a concentration of .11% for at least 10 weeks. This treatment corresponded, in the case of saccharin sodium and sodium cyclamate, to an active substance intake of approximately 2000 mg/kg/day (corresponding to approximately 136 mg cyclohexylamine/kg/day). These doses affected neither the females nor the males in respect to appearance, behavior, weight gain, or fertility. Treatment did not cause a biologically important increase of preimplantative and postimplantative losses. The dominant lethal tests did not indicate a mutagenic action of saccharin sodium or sodium cyclamate (1% in the food) and of cyclohexylamine sulfate (.11% in the food) after 10 weeks' treatment of male and female mice. These results, obtained after long-term treatment, corresponded generally to the findings of dominant lethal examinations after acute and subacute application of these 3 substances.

[The inhalation toxicity of tertiary butylisonitril in rats and mice. Acute toxicity and evaluation of embryotoxic and mutagenic effects (author's transl)]. / Inhalationstoxikologische Untersuchungen mit tertiärem Butylisonitril an Ratten und Mäusen. Akute Toxicität und Prüfung auf embryotoxische und mutagene Wirkungen

The inhalation toxicity of tertiary butylisonitril (TBIN) was evaluated in rats and mice. In addition, pregnant rats were exposed to TBIN aerosols to test its embryotoxic effects and male mice were exposed to TBIN aerosols to evaluate its mutagenic effects using the dominant lethal test. Inhalation of TBIN aerosols caused death in rats only at high concentrations. The following inhalation LC50 values were determined for rats and mice following inhalation of TBIN aerosols: 4-hr exposure: male rats 715, female rats 710, male mice 377; five 4-hr exposures: male and female rats between 356 and 583 mg/m-3 air. The animals were impaired for a long period of time, and death occurred up to 10 days after the exposure. Female rats were exposed to TBIN aerosols from the 6th to the 15th day of gestation daily for 4 hrs. The used concentrations were not toxic to the pregnant rats. It became evident that only the lowest concentration (14 mg/m-3) was not effective to the development of the fetus. A TBIN concentration of 36 mg/m-3 definetely increased the resorption in embryos, and with 71 mg/m-3 a complete loss of the fetus occurred due to resorption. A teratogenic effect could not be determined. A single 4-hr inhalation of 125 mg TBIN/m-3 air caused changes of the sperms of male mice. There was a decreased fertilization capability during the first week of mating after the exposure and a decreased implantation rate with a simultaneous increase of pre-implantation losses in the females. With respect to industrial hygiene it is important that concentrations of TBIN in the air show embryotoxic and anti-spermatogenic effects in animals, which they tolerated without symptoms of poisoning.