Invited contribution: Acute toxicity testing,public responsibility and scientific challenges

Knowledge of the acute toxic effects of chemicals is important for the protection of exposed humans. Since sufficient information in humans is often lacking, experiments on laboratory animals must be performed. The LD₅₀ test, which requires large numbers of animals, has become the preferred procedure. It is now widely criticized on scientific and ethical grounds. This paper reviews the possibilities of using fewer animals to obtain relevant information on the acute hazards of chemical substances, but it also identifies the reasons why the traditional testing approaches cannot be changed immediately. An important problem is the practice of basing legal decisions on classification of chemicals in official lists of hazardous substances and for labeling purposes on LD₅₀ values. Proposals are presented on how pain and suffering of the animals included in acute toxicity tests can be reduced. The use of in vitro systems for the evaluation of the hazardous properties of chemicals is discussed.Abbreviations LD₅₀ lethal dose 50%, median lethal dose     

Pharmacological activity and toxicity of Phencyclidine (PCP) and Phenylcyclohexene (PC), a pyrolysis product

Initial acute behavioral studies in mice indicated that phencyclidine (PCP) produced marked motor impairment as measured by the inverted screen technique with an ED₅₀ value of 4.1 μMole/kg (i.v.). Phenylcyclohexene (PC) was considerably less active with an ED₅₀ value of 325 μMole/kg (i.v.). PCP was also shown to be more lethal than PC as acute (24 hr; i.v. injection) LD₅₀ values (μMoles/kg) in males were 57 and 448, and in females were /6 and 425, respectively. A greater acute lethality was also produced by PCP after i.p. and p.o. administration. Subchronic (14-day) exposure (i.p.) to PCP at doses up to ≈40 percent of the acute LD₅₀ value (123.6 μMole/kg, i.p., daily) was without significant effect on body and organ weights, hematology and clinical chemistry, and humoral and cell-mediated immunity. Higher doses of PCP were not possible because of acute lethality. Subchronic exposure to PC (63.4, 317, and 634.5 μMoles/kg; 4 percent, 20 percent and 40 percent of acute i.p. LD₅₀ value, respectively) produced several marked effects. At the highest dose tested, body weight and thymus weight in both males and females, and liver weight in males were significantly decreased. The spleen weight of males exposed to 317 μMole/kg PC was also significantly decreased. Humoral immunity (production of antibody forming cells) was significantly inhibited in both males and females exposed to PC. In contrast, cell-mediated immunity (development of a delayed-type hypersensitivity response) was only significantly inhibited in females. As PCP has no measurable toxicity under these conditions and PC produced significant effects at relatively high doses, the results suggest that neither chemical is exceptionally toxic following subchronic exposure.     

Effect of zinc on the toxicity of the mycotoxin sporidesmin to the rat

The toxicity of sporidesmin to rats fed diets containing either 4 ppm or 200 ppm Zn was determined. The high zinc diet doubled the LD₅₀ level for sporidesmin given as a single dose, but the LD₅₀ dose for rats on a zinc deficient diet was not significantly different from that for rats on a diet containing zinc at levels sufficient for normal growth. Subsequent trials demonstrated that large doses of ZnSO₄ given orally protected rats from sporidesmin poisoning. The protection was short lived as zinc needed to be given at or soon after sporidesmin challenge.     

An assessment of the developmental,reproductive, and neurotoxicity of endosulfan

BACKGROUND : Endosulfan has been used for over 50 years. Although most analogs have been discontinued, endosulfan has less environmental persistence. Nevertheless, pressure groups are lobbying for a worldwide ban. The reasons are: possible rodent male reproductive toxicity, other endocrine effects and cancer; human epidemiology, and exposure studies; residues appearing in remote areas of the world, e.g., the Arctic. METHODS : The endosulfan toxicology database is described and risks of its use assessed. RESULTS : Endosulfan is an antagonist at the GABA_A receptor Cl^? ionophore in mammalian CNS. Rat acute toxicity is moderate, LD₅₀=48 (M) or 10 mg/kg/d (F), oral gavage; 130 (M), 70 mg/kg/d (F) dermal; LC₅₀=34.5 µg/L (M), 12.6 µg/L (F), inhalation. Critical NOELs for risk assessment: acute oral (gavage)=0.7 mg/kg/d (rabbit developmental); Subchronic oral (diet)=1.2 mg/kg/d (rat reproduction); Chronic oral (diet)=0.6 mg/kg/d. There were no acceptable dermal toxicity studies. The critical acute and subchronic inhalation NOELs=0.001 mg/L, chronic inhalation=0.0001 mg/L (estimated). Toxicity to rat sperm occurred at doses causing neurotoxicity. Endocrine effects, resulting from P450 oxygenase(s) induction, were reversible. Increased cancer, genotoxicity, or histopathology in rodents was not observed in any organ. Possible effects on brain biogenic amine levels were probably secondary. CONCLUSIONS : Epidemiology and rodent studies suggesting autism and male reproductive toxicity are open to other interpretations. Developmental/ reproductive toxicity or endocrine disruption occurs only at doses causing neurotoxicity. Toxicity to the fetus or young animals is not more severe than that shown by adults. Birth Defects Res (Part B)86:1‐28, 2009. © 2009 Wiley‐Liss, Inc.     

Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides

Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidacloprid (IMI), have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over‐expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IMI and nitromethyleneimidazole (NMI), in two planthopper species by IMI lethal dose that kills 85% of the population (LD₈₅), and in N. lugens among three IMI doses (LD₁₅, LD₅₀ and LD₈₅). When IMI and NMI at the LD₈₅ dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up‐regulation of nine genes and down‐regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD₈₅ dose, 10 genes had very similar patterns, such as up‐regulation in seven genes, down‐regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up‐regulation in four genes at all doses and dose‐dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up‐regulated by the LD₅₀ dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose‐dependent.     

An evaluation of the genotoxic properties of some chosen dyes using the micronucleus test in vivo

The frequency of micronucleated polychromatic erythrocytes and the polychromatic to normochromatic erythrocyte ratio was studied in BalbC mice treated with four azo dyes: Direct Blue 74, Direct Blue 296, Direct Blue 297 and Direct Green 98 at two (40and 80% LD₅₀/kg body weight) concentrations. None of the studied compounds revealed a genotoxic activity in the micronucleus test. However, it was found that two dyes, Direct Blue 297 at doses 40% and 80% LD₅₀ and Direct Green 98 at dose 80% LD₅₀, cause a significant decrease in the ratio of polychromatic to normochromatic erythrocytes in bone marrow of mice, which means that at the doses specified above they can affect the proliferation of the blood cells.     

Effect of L-carnitine against acute mitoxantrone toxicity in mice

Various experiments were performed in our laboratory to define a possible role for carnitine derivatives in mitoxantrone (MX) therapy. We report here the results of the effect of L-carnitine (LCAR) on the lethal toxicity of MX in mice. MX was administered intravenously at doses of 7, 9, 11, 13, 15 or 17 mg kg⁻¹ either alone or in combination with LCAR at a single intravenous dose of 200 mg kg⁻¹. The dependence of the probability of death on various doses was evaluated for the MX-LCAR combination compared to MX alone. From these experiments, the following lethal dose fifty (LD₅₀) values were calculated: LD₅₀ for MX alone was 15.2 mg kg⁻¹, whereas in combination with LCAR it increases to 21.8 mg kg⁻¹. The relative toxicity given as the ratio of the LD50 of both MX alone and the combination of MX-LCAR was 69.7%. The results of our experiments unequivocally show the effect of LCAR on acute toxic doses of MX.     

Efficacy and safety of catnip (Nepeta cataria) as a novel filth fly repellent*

Catnip (Nepeta cataria) is known for its pseudo‐narcotic effects on cats. Recently, it has been reported as an effective mosquito repellent against several Aedes and Culex species, both topically and spatially. Our laboratory bioassays showed that catnip essential oil (at a dosage of 20 mg) resulted in average repellency rates of 96% against stable flies, Stomoxys calcitrans (L.) and 79% against houseflies, Musca domestica (L.), respectively. This finding suggested that the application of repellent could be used as part of filth fly management. Further evaluations of catnip oil toxicity were conducted to provide a broad‐spectrum safety profile of catnip oil use as a potential biting and nuisance insect repellent in urban settings. Acute oral, dermal, inhalation, primary dermal and eye irritation toxicity tests were performed. The acute oral LD₅₀ of catnip oil was found to be 3160 mg/kg body weight (BW) and 2710 mg/kg BW in female and male rats, respectively. The acute dermal LD₅₀ was > 5000 mg/kg BW. The acute inhalation LD₅₀ was observed to be > 10 000 mg/m³. Primary skin irritation tested on New Zealand white rabbits showed that catnip oil is a moderate irritant. Catnip oil was classified as practically non‐irritating to the eye. In comparison with other U.S. Environmental Protection Agency‐approved mosquito repellents (DEET, picaridin and p‐menthane‐3,8‐diol), catnip oil can be considered as a relatively safe repellent, which may cause minor skin irritation.     

Variation in the time‐mortality responses of laboratory and field populations of Helicoverpa armigera infected with nucleopolyhedrovirus

The virulence of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) to South African laboratory and field populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was evaluated. After determining the volume imbibed by each population, bioassays were performed using the droplet feeding method and gradient‐purified HearNPV occlusion bodies. Evaluation of dose‐ and time‐mortality data showed that the median lethal doses (LD₅₀s) did not differ significantly between the populations but that the median lethal time (LT₅₀) of the laboratory population was significantly shorter than the LT₅₀s of the field populations. The study shows the importance of considering both the dose‐ and time‐mortality responses when comparing the response variability of populations to a nucleopolyhedrovirus infection.     

X-ray irradiation of excised embryos of mesta (Hibiscus cannabinus L. and H. sabdariffa L.)

Summary Excised embryos of Hibiscus spp. were treated with 1 kR to 6 kR of X-ray. Results indicate that germination was unaffected at this level of employed doses in both species, which in turn implies that the factors responsible for inhibition of germination are not present in the embryo. LD₅₀ values differed between varieties and species. Early varieties of both species were more sensitive to radiation than late varieties. Strikingly similar effects were observed for the varieties with smaller embryos over those with larger ones. Allopolyploid H. sabdariffa (2n=72) was more susceptible than diploid H. cannabinus (2n=36).Differences in mutation frequency exist between species with different levels of ploidy and between varieties within the same species. Most of the HC mesta varieties yielded higher mutation frequencies than those of HS mesta. Optimal dose for triggering mutations in all varieties (except the chlorophyll mutation variety of HC mesta) of the two species lies within a narrow range of 1 kR to 2 kR. Cent per cent seedling abnormalities is concomitant to LD₅₀; nevertheless, optimum dose for mutation frequency is independent of LD₅₀. Hence, the response should be viewed in terms of respective genotype. The advantages of the embryo irradiation technique are mentioned.     

Permethrin and malathion LD90 values for Culex quinquefasciatus vary with topical application site

Prior research in multiple insect species has demonstrated that insecticide‐induced mortality varies according to the body region exposed on the insect. This variation has been demonstrated in Culex quinquefasciatus Say (Diptera: Culicidae), but has not been quantified using dose–response curves. Applications of technical permethrin or malathion to one of three body regions on Cx. quinquefasciatus resulted in dose–response curves that were not equivalent to one another. The generated LD₉₀ values and curves for each body region were compared with previously reported LD values for analogous sites in several mosquito species, specifically the mesothorax. Based on the present results, the permethrin and malathion LD₅₀ and LD₉₀ concentrations required for droplets impinging on the abdomen and mesothorax of Cx. quinquefasciatus when applied through ground‐based spray systems utilized by mosquito control programmes were calculated.     

Resistance ofSpodoptera frugiperda [Lep.: Noctuidae] to a nuclear polyhedrosis virus in the field and laboratory

Median lethal doses (LD₅₀s) of nuclear polyhedrosis virus (NPV) were determined in neonatal offspring ofSpodoptera frugiperda (J. E. Smith) (Sf) larvae captured in southeastern Louisiana in 1981, 1982, and 1984. These LD₅₀s ranged from 1.8 to 16.3 polyhedral inclusion bodies (PIB)/insect. The LD₅₀s significantly (P<0.05) increased during the season of 1982 but had no pattern in 1981 or 1984. However, the Sf populations increased in heterogeneity of response to the NPV during all 3 years. The LD₅₀ increased from 4.1 to 18.7 PIB/insect in a Sf laboratory colony exposed to the NPV LD₈₀ for 7 generations, whereas in a control colony not exposed to NPV the LD₅₀ was 5.9 PIB/insect after 7 generations.     

Comparison of in vivo acute lethal potency and in vitro cytotoxicity of 48 chemicals

The cytotoxicity of 48 compounds included in the MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) list was determined in cultures of rat hepatocytes, McCoy, and MDBK cells. The average minimum concentration of each compound inducing cytotoxicity was measured in each cell type. The cytotoxicity values were then compared with published oral LDS p values for rats and mice. The logarithmic transformation of in vivo toxic doses and the corresponding in vitro cytotoxic concentrations showed a statistically significant correlation between the in vitro and in vivo values. The results show that an accurate in vivo LDS p dose could be predicted from in vitro data for at least 75% of the selected compounds. It is hoped that this finding will not only stimulate others to pursue in vitro technique but will eventually lead to elimination of the in vivo LD₅₀ test.Abbreviations CT₅₀ 50% cytotoxic concentration - CT₁₀₀ 100% cytotoxic concentration - DMSO dimethyl sulfoxide - LD₅₀ 50% lethal dose - LDH Lactate dehydrogenase - MEIC Multicenter Evaluation of In Vitro Cytotoxicity     

Chick embryo chorioallantoic membrane (CAM): an alternative predictive model in acute toxicological studies for anti-cancer drugs

The chick embryo chorioallantoic membrane (CAM) is a preclinical model widely used for vascular and anti-vascular effects of therapeutic agents in vivo. In this study, we examine the suitability of CAM as a predictive model for acute toxicology studies of drugs by comparing it to conventional mouse and rat models for 10 FDA-approved anticancer drugs (paclitaxel, carmustine, camptothecin, cyclophosphamide, vincristine, cisplatin, aloin, mitomycin C, actinomycin-D, melphalan). Suitable formulations for intravenous administration were determined before the average of median lethal dose (LD₅₀) and median survival dose (SD₅₀) in the CAM were measured and calculated for these drugs. The resultant ideal LD₅₀ values were correlated to those reported in the literature using Pearson’s correlation test for both intravenous and intraperitoneal routes of injection in rodents. Our results showed moderate correlations (r²=0.42 − 0.68, P<0.005–0.05) between the ideal LD₅₀ values obtained using the CAM model with LD₅₀ values from mice and rats models for both intravenous and intraperitoneal administrations, suggesting that the chick embryo may be a suitable alternative model for acute drug toxicity screening before embarking on full toxicological investigations in rodents in development of anticancer drugs.     

Effect of UVB radiation on the survival, feeding, and egg production of the brackish-water copepod, Sinocalanus tenellus, with notes on photoreactivation

We examined the effects of UVB radiation on hatching success of eggs, survival of various naupliar and copepodite stages, and feeding and egg production of adult females of the brackish-water copepod, Sinocalanus tenellus, by exposure to varying doses of UVB irradiance in the laboratory. Artificial UVB radiation resulted in an increased mortality of eggs, nauplii and copepodites with increasing UVB doses. UVB induced damage was stage-specific with eggs being most susceptible (LD₅₀= 4.1 kJ m^–2 ) and adult females being least susceptible (LD₅₀= 16.7 kJ m^–2). Effects on feeding and egg production of adult females were significantly evident at UVB doses higher than 11.0 kJ m^–2 and 7.0 kJ m^–2, respectively. We also examined the photorepair response of eggs and various developmental stages in simultaneous irradiation of UVB and enhanced PAR. With enhanced PAR there was a considerable recovery against UVB damage, being higher for younger animals than older ones. In nature, however, solar UVB radiation may rarely cause appreciable damage to S. tenellus population due to optically high attenuation properties of their habitat waters.     

Cytophotometric analyses of thalamic neuronal RNA in soman intoxicated rats

Quantitative azure B-RNA cytophotometry was used to monitor metabolic responses of individual neurons within the ventrobasal nuclear complex (VBC) and nucleus reticularis (NR) of the rat thalamus following administration of soman (0.5, 0.9 or 1.5 LD₅₀, sc). A dose-dependent depression in brain acetylcholinesterase (AChE) was evidenced. With respect to thalamic RNA responses, a complex pattern of RNA alterations was evidenced, with these two regions generally exhibiting opposite patterns of dose-related RNA changes. With sub-lethal dosages of soman, RNA accumulation was evidenced in the acetylcholine (ACh) mediated excitatory VBC region and RNA depletion in the ACh mediated inhibitory NR neurons. With a lethal dose, an opposite RNA response pattern observed in both thalamic regions. It is postulated that the observed RNA response pattern with sub-lethal dosages of soman is what one would anticipate with cholinergic brainstem reticular formation activation. The absence of such a response with lethal doses strongly suggests some disruption of functional excitatory cholinergic activity and perhaps also an impairment of inhibitory cholinergic synaptic activity.     

Informative value of a mouse model ofKlebsiella pneumoniae infection used as a host-resistance assay

To obtain a host-resistance assay (HRA) for quantitative evaluation of immunostimulatory effects of various substances, an experimental model ofK. pneumoniae inhalatory infection was elaborated. The highly virulent bacterial strain (inhalation LD₅₀=400 CFU), appliedvia the natural route into the respiratory tract elicits an acute infectious process possessing characteristic dynamics. Although the intensity of clearance in the bronchoalveolar lavage after challenge or the mean survival time can be used in individual cases for quantitative resistance determination, the inhalation LD₅₀ values yielded the most standard results. Systemic immunization with the corpuscularK. pneumoniae vaccine provided a high protection expressed by increasing the inhalation LD₅₀ by two orders of magnitude. The antibodies formed, detectable by the ELISA test, are specific for capsular polysaccharide. The type-specific immunity was also found in the protection test. The nonspecific stimulatory effect of the peptidopolysaccharide complex isolated fromListeria monocytogenes (EiF) was manifested at the level of one LD₅₀ only while with higher infectious doses it was absent. However, the adjuvant activity of EiF was significant. The HRA can distinguish and quantitatively determine both nonspecific and specific stimulatory effects of immunomodulatory substances. Translated by I. Miler     

In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

Anticonvulsant Activity and Toxicity of Essential Oil and Methanolic Extract of Zhumeria majdae Rech, a Unique Iranian Plant in Mice

Drug-resistance and adverse effects of current drugs are the most obstacles in the treatment of epilepsy. In a plan for finding new natural anticonvulsant agents, we studied the anticonvulsant effects of essential oil (ZMEO) and methanolic extract (ZMME) of Zhumeria majdae in pentylene tetrazol (PTZ) and maximal electro-shock (MES) models in mice. Mice received different doses of ZMEO and ZMME, 30?min before induction of chemical and electrical convulsions. Neurotoxicity (movement toxicity and sedation) was evaluated using rota-rod test. The mortality was determined after 24?h following injection of different doses of the ZMEO and ZMME. The obtained results show that ZMEO dose-dependently protected mice from tonic convulsions induced by PTZ and MES with effective doses (ED₅₀) of 0.26 (0.13?C0.39) and 0.27 (0.17?C0.37)?ml/kg respectively. Toxic doses (TD₅₀) in rota-rod test for ZMEO was 0.55 (0.42?C0.70)?ml/kg. ZMME at dose of 2?g/kg decreased tonic convulsions as much as 40?%. For ZMEO, TD₅₀ of 0.55 (0.45?C0.69)?ml/kg was obtained. ZMME significantly decreased the walking time in rota-rod test at dose of 2?g/kg. Lethal dose (LD₅₀) of ZMEO was determined as 2.35 (1.98?C2.65)?ml/kg. ZMME showed about 34?% death of the animals at dose 5?g/kg. The essential oil of Z. majdae could be a good candidate for further anticonvulsive studies.     

Histopathological and biological studies of the effect of cadmium on Rhinella arenarum gonads

This study was to determine the lethal dose 50 (LD₅₀) of CdCl₂ in adult Rhinella arenarum and analyzed the effect of two sublethal doses (0.5 and 5 mg/kg) of the xenobiotic in gonads. The 48 h LD₅₀ were 50.0 and 49.8 mg/kg for males and females respectively. Alterations in the ovary were evidenced by nuclear pleomorphism and cytoplasmic vacuolization of the oocytes at the early stages of development with the highest dose and an increase in the population of atretic oocytes. In the interstitial tissue we noticed congestion, edema and fibroblast proliferation. The nuclear maturation of the oocytes was affected by the xenobiotic in a dose- and time-dependent manner. In males, treatment with 5 mg/kg of cadmium (Cd) caused a decrease in the concentration, viability and straight progressive motility of sperm while there was an increase in immotile sperm. Testis histopathology revealed dilated seminiferous tubules, disappearance of cysts, tissue disorganization and leukocyte infiltration. Numerous germ cells showed hydropic tumefaction or signs of focal necrosis. The Cd content in animals intoxicated gonads with the highest sublethal dose was significantly higher than in the control. Results indicate that R. arenarum gonads are target for the xenobiotic, compromising the formation of gametes competent for fertilization, the effective CdCl₂ dose being 5 mg/kg.