Determination Of LD50 Research Articles

Relative toxicity of native Chilean Bacillus thuringiensis strains against Scrobipalpuloides absoluta (Lepidoptera: Gelechiidae)

L. MEZA-BASSO. 1997. The larva of Scrobipalpuloides absoluta, a South American moth, is the most devastating insect pest of tomato production in Chile. The potential for using bacterial insecticides was studied analysing the relative toxicity of native Bacillus thuringiensis (BT) isolates belonging to the Chilean collection. The polymerase chain reaction (PCR) technique was used in order to facilitate the prescreening. Mixtures of homologous specific primers to regions within genes encoding CryI, CryIII and CryIV crystal proteins were employed to generate a PCR product profile of each BT isolate. Four isolates were selected and further characterized by means of SDSPAGE, Western blot and bioassays on fourth-instar S. absoluta larvae. Relative toxicities were evaluated by LD50 determinations. The entomocidal activity of isolate 121e, an autoagglutinating strain, was threefold higher than toxin synthesized by B. thuringiensis var. kurstaki. This native strain was also active against Culex pipiens larvae, although much less than towards S. absoluta.

Effect of sample size and number of doses for the determination of LD50

AbstractThe effects of the LD50 value of the number of applied doses and the number of individuals submitted to each dose were studied. Simulated data were used and the lognormal distribution was assumed. Increasing the number of individuals submitted to the doses results in a much larger effect than increasing the number of doses. To obtain an accuracy of 50% of the true value, with a chance of .85 that this should happen, at least 20 individuals per dose should be used.

Computer programs for calculation of median effective dose (LD50 or ED50) using the method of moving average interpolation.

Over the past 40 years, toxicologists and pharmacologists have used tables published by Weil for the determination of LD50 (or ED50) values and their associated 95% confidence intervals. With the advances in computer technology, it is now common for investigators to have personal computers in their laboratories. Therefore, two identical programs were developed for determination of the LD50 (or ED50) which may be run on a personal computer. One of these programs was written in BASIC, and the other in FORTRAN. The programs are easy and rapid to use, requiring minimum computer hardware and little, if any, knowledge of programming. They also offer more user flexibility than the previously published tables of Weil, in that there are fewer restrictions on the number of animals and number of dosage levels used in an experiment. The output of the programs may be typed on the screen of a computer monitor, and may be sent to a printer. The two programs calculate the LD50 and 95% confidence intervals for the LD50. These programs should be valuable for many investigators.

Determination of the LD50: choice of the underlying model

AbstractDetermination of the LD50 assumes a (log)normal distribution for the tolerance concentrations of a population. Two alternative distributions, the exponential and the Pareto, are considered as the true underlying models. The effect on the LD50 value is examined when one of these distributions is the true underlying model although the classic assumption (normality) is made. It is shown that important discrepancies between the LD50 values can occur, using a not adequate model. Graphing the data and including the tails of the distribution can be helpful to determine the model to be used.ZusammenfassungZur Bestimmung der LD50: Wahl des zugrundeliegenden ModellsDie Bestimmung der LD50 impliziert eine (log) normale Verteilung der Konzentrationstoleranz einer Population. Zwei alternative Verteilungen, die exponentielle und die Pareto, werden als die zugrundeliegenden Modelle in Betracht gezogen. Der Einfluß auf den LD50‐Wert wurde untersucht, wenn eine dieser Verteilungen das vorliegende Modell ist, ungeachtet der klassischen Voraussetzung (Normalität). Es wurde gezeigt, daß wichtige Diskrepanzen zwischen den LD50‐Werten vorkommen können, wenn kein geeignetes Modell verwendet wird. Die graphische Darstellung der Daten und die Berücksichtigung der Verteilungsspitzen kann die Auswahl des zu verwendenden Modells erheblich unterstützen.

Antigen-specific deletion of cloned T cells using peptide-toxin conjugate complexed with purified class II major histocompatibility complex antigen.

In a previous report, we showed that cloned T cells incubated with soluble, cognate major histocompatibility complex (MHC) II-peptide complex internalized the peptide moiety of the complex. Here, we report antigen-specific deletion of cloned T cells by treatment with soluble, cognate MHC II-(peptide-toxin) complexes. Toxin (doxorubicin or mycophenolic acid) was attached to synthetic AcMBP(1-14)Ala4 peptide, an analog of the natural acetylated NH2-terminal segment, AcMBP(1-14), of rat myelin basic protein (MBP). IAk-restricted, AcMBP(1-14)-Specific AJ1.2 and 4R3.9 cloned murine T cells were killed by IAk-(AcMBP(1-14)Ala4-toxin). No killing resulted from incubating AJ1.2 and 4R3.9 cells with irrelevant MHC II-(peptide-toxin) or treating IEk-restricted, pigeon cytochrome c-specific A.E7 cloned murine T cells with IAk-(AcMBP(1-14)Ala4-toxin). T cell receptor-mediated T cell uptake of the peptide-toxin moiety of relevant complex was blocked by anti-T cell receptor-alpha/beta antibody and by excess toxin-free complex. LD50 determinations revealed that cognate MHC II-(peptide-toxin) killed T cells much more effectively than did peptide-toxin conjugate alone. Finally, T cell uptake of peptide-toxin and intracellular release of toxin occurred after incubation with relevant MHC II-(peptide-toxin) containing radiolabeled toxin. These findings, which provide the first evidence that cloned T cells can be deleted with soluble, cognate MHC II-(peptide-toxin) complexes, may have significant clinical relevance for antigen-specific therapy of autoimmune or other T cell-mediated diseases.

The effect of an electrical current on snake venom toxicity

An electric current (twenty 11 A, 7000 V spikes s−1 for 90 s) from a commercial stun gun was applied directly to a rattlesnake venom solution in an electrolysis cell with 2 electrode compartments and a central compartment, in order to evaluate the effect that high voltage electroshock might have on the lethality of the venom. The venom was electroshocked for 18 times longer than recommended by stun gun manufacturers. There was no measurable inactivation of the venom using LD50 determinations in mice. A venom sample was electrolyzed at a voltage lower than that from the stun gun, but with 4–5 times the total charge delivered from the stun gun. This inactivated the venom at the electrodes, but not within the central compartment, demonstrating that there was no direct effect of the electric field on the activity of the snake venom.

Comparison of lethalities in mouse versus goldfish for clinical and food isolates ofAeromonas hydrophila

The lethality of 16 clinical or food isolates ofAeromonas hydrophila was assessed by determination of LD50 (i.p.) in mice and goldfish. In mice LD50 values for the variousA. hydrophila strains were similar, ranging from 1.2–21.0×108 cells/animal. A wider range of LD50 values, 0.03–11.8×108 cells/animal, was observed with goldfish. Lethality was not correlated between the two test animals. Further, cytotoxic response in Y-1 adrenal cells did not correlate with lethality in either test animal. It appears that lethality is not a good measure of potential enterotoxigenicity, but may be useful in assessing the invasive character of isolates causing systemic infections in immunocompromised hosts.

LD50 determination: Objections to the method of Beccari as modified by Molinengo

C. Sevcik. LD 50 determination: objections to the method of Beccari as modified by Molinengo. Toxicon 25, 779 – 783, 1987.—The effiçiency of the method of Beccari as modified by Molinengo is compared with the sequential technique of Dixon and Mood. It is concluded that the sequential technique is less constrained, gives estimates that are less disperse and is simpler to analyze statistically than the regression of Beccari - Molinengo. Both techniques are comparable in the number of animals required, as well as in the time invested in each assay.

Acute toxicity of pesticides in adult and weanling rats

LD50 values were determined for 57 pesticides administered by the oral or dermal route to adult male and female Sherman rats. Thirty-six of the chemicals were also tested by the oral route in one sex of weanlings. Nine pesticides tested by the oral route (bufencarb, cacodylic acid, dialifor, deltamethrin, dicamba, diquat, quintozene, phoxim, pyrazon) and four tested by the dermal route (bufencarb, chlordimeform, dichlofenthion, leptophos) were more toxic to females than to males whereas famphur and 2,4,5-T (oral route) were less toxic to females. Eighteen of the test chemicals were more toxic to the adult than to the weanling and four compounds (leptophos, methidathion, pyrazon, and sulfoxide) were more toxic to the weanling. In additional studies the variability of the LD50 value over a 1-year period was examined for two typical insecticides. Six consecutive bimonthly oral LD50 determinations for parathion and DDT in adults of both sexes indicated that the LD50 values were little affected by the time of year that the tests were done.

Acute Toxicity of Pesticides in Adult and Weanling Rats

Acute Toxicity of Pesticides in Adult and Weanling Rats. GAINES, T. B., AND LINDER, R. E. (1986). Fundam. Appl. Toxicol. 7, 299-308. LD50 values were determined for 57 pesticides administered by the oral or dermal route to adult male and female Sherman rats. Thirty-six of the chemicals were also tested by the oral route in one sex of weanlings. Nine pesticides tested by the oral route (bufencarb, cacodylic acid, dialifor, deltamethrin, dicamba, diquat, quintozene, phoxim, pyrazon) and four tested by the dermal route (bufencarb, chlordimeform, dichlofen-thion, leptophos) were more toxic to females than to males whereas famphur and 2,4,5-T (oral route) were less toxic to females. Eighteen of the test chemicals were more toxic to the adult than to the weanling and four compounds (leptophos, methidathion, pyrazon, and sulfoxide) were more toxic to the weanling. In additional studies the variability of the LD50 value over a 1-year period was examined for two typical insecticides. Six consecutive bimonthly oral LD50 determinations for parathion and DDT in adults of both sexes indicated that the LD50 values were little affected by the time of year that the tests were done.

Natural Resistance to Salmonellae in Mice: Control by Genes Within the Major Histocompatibility Complex

Determinations of 50% lethal dose (LD50) values in H-2 congenic B10 lines showed that late-emerging resistance (postimmune response phase) to salmonellae of intermediate virulence was less in H-2b and H-2d than in H-2a, H-2k, and H-2f mice. Association of resistance to H-2 was confirmed by backcross analysis, and LD50 determinations on H-2 recombinant haplotype strains showed that resistance maps to the I-E subregion. Bacterial growth curves in liver and spleen showed that susceptible mice carried bacteria for longer in the reticuloendothelial system than did resistant mice and that susceptible mice showed greater splenomegaly. Association of resistance and susceptibility to H-2 was not different when sister transductant salmonellae expressing somatic antigens O4 and O9 were used. Thus a gene(s) within the major histocompatibility complex controls natural resistance to salmonellae in mice by influencing the ability to clear bacteria from the reticuloendothelial system in the later phase of the infection, and the immunodominant O antigen cannot be solely involved.

Relationship between agglutinative properties of Aeromonas salmonicida strains isolated from fish in Japan and their resistance to mechanisms of host defense.

Phenotypic variation in spontaneous agglutination of Aeromonas salmonicida was investigated using 114 strains isolated from naturally epizootics of furunculosis in salmonid fish in Japan from 1967 through 1982. These strains were categorized into three different types: an agglutinating (AG) type which produced cell flocks resulting in precipitation and loss of turbidity in cell suspensions; a nonagglutinating (NAG) type, representing no loss in turbidity; and an intermediate (IM), weakly autoagglutinating type. Based on the occurrence of the AG, NAG and IM types determined in 1978 and again 1982, it was shown that a transition occurs progressing from the AG to the NAG via the IM type. The strains involved in the AG and NAG types were virulent and avirulent, respectively, based on LD50 determination in rainbow trout (Salmo gairdneri). The NAG type was much less susceptible than the AG to the bactericidal activity of fresh normal rainbow trout serum, to immune bacteriolysis and to phagocytosis by cells from pertioneal exudate. When growth properties of AG and NAG types were compared under mixed culture conditions, the NAG type predominated in the culture broth 2-5 days after inoculation. In contrast, when rainbow trout were inoculated intraperitoneally with cells of both types, the NAG-type cells were selectively eliminated from the kidneys within a period of a few days. The AG-type cells, however, greatly proliferated in the kidneys and brought about clinical furunculosis. These results indicate that the AG type possesses a capacity to escape the defense mechanisms of the host by resisting the bactericidal activity of serum and phagocytosis by leucocytes.

Toxicology of 1,1,2-trichloroethane in the mouse.

1,1,2-Trichloroethane (TCE) was administered to male and female CD-1 mice to evaluate its effect on standard toxicological parameters. Following determination of the acute LD50 (378 mg/kg in males and 491 mg/kg in females), and a 14-day range-finding study, a 90-day drinking water study was performed in which the doses consumed were 4.4, 46, and 305 mg/kg for males and 3.9, 44, and 384 mg/kg for females. The liver was a target of TCE toxicity in both sexes as demonstrated by dose-dependent alterations in hepatic microsomal enzyme activities and serum enzyme levels. The erythroid element of the female mice was also affected, as indicated by significantly decreased hematocrit and hemoglobin levels.

Toxicology of trans-1,2-dichloroethylene in the mouse.

Trans-1,2-dichloroethylene (DCE) was administered to male and female CD-1 mice in order to evaluate its effects on standard toxicological parameters. Following an acute LD50 determination (2122 mg/kg in males and 2391 mg/kg in females) and a 14-day range-finding study, a 90-day drinking water study was performed using levels of DCE calculated to deliver approximately 1/100, 1/10, and 1/5 the LD50. Various toxicological assessments were made, including body and organ weights, hematology, serum chemistries, and hepatic microsomal activities. Few alterations were observed in either sex following 90 days of exposure. The most noteworthy changes occurred in the males exposed to the highest level of DCE, where there was a significant decrease in glutathione levels, and in the females exposed to all three DCE levels, where there was a significant decrease in aniline hydroxylase activity. These data served as background for the immunotoxicological evaluation presented in the following manuscript.

Role of haemolysin and temperature in the pathogenesis of Listeria monocytogenes in fertile hens' eggs.

The haemolysin produced by Listeria monocytogenes at 37 degrees C and 4 degrees C was examined in fertile hens' eggs. Organisms, cell free extracts and purified haemolysin derived from broth cultures grown at the lower temperature were more pathogenic for chick embryos, induced higher mortality with toxic changes in the embryos. These effects were most pronounced with the purified haemolysin as shown by LD50 determinations and following inoculation of constant haemolytic doses. Pathological changes induced by the haemolysin included sub-cutaneous haemorrhage due to endothelial damage, hepatosplenomegaly with macroscopic and histological lesions in heart, spleen and liver in the absence of an inflammatory response. At the cellular level, the myocardial tissue, and hepatocyte structure were destroyed with intravascular haemolysis, fatty degeneration of mitochondria, dilation of endoplasmic reticulum and distortion of liver cell nuclear membranes evident. The mortality and morphological data showed an increase in virulence for Listeria after culture at 4 degrees C compared with 37 degrees C and suggested a more cytotoxic component of the haemolysin which was activated at lower temperatures.

Methylene blue: effects and disposition in sheep

The disposition and urinary excretion pharmacokinetics of methylene blue were determined after its intravenous administration at 15 mg/kg to mature female sheep. Comparisons were made between methylene blue administered alone or subsequent to 50 mg/kg sodium nitrite. The overall elimination rate constant (beta) of methylene blue, 0.0076 +/- 0.0016 min-1, was not influenced by prior administration of sodium nitrite. However, the distribution rate was significantly altered by sodium nitrite. Very little of the methylene blue was eliminated in the urine either intact or as leucomethylene blue in spite of its relatively short half life. Toxicologic assessment was carried out using LD50 determination, methemoglobin production and hematologic effects as evaluation parameters. Methemoglobin production was minimal with doses as high as 50 mg/kg and no significant hematologic changes were seen up to 4 weeks after a total dose of 30 mg/kg methylene blue. The 24 h LD50 for intravenous methylene blue administered as a 3% solution was 42.3 mg/kg with 95% confidence interval limits of 37.3 to 47.9 mg/kg. From these data it appears that as conditions may warrant, the dosage of methylene blue may be safely increased up to at least 15 mg/kg in therapy of severe methemoglobinemias.

Virulence of beta-hemolytic and non-hemolytic Streptococcus mutans: lethal dose determinations in neonatal mice.

The virulence of beta-hemolytic and non-hemolytic strains of Streptococcus mutans was studied in neonatal mice by LD50 determinations after intracerebral injection of the bacteria. Although the differences in LD50 values are small the results may indicate that beta-hemolytic S. mutans strains (mean LD50 of 6.3 X 10(7) c.f.u.) were more virulent than non-hemolytic S. mutans strains (mean LD50 of 47.7 X 10(7) c.f.u.). The LD50 values of other viridans streptococci varied between 4.8 and 10(7) c.f.u. Strains of S. pyogenes and S. agalactiae were highly virulent in this animal model with LD50 values lower than 10(5) c.f.u.

Virulence comparée des cinq groupes génomiques de Listeria monocytogenes (sensu lato)

Virulence of the five genomic groups of Listeria monocytogenes (sensu lato) was tested toward mice by determination of LD 50 and growth or survival kinetics in vivo . Virulent strains included L. monocytogenes (sensu lato) (genomic group 1) and « L. bulgarica (genomic group 2). The three other genomic groups, «L. innocua, «L. welshimeri and « L. seeligeri , were apathogenic and avirulent under these experimental conditions. La virulence des cinq groupes génomiques de Listeria monocytogenes (sensu lato) étudiée chez la souris par détermination de la DL 50 et par la courbe de croissance ou de survie in vivo . Les souches pathogènes et virulentes appartiennent aux groupes génomiques 1 ( L. monocytogenes ) et 2 (« L. bulgarica ). Les trois autres groupes génomiques (« L. innocua, «L. welshimeri et « L. seeligeri ) sont composés de souches apathogènes et avirulentes dans les conditions expérimentales utilisées.

Methodological aspects of acute toxicity testing particularly LD50 determinations present use in development of new drugs.

It is essential to have an adequate understanding of the acute toxic effects of a new drug. It may not be necessary however to blindly follow the full classical median lethal assay on every occasion. The LD50 was developed to obtain the maximum accuracy from a modest experimental size. The objective of the study and the limitations of the assay should not be overlooked however and undue precision should not be sought. Methodological aspects including species and maximum and minimum doses will be discussed. Other methods and approaches to acute toxicity will be recommended with a more broad approach to acute toxicity than lethality alone.

Economical LD50 and slope determinations.

Many regulatory standards and guidelines required LD50 determinations and dose-response curve slopes, using at least 5 animals per sex per dosage level and at least 3 levels. In contrast, the current trend is to use as few animals as necessary for toxicological studies. The moving average method for calculation of the LD50 has been available since 1947 but, as it does not involve curve fitting, has not been used to determine the slope. An accurate adaptation of this is presented so that both the LD50 and slope can be determined using very few animals and using levels with 0 or 100% kills; not usable with probit determinations.