Determination Of LD50 Research Articles

Retrospective study on the reliability of an “approximate LD50” determined with a small number of animals

Based on 364 LD50 determinations in mice and rats after intravenous and oral administration of drugs, the reliability of an “approximate LD50” was retrospectively tested. The difference between “approximate LD50” and LD50 is — independent of species and route of administration — not greater than ± 20% of the LD50 in 90% of the cases. Four to five doses — uniformly distributed over the dose-mortality range can suffice in reliably determining the “approximate LD50”. The probability is 10% that an “approximate LD50” and LD50 are significantly different from each other. 152 parallel studies on male and female animals show that the LD50 or “approximate LD50” must not be determined for both sexes. It is sufficient to test a dose near the LD50 in the opposite sex. A 50–75% reduction of expenditure in animal material is possible in most of LD50 determinations.

Toxicity of Gonyaulax tamarensis var. excavata Cells to the Brine Shrimp Artemia salina L.

Brine shrimp {Artemia salina L.) were exposed to concentrations of live Gonyaulax tamarensis var. excavata cells ranging from 0 to 13,400 cells/ml in unialgal culture. The shrimp ingested the dino-flagellate cells and an LD50 determination was made. The shrimp had a 72-hr LD50 of 1258 G. tamarensis cells/ml, which was calculated to correspond to 73,385 μg/kg of body weight. This figure can be compared with an oral LD50 value of 2100 μg/kg for the mouse. Since it requires one-tenth as much toxin to kill a mouse (LD50 approximately 0.04 mg) as to kill an Artemia (LD50 approximately 0.4 mg), the whole cell G. tamarensis bioassay is a poor alternative to the current mouse assay.

The biophysical approach to excitable systems

In comparison with adult rats, the cardiac glycosides (digitoxin and proscillaridin) have been shown by LD50 determinations to be 200–1000 times more toxic to 7-day-old rats. This was not due to major differences in the rate of intestinal absorption. Reversal of toxicity with respect to sex was observed in adult rats, males being more sensitive to proscillaridin and females more sensitive to digitoxin.

Evaluation of virulence test procedures for Yersinia enterocolitica recovered from foods.

Recently a heat stable toxin (ST) and the vw factors of the plague bacteria were identified in Yersinia enterocolitica recovered from human infections. The vw factors were reported to be associated with a plasmid of 42‐46 M Daltons in size and were essential for the expression of virulence. With this knowledge virulence tests were developed which allowed us to assess the virulence potential of food‐borne Y. enterocolitica regardless of biotypes or serotypes. The tests evaluated were: (1) rapid presumptive test for the virulence plasmid; (2) gel electrophoretic confirmation of the virulence plasmid; (3) Laird's qualitative oral feeding test with thirst stressed mice; (4) quantitative LD50 determination by i.p. injection of the mouse lethal (i.e. serotype 0:8) strains in saline; (5) quantitative LD50 determination of mouse non‐lethal (i.e. serotype 0:3) strains by i.p. injection of these strains suspended in 1 ml of 10% iron dextran saline solution for virulence enhancement. These tests were evaluated with the serotypes 0:3 and 0:8 strains associated with human infections with and without the virulence plasmid with reproducible results. Then the virulence tests procedures were applied to 79 food isolates. The virulence plasmid was detected only in the Nilehn biotype 2, 3 and 4 strains, but it was absent in Nilehn biotype 1 or the atypical strains that ferment rhamnose. The virulence of food and clinical isolates of Y. enterocolitica can be assayed fairly accurately with the above tests.

Significance of the LD50-test for the toxicological evaluation of chemical substances.

The LD50-test was developed in 1927 for the biological standardization of dangerous drugs. Then it was incorporated into the routine toxicological protocol of other classes of chemical compounds and is now part of practically all governmental guidelines which regulate toxicological testing of chemicals. For scientific, economic, and ethical reasons it is necessary to periodically reassess all toxicological test procedures, including the LD50-test. Tests which are not optimal or that have become obsolete because of new scientific knowledge, must be changed or eliminated. The review of the LD50-test shows that the precision of the procedure is dependent on the number of animals used. But even with large numbers of animals there are considerable variations of the test results, because the numerical value of the LD50 is influenced by many factors, such as animal species and strain, age and sex, diet, food deprivation prior to dosing, temperature, caging, season, experimental procedures, etc. Thus, the LD50 value cannot be regarded as a biological constant. Through standardization of the test animals and the experimental conditions the variability of the LD50 determinations can be reduced but never fully eliminated. There are several tests with which an approximate LD50 can be determined. These methods use fewer animals than the classical LD50-test, but their precision and reproducibility are sufficient for most purposes of acute toxicity testing. Through incorporation of physiological, hematological, biochemical, pathological, and histopathological investigations in the simplified test procedures with small numbers of animals, it is possible to markedly increase the informational content of the results with regard to the toxicological spectrum and the target organs of toxicity. Such studies have already replaced the LD50-test in large animals, such as dogs and monkeys. It is also desirable to replace the LD50 in rodents with such a procedure. With pharmacologically inert compounds that have no acute effects with single administration the classical LD50-test does not provide relevant toxicological results. For the prediction of the human lethal dose and for the prediction of the symptomatology of poisoning after acute overdosing in man the LD50-test is of limited usefulness. An acute toxicity test with small numbers of animals combined with comprehensive studies of physiological functions, biochemical and histopathological examinations often provides more important information for emergency physicians and poison control centers. For the selection of doses to be used in subacute and chronic toxicity experiments the LD50-test does not provide consistent and reliable results. A simple pilot experiment with few animals but repeated dosing gives more useful information. For the evaluation of special risks for the human newborn and infant the LD50-test is poorly suited. For the appraisal of pharmacokinetic behavior and bioavailability the LD50-test gives only semi-quantitative, often ambiguous information. In all cases where the acute toxicity testing is mainly concerned with the evaluation of toxicological potential of the test substances, the symptomatology following acute overdosing, and the knowledge of target organs of toxicity, the classical LD50-test should be replaced by a more comprehensive short term test that can be done with small numbers of animals. The classical LD50-test should only be permitted in those rare instances where a high precision of the LD50 determination is indispensable.

Neriifolin and 2’-Acetylneriifolin: Insecticidal and Cytotoxic Agents of Thevetia thevetioides Seeds1

A bioassay procedure utilizing the European corn borer, Ostrinia nubilalis (Hubner), has been used to guide the phytochemical fractionation of active extracts of the seeds of a yellow oleander, Thevetia thevetioides (HBK.) K. Schum. (Apocynaceae). The known cardiotonic glycosides, neriifolin and 2’-acetylneriifolin, were crystallized as the active insecticidal agents, giving LD50 determinations of 30 ppm and 192 ppm, respectively, when incorporated into the corn borer diet. These compounds also exhibited cytotoxic activities of 2.2×10−2 and 3.3 × 10−2 μg/ml, respectively, in the KB (human nasopharynx epidermoid carcinoma) in vitro system. The glucoside of β-sitosterol also was isolated but it lacked insecticidal activity.

Effect of experimental dengue virus infection on humoral and cell-mediated immune response to thymus-dependent antigen.

Mice injected with dengue virus showed a depressed primary and secondary humoral immune response to SRBC as measured by the number of antibody-forming cells, haemagglutinin and haemolysin titres. The time of administration of SRBC during the infection was found to be an important factor determining the response to SRBC. The cell-mediated immune response was also suppressed as measured by the delayed hypersensitivity reaction to SRBC in the footpad. While the immunosuppression of humoral immunity was only transient and the animals recovered within 30 days following infection, the cell-mediated immunity remained suppressed throughout this period. Immunofluorescent studies and LD50 determination of the infected brains demonstrated significant virus titres from 7 to 15 days postinfection. This period coincided with maximal immunosuppression.

The acute intravenous toxicity of biological stains, dyes, and other fluorescent substances

Sodium fluorescein and indocyanine green are currently used as indicators for angiography of the retinal and choroidal vasculatures. This study was conducted to suggest additional indicator substances for this technique. Fifty-three dyes and biological stains were evaluated by the following criteria: intravenous LD50, histopathological damage from intravenous administration, binding properties in blood, in vivo metabolism of the substances, fluorescent and absorption spectra, and impurities detected with thin-layer chromatography. Male Swiss-Webster mice were used for the intravenous toxicity studies and for the metabolism and LD50 determinations. Several cyanines were modified by recrystallization with sodium iodide and an oxazine was sulfonated to improve their solubility and biocompatibility. Both techniques were successful in improving the intravenous toxicity and the solubility of the dyes in water. Iodination was not successful on the merocyanines. Patent blue VF and 8-hydroxy-1,3,6-pyrene trisulfonic acid trisodium salt were promising since both had impurities yet their LD50s exceeded 1000 mg/kg. Fluorones with two salt sites (NaO and COONa) were soluble at 10% or more in water. Nonhalogenated fluorones were excreted totally in the urine, while those with eight halogen atoms were removed via the biliary system. Those fluorones with six or less halogen atoms were excreted by both systems. Twenty-three of the fifty-three dyes were tolerated well enough for use in angiography of the ocular fundus. The majority of the substances had at least one impurity. In general, the cationic dyes were poorly tolerated; only 2 of the 23 acceptable substances were cationic. Those dyes that were anionic or dipolar were the best tolerated, with the dipolar molecules being cleared, at least partially, by the liver.

Use of synthetic analogs for a study on the structure-activity relationship of apamin.

[Lys13,Lys14]Apamin, [Lys13]apamin and [Lys14]apamin, three structural analogs of the bee venom neurotoxin, have been obtained by solid-phase peptide synthesis while an attempt to obtain [Cit13]apamin failed, probably at the step of reoxidation of cysteines. After the chemical purity of these three derivatives had been assessed, further chemical modifications led to three new peptides: [Ac-Cys1,Lys(Ac)4,Lys(Ac)13]apamin, [Ac-Cys1,Lys(Ac)4,Lys(Ac)14]apamin and [Har4,Har13,Har14]apamin. These six analogs have been tested for their neurotoxicity, i.e. determination of LD50 for mouse by subcutaneous injection. A lethal potency is observed only when the region 13-14 of the sequence contains a double positive charge. One arginyl residue is necessary for a high biological activity, while its location in position 13 or 14 is of minor importance. When homoarginine (Har) replaces arginyl residues the neurotoxicity is lowered.

Antineoplastic agents. 1. N-Protected vinyl, 1,2-dihaloethyl, and cyanomethyl esters of phenylalanine.

A series of N-protected vinyl, 1,2-dihaloethyl, and cyanomethyl esters of phenylalanine was synthesized and these compounds were evaluated for antitumor activity against the growth of Ehrlich ascites carcinoma in CF1 male mice (33 mg/kg/day), Walker 256 carcinosarcoma in Sprague-Dawley male rats (2.5 mg/kg/day), and P388 lymphocytic leukemia in DBA/2 mice (20 mg/kg/day). Structure-activity relationships were evaluated and acute toxicity studies (LD50 determinations) in male CF1 mice were also carried out on selected compounds. Carbobenzoxy-L0phenylalanine vinyl ester (5), N-carbobenzoxy-L-phenylalanine 1,2-dibromoethyl ester (12), and N-carbobenzoxy-L-phenylalanine cyanomethyl ester (8) were found to be very potent inhibitors of Ehrlich ascites tumor growth at nontoxic doses cited above. Compounds 5 and 12 also tripled survival time in the Walker 256 system. LD50 values for compounds 5, 12, and 8 were greater than 2000 mg/kg (greater than 6.15 mmol/kg), 74 mg/kg (0.15 mmol/kg), and 150 mg/kg (0.44 mmol/kg), respectively.

Influence of pH and route of injection on acute toxicity of tetracycline in mice

AbstractSome LD50 determinations for tetracycline hydrochloride in mice were carried out over a range of pH values, using both the intraperitoneal and the subcutaneous routes of injection. Depending on the pH of the formulation, either water or a solvent system of water and 60% (v/v) propylene glycol was employed thus ensuring complete solution of the drug at all pH values tested. Higher LD50 values were obtained with the subcutaneous route than with the intraperitoneal route. With either route of administration, there was a trend toward lowest LD50 value occurrence at the isoelectric pH of tetracycline. Actual statistical significance was achieved for the intraperitoneal route only when LD50 values obtained at the isoelectric pH were compared with either of the more acidic pH values.

LD50 determination and hypotensive effect of 3-methyl-2-benzothiazolone hydrazone

3-Methyl-2-benzothiazolone hydrazone (MBTH) is a compound employed in the colorimetric determination of aliphatic aldehydes. LD50 values based on ip injections of MBTH in rats and mice were found to be 135 and 119 mg/kg, respectively. The induction of a lethargic state followed by hyperactivity and excitability was also observed in these experiments. Intravenous injections of MBTH produced a brief fall in mean arterial blood pressure. The hypotensive effect was not blocked by the beta antagonist, propranolol. The amount of blockade seen in the presence of diphenhydramine was not of sufficient magnitude to indicate clearly that MBTH releases histamine to cause vasodilation and a reduction in the blood pressure. The degree of blockade seen with atropine indicates that the hypotensive effect of MBTH may be partially mediated through cholinergic receptors. These data indicate also that MBTH may possess direct smooth muscle vasodilator properties.

Immunopathology of Mouse Hepatitis Virus Type 3 Infection

Abstract Humoral and cell-mediated immune responses were studied in resistant and susceptible strains of mice infected with mouse hepatitis virus type III (MHV3). Virus was maintained by regular passages in susceptible DBA/2 mice and assayed in DBA/2 mice by LD50 determination. Normal resistant A strain mice were able to clear the virus from liver, brain, and serum within 7 days after infection. No neutralizing antibody was found. Transfer of serum from immunized A strain mice was not effective in protecting susceptible DBA/2 mice against challenge with virus. In A strain animals resistance to MHV3 developed rapidly during the 3rd week of life. During the period of susceptibility, newborns were protected neither by transplacental passages of anti-MHV3 antibodies nor by injection of “educated” thymus cells. These results suggest that the development of resistance to MHV3 infection in mice cannot be explained solely on the basis of humoral or cell-mediated immunity.

Virulence measurements of Pseudomonas aeruginosa based on plaque and toxin Z formation in cell culture.

SummaryAn in vitro system was devised to measure the virulence of Pseudomonas aeruginosa based on the organism's ability to form virus-like plaques and toxin Z in cell culture monolayers.Plaquing efficiency varied among clinical strains and also between the original isolates and their substrains after 10 and 20 subcultures. These variations were significant at the 99.9% confidence level.The data on the potency of toxin Z produced by these strains as measured by cytopathogenic effect in cell culture and by mouse lethality generally agreed with the data on their plaquing efficiency. Conversely no differences between the tested strains were detected in LD50 determinations in mice. This latter result is in agreement with the findings of other investigators and reemphasizes the need for a more reliable indicator system to assess the virulence of P. aeruginosa. Plaquing efficiency and toxin Z formation in cell culture appear to hold considerable promise as indicators that may satisfy this need.

Interactions of chlorpheniramine ethanol combinations: Acute toxicity and antihistaminic activity

Significant toxicologic and pharmacologic interactions occurred between chlorpheniramine maleate and 25% v v ethanol at several dosage combinations. Conditions of both independence and antagonism of acute toxicity were observed in LD50 determinations of chlorpheniramine-ethanol combinations in mice, and confirmed expected results based on the toxicologic pattern of the agents when administered singly. Enhancement of the antihistaminic action of chlorpheniramine by ethanol was demonstrated during histamine-aerosol challenge studies in guinea pigs.

AGE DEPENDENCE OF SENSITIVITY TO MHV3 INFECTION IN RESISTANT MICE

Mechanisms of newborns susceptibility to viral infection was studied in A strain mice infected with mouse hepatitis virus type 3(MHV3). Virus was regularly passaged in susceptible adult DBA/2 mice, prepared by liver homogeneization and titered by LD50 determination. Normal adult A strain mice were always resistant to virus. Young isogenic mice however were highly susceptible and 100% of them died when infected during the first two weeks of age. Resistance to disease developed suddenly during the 3d week of life. Passive transfer of serum from immune A mice failed to protect young animals. When newborns from immunized A strain mothers were injected with MHV3 the mortality was similar to that of control groups. In addition A strain newborns were not protected against virus infection either by injection of isogenic spleen cells or by means of educated thymus cells. However when A strain mice were thymectomized at birth they failed to resist MHV3 infection when tested at adult age. Therefore, along with cell mediated immunity it appears that other mechanism(s), e.g. viral replication in susceptible target cells may play an important role in resistance of mice to MHV3.(Supported by INSERM ATP 5).

Toxicological Studies of 2-Mercaptoethanol

The toxicity of 2-mercaptoethanol, a potential reaction product between residual ethylene oxide and sulfides in rubber medical devices sterilized by ethylene oxide, was investigated. This report includes LD50 determinations for mice by both the intraperitoneal and oral routes of administration, as well as sub-chronic dosage studies. Both ethanol and sodium pentobarbital significantly decreased the acute toxicity potential of 2-mercapto-ethanol, although pretreatment with atropine and with a combination of metal ions had no beneficial effects. 2-Mercaptoethanol was found to be more toxic to all tissues than ethanol but showed a marked decrease in such activity upon dilution.

Determination of LD50 of barium chloride and allied agents

The present study reports in detail the LD50 determination in white mice of ICR strain from iv administration of barium in the form of chloride. The LD50 value was found to be 19.2 mg Ba2+/kg. The results for nitrate and acetate were close to chloride, but in Swiss-Webster strains these values were lower by a factor of almost 2. Further, LD50 values for Mg, Ca and Sr (using chlorides in ICR-mice) were 14.4, 42.4 and 147.6 mg/kg, respectively.

The comparative acute toxicity of two cardiac glycosides in adult and newborn rats

In comparison with adult rats, the cardiac glycosides (digitoxin and proscillaridin) have been shown by LD50 determinations to be 200–1000 times more toxic to 7-day-old rats. This was not due to major differences in the rate of intestinal absorption. Reversal of toxicity with respect to sex was observed in adult rats, males being more sensitive to proscillaridin and females more sensitive to digitoxin.

Laboratory evaluation of malathion for controlling the desert locust, Schistocerca gregaria (Forsk).

Experiments were carried out to remove existing doubts about the toxicity (and therefore cost per unit killing power) of malathion to adults of Schistocerca gregaria (Forsk.) which have arisen from earlier field and laboratory tests. Malathion is shown to be almost as toxic to these insects as γ-BHC, but superior in speed of action. These observations are in agreement with the performance of these two compounds in the field.Subdivision of a dose of malathion with simultaneous application at two loci increased its toxicity. This, as well as other factors, brings the value of LD50 determinations into question. Recommendations for large-scale use of new insectsicides, based only on laboratory data, may not always be reliable.