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PubMed1983

Complete programme for acute toxicity testing - not only LD50 determination.

The objective of any experimenter should be to obtain the maximum amount of useful information in the most economical manner possible. In this context, 'useful' means study both of the principal or anticipated actions of the compound under examination, and also detection of additional responses to it. For efficient evaluation, including greater statistical rigour, a continuous or graded response and quantitative measurement is preferable, although much can be done by simply analysis of the presence or absence of responses, even if these are complex or dependent upon subjective criteria. Consistency of experimental conditions, and of diagnostic criteria are essential, whether in single, exploratory or comparative tests. Acute toxicity tests, like other experiments, should be designed pragmatically, therefore, confirming to the properties of the substance under examination, but also with general or holistic coverage, albeit less specific, to detect unanticipated actions. Greater or lesser weight may be given to these two classes of method, depending on whether the test is done as part of a general screen of a previously unknown substance, or as a specific control measure done to assay one particular property. Extrapolation of the results to man or other species is only possible with the caution proper to any experiment done under restricted and highly artificial conditions, but the value of acute toxicity data has often been shown by experience gained in man.

Animals↗
PubMed1965

Laboratory toxicity test of field trial typhoid vaccines.

It is important for the control of vaccines to develop methods for testing their toxicity in the laboratory. Two typhoid vaccines that had given different rates of reactions among vaccinated persons in extensive field trials provided a unique opportunity for exploring the validity of the laboratory assessment of their toxicity. An elaborate design of a toxicity test based on weight changes in intraperitoneally vaccinated mice is described in this paper. The results of the laboratory test reflected the experience gained in field studies.

Animals↗
PubMed2001

Statistical comparisons of the no-observed-effect concentration and the effective concentration at 10% inhibition (EC10) in algal toxicity tests.

Batch-type algal toxicity tests were conducted to evaluate the low toxic effects of metal toxicants. Both the no-observed-effect concentration (NOEC) and the effective concentration at 10% inhibition (EC10) were calculated and compared. The results of this study indicate that, for algal toxicity tests, NOEC offers better protection to the test organisms than EC10. In addition, the consistency of NOEC is found to depend on the response endpoints measured. A cut-off-value approach is proposed to determine whether NOEC or EC10 should be chosen for estimating low toxic effects. For a specific toxicity test, the average cut-off value directly indicates the magnitude of the square root of the within-group-variance. A precise test associated with small within-group-variances also produces relatively small average cut-off value (say, less than 10% of the growth reduction). This average value provides useful information on the minimum protection that can be offered by the NOEC.

Dose-Response Relationship, Drug↗
PubMed2002

A field validation of two sediment-amphipod toxicity tests.

A field validation study of two sediment-amphipod toxicity tests was conducted using sediment samples collected subtidally in the vicinity of a polycyclic aromatic hydrocarbon (PAH)-contaminated Superfund site in Elliott Bay (WA, USA). Sediment samples were collected at 30 stations with a 0.1 m2 grab from which subsamples were taken for sediment toxicity testing and geochemical and macrofaunal analyses. Standard 10-d sediment-amphipod toxicity tests were conducted with Rhepoxynius abronius and Leptocheiros plumulosus. Sediments were analyzed for 33 PAHs, pentachlorophenol, polychlorinated biphenyls, acid-volatile sulfide, simultaneously extracted metals (Cd, Cu, Zn, Pb, Ni), total organic carbon, and grain size. Sediment temperature, oxygen-reduction potential, water depth, and interstitial water salinity were also measured. Polycyclic aromatic hydrocarbons, quantified as total PAH toxic units (TU(PAH)), were confirmed to be an important common causal agent of the changes in the two toxicity test (% survival R. abronius, % survival L. plumulosus) and five macrofaunal community (number of species, S; numerical abundance, A: total biomass, B: Swartz's dominance index, SDI; Brillouin's index, H) endpoints. Two other macrofaunal community metrics (the complement of Simpson's index, 1 - SI, and McIntosh's index, MI) were less sensitive to TU(PAH) than the two toxicity test endpoints. The sensitivities of R. abronius and L. plumulosus to TU(PAH) were statistically indistinguishable. Field validations were conducted by testing the association between or among each toxicity test endpoint, each of seven macrofaunal community metrics (S, A, B, SDI, H, 1 - SI, MI), and TU(PAH) by (1) Spearman's coefficient of rank correlation, (2) Kendall's coefficient of concordance, (3) G tests of independence, and (4) regression analysis. Some field validations based on multivariable tests of association (e.g., points 2 and 3) among toxicity test, field, and stressor endpoints produced false positive results. Both toxicity test endpoints were validated as indicators of changes in S, A, SDI, and H by all the methods tested. The resolution power of the relationships between the laboratory toxicity test and macrofaunal field endpoints was low (< or = three classes) but sufficient to discriminate ecologically important effects. We conclude that standard sediment-amphipod toxicity tests are ecologically relevant and that, under the proper conditions, their results can be used for lab-to-field extrapolation.

Amphipoda↗
PubMed2003

Stable isotopes and fatty acids reveal that Chironomus riparius feeds selectively on added food in standardized toxicity tests.

During long-term standardized toxicity tests with Chironomus riparius, food additions are a prerequisite for normal development and to avoid false-positive results. Consequently, larvae may selectively feed on added food rather than on contaminated sediment, which may confound toxicity test results. We designed a feeding study and estimated the degree of feeding on different food resources by using stable isotope and fatty acid (FA) analyses. In one treatment, larvae were offered both artificial sediment (peat, kaolin clay, sand, and calcium carbonate) and added food (TetraPhyll), whereas larvae in the two other treatments had access to either one of these potential food items. The highest biomass and survival were found among larvae with access to both artificial sediment and TetraPhyll. Two-source mixing models revealed that larval Chironomus that were offered both TetraPhyll and artificial sediment obtained 94 +/- 6.9% of their carbon and 90 +/- 4.3% of their nitrogen from added TetraPhyll. Larvae with access to only sediment had lower delta13C and delta15N (-23.34 +/- 0.56 per thousand and 0.33 +/- 0.52 per thousand) than those that were offered both sediment and TetraPhyll (-20.95 +/- 0.13 per thousand and 7.45 +/- 0.36 per thousand) or only TetraPhyll (-20.17 +/- 0.20 per thousand and 7.82 +/- 0.15 per thousand). In addition, FA composition of larvae that were offered both artificial sediment and TetraPhyll closely resembled that of those fed exclusively TetraPhyll. These results show that larval Chironumus strongly prefer added food, rather than artificial sediment in long-term toxicity tests. This preferential feeding behavior affects exposure pathways and ultimately toxicity test results.

Animal Feed↗
PubMed2002

Silver speciation during chronic toxicity tests with the mysid, Americamysis bahia.

A 28-day chronic toxicity test and two 7-day chronic estimation toxicity tests were conducted with silver nitrate (AgNO(3)) and the marine invertebrate, Americamysis bahia, in 20 per thousand (parts-per thousand) salinity seawater. One 7-day test was initiated with 7-day-old mysids and the second was initiated with <24-h-old mysids. There was very good agreement between the three toxicity tests. The no-observed-effect concentration (NOEC) values from the 28-day test, the 7-day test initiated with 7-day-old mysids, and the 7-day test initiated with <24-h-old mysids were 34, 65 and 38 microg/l silver, respectively. The 96-h LC50 values from the 28-day toxicity test and the 7-day toxicity test initiated with 7-day old mysids were 260 microg/l, and the 96-h LC50 value from the 7-day toxicity test initiated with <24-h-old mysids was 280 microg/l. Free ionic silver, Ag(+), concentrations measured with a silver electrode were in good agreement with concentrations calculated using total dissolved silver and chloride concentrations. Mean measured concentrations of Ag(+) in the test solutions ranged from 0.99 to 25 ng/l for the dissolved silver concentrations that ranged from 34 to 410 microg/l silver, indicating that free ionic silver varied from 0.003 to 0.006% of the silver dissolved in the 20 per thousand salinity seawater. Understanding the relationship of salinity and silver speciation, and the effect of this relationship on chronic invertebrate toxicity, will be useful for development of a marine biotic ligand model (BLM) and a water quality criterion for silver. This model could provide an important tool for improving the relationship of laboratory toxicity test results and predicted effects in natural environments, where variations in salinity may act to modify the toxicity of silver and other metals.

Animals↗
PubMed2004

A field test and comparison of acute and chronic sediment toxicity tests with the estuarine amphipod Leptocheirus plumulosus in Chesapeake Bay, USA.

A 28-d partial life-cycle test with the estuarine amphipod Leptocheirus plumulosus was developed in response to the need for an assay to mimic chronic exposure to sediment-associated contaminants. To ensure that toxicity tests have environmental relevance, it is essential to evaluate the relationship between laboratory responses and field measures of contamination. Consequently, one objective of the study was to compare the results of the chronic sediment toxicity test with L. plumulosus to gradients of sediment contamination and the in situ benthic community in its native Chesapeake Bay. Chronic tests were conducted by two laboratories, the Army Corps of Engineers Waterways Experiment Station ([WES]; Vicksburg, MS, USA) and the University of Maryland ([UM] College Park, MD, USA) using different feeding regimes, providing the opportunity to evaluate the effect of this variable on response sensitivity. A second objective was to compare the relative sensitivity of acute and chronic tests with L. plumulosus with field-collected sediments. Overall, there was good agreement between the toxicological response of acute and chronic tests with L. plumulosus and field measures of contamination. Survival in the acute test and chronic test conducted by WES was negatively correlated with concentrations of sediment-associated contaminants. Survival in acute exposures was significantly reduced in sediments from 8 of 11 stations. Indigenous L. plumulosus were found only at two of the three stations that did not exhibit acute toxicity. An unexpected finding was the difference in responsiveness of the two chronic tests. Survival in tests conducted by UM and WES was significantly reduced in sediments from 4 and 6 of 11 stations, respectively. No additional sublethal toxicity was detected in the UM chronic test, but the WES test detected reproductive effects at two additional stations. We believe the observed differences were related to the test diet used. Partly as a result of our findings, the recommended diet for the L. plumulosus chronic test was changed in the final methods document.

Acute Disease↗
PubMed2003

Enhancing toxicity test performance by using a statistical criterion.

Aquatic toxicity tests are laboratory experiments that measure the biological effect (e.g., growth, survival, reproduction) of effluents, receiving waters, or storm water on aquatic organisms. These toxicity tests must be performed using the best laboratory practices, and every effort must be made to enhance repeatability of the test method. We evaluated the generated reference toxicant test data for insurance of a level of quality assurance for tests over time within a laboratory and among laboratories. We recommend the reporting and evaluation of the percent minimum significant difference (PMSD) value for all toxicity test results. The minimum significant difference (MSD) represents the smallest difference between the control mean and a treatment mean that leads to the statistical rejection of the null hypothesis (i.e., no toxicity) at each concentration of the toxicity test dilution series. The MSD provides an indication of within-test variability, and smaller values of MSD are associated with increased power to detect a toxic effect. We recommend upper and lower PMSD bounds for each test method in order to minimize within-test variability and increase statistical power. To ensure that PMSD does not exceed an upper bound, testing laboratories may need to increase replication, decrease variability among replicates, or increase the control mean performance.

Animals↗
PubMed2002

A modified toxicity testing method using tropical marine microalgae.

Toxicity testing of four heavy metals (Cd, Cu, Mn and As) using four species of tropical marine phytoplankton, Chaetoceros calcitrans, Isochrysis galbana, Tetraselmis tetrahele and Tetraselmis sp., was carried out in multiwell plates with test volumes of 2 mL and the results compared to those of standard, large volume, shake-flasks. IC50 values (concentrations of metals estimated to inhibit 50% growth relative to the control) were determined after 96 hours based on automated O.D. readings measured in Elisa microplates by a Multiskan spectrophotometer. Good agreement was achieved between O.D. readings and cell counts indicating that this new method is a simple, economical, practical and rapid technique for toxicity testing, and provides good reproducibility of IC50 values. Results of the toxicity tests indicate that Cu was the most toxic metal (average IC50 values ranging from 0.04 to 0.37 mg L(-1)), followed by Cd (0.06-5.7 mg L(-1)), Mn (7.2-21.4 mg L(-1)) and As (33.9-319.3 mg L(-1)). Test species had different degrees of sensitivity to the metals tested, with I. galbana and C. calcitrans the most sensitive to Cu, Cd and Mn. Based on these findings it is recommended that the existing Malaysian Interim Standards for Marine Water Quality for Cd and Cu be reviewed.

Environmental Monitoring↗
PubMed1998

[Microbial test combination with Vibrio fischeri as a screening toxicity test]

Two different microbial toxicity tests were investigated concerning the possibility to reduce animal experiments in product testing. In the acute bioluminescence test the inhibition of the bacterial luminescence was measured. In the chronic DMS test the inhibition of the microbial metabolism of DMSO to DMS was recorded. The combination of both tests with the same organism Vibrio fischeri enhances the power as a toxicological indicator. We used well known alcohols and tensides. For 15 substances, the correlation between the experimental results and the Draize eye irritation test (rs = 0,943) and the intravenous toxicity (rs = 0,958) was statistically significant. If both tests gave EC50-values >10000mg/L, the substance could be categorised as non or mild toxic. If the EC50-values were <5mg/L, an extremely toxic substance could be expected. Neither false positive nor false negative results have been found, therefore the application of the method as a screening test or as a part in a test battery is conceivable.

Journal Article↗
PubMed2006

The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test.

The most commonly used toxicity test worldwide is the acute Daphnia magna test. The relevance of acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity in D. magna exposed to chromium, cadmium, and diazinon was evaluated in connection with this standard test. We found no link between enzyme activities and immobility. Concentrations of Cr(6+) up to 280 microg/L had no effect on AChE and GST activities, while 20% immobility was observed. At concentrations of 20-25 microg/L of Cd(2+) AChE activity was increased by about 50%. The effect of diazinon on both enzymes was insignificant up to concentrations that caused 27% immobility. Consequently, while the use of AChE and GST activities is recommended when the mode of action of chemicals is studied, the value of these biomarkers in routine acute toxicity tests is limited because the relationship between enzyme activities and immobility of D. magna exposed to different chemicals is unclear.

Acetylcholinesterase↗
PubMed2006

Ecotoxicological assessment of effluents in the Basque country (Northern Spain) by acute and chronic toxicity tests using Daphnia magna straus.

Acute pass/fail, multi-concentration tests, and 3-brood chronic toxicity tests with Daphnia magna Straus (Cladocera, Crustacea) were used to characterise industrial and municipal effluents from various sources. The effluents that "passed" the pass/fail tests had 48-h EC50 values >100% and reproduction No Observed Effect Concentration (NOECs) > or =100%, except for one effluent that had a reproduction NOEC of 31.6%. The acute multi-concentration toxicity tests allowed a rapid classification of effluents from Very Toxic (48-h EC50 < 25%), to Non-Toxic (48-h EC50 >100%). The acute-to-chronic ratio (ACR: 48-h EC50 divided by the NOEC for reproduction) in the studied effluents ranged from 5 to about 100. From these results, we propose a step-wise protocol for assessing effluent toxicity. First, effluent is evaluated by means of simple and rapid pass/fail acute toxicity tests, to discriminate Non-Toxic from potentially Toxic effluents, thus facilitating the establishment of priority actions. Second, 48-h ECx is estimated to classify effluents on a toxicity scale from Non-Toxic to Very Toxic. Third, chronic multi-concentration tests are used to calculate reproduction NOECs. These parameters combined with data on effluent chemical composition, chemical and hydrological characteristics of receiving waters, and biological quality criteria can be jointly used for more rational regulatory practices and risk assessment of effluents.

Animals↗
PubMed2002

Ecotoxicological studies of environmental samples from Buenos Aires area using a standardized amphibian embryo toxicity test (AMPHITOX).

The toxicity of 34 environmental samples from potentially polluted and reference stations were evaluated by means of the AMPHITOX test from acute to chronic exposure according to the toxicity found in each sample. The samples were obtained from surface and ground water, leaches, industrial effluents and soils. The data, expressed in acute, short-term chronic and chronic Toxicity Units (TUa, TUstc and TUc) resulted in a maximal value of 1000 TUc, found in a leach, while the lower toxicity value was 1.4 TUa corresponding to two surface water samples. In five samples (four providing from reference places) no toxicity was detected. The results point out the possibility of evaluating the toxicity of a wide diversity of samples by means of AMPHITOX as a customized toxicity test. The fact that almost all samples with suspected toxicity in rivers and streams from the Metropolitan area of Buenos Aires city resulted toxic, indicates the need of enhanced stewardship of chemical substances for environmental and human health protection purposes.

Animals↗
PubMed2003

Use of multidimensional scaling in the selection of wastewater toxicity test battery components.

In aquatic toxicity testing, no single test species is sensitive to all toxicants. Therefore, test batteries consisting of several individual assays are becoming more common. The organisms in a test battery should be representative of the entire system of interest. The results of the assays should be complementary to other components in the test battery to avoid redundancy. With the aid of multidimensional scaling (MDS), a multivariate statistical method, we examined the toxicity data of five bioassays (the continuous Shk1, Polytox, activated sludge respiration inhibition, Nitrosomonas, and Tetrahymena assays) that could serve as test battery components for the assessment of wastewater toxicity to activated sludge. MDS mapped the five assays into a two-dimensional space and showed that the Nitrosomonas assay should be included in test batteries plus one of the remaining four assays for assessing wastewater toxicity to activated sludge.

Animals↗
PubMedDate not supplied

The Registry of Cytotoxicity: toxicity testing in cell cultures to predict acute toxicity (LD50) and to reduce testing in animals.

This is a translation of a report on the Registry of Cytotoxicity (RC), originally published in German in 1998. The report presented an advanced in vitro method, which can significantly reduce the number of animals needed for the toxicity testing of a broad range of compounds/xenobiotics. With the RC method, it was possible to predict the oral or intravenous acute toxicity (LD50)--which is a regulatory requirement for newly developed pharmaceuticals and industrial and household chemicals--from the cytotoxicity data (mean IC50 = IC50X) obtained with mammalian cells. The RC method can be used before the in vivo test, and it does not pose any additional harm or suffering to laboratory animals. The RC method is of broad practical use: it can be applied, for example, in the pharmaceutical industry or the chemical industry in regulatory testing or in research. It is ready for validation, and could then be incorporated into OECD guidelines, thus reducing the total number of animals needed for regulatory toxicity testing. The RC method is based on the comparison of the IC50X values and the LD50 values by using linear regression analysis. With the RC method, it was possible to predict, within a predefined dose range, the acute oral LD50 for 252 of 347 xenobiotics, and the intravenous LD50 for rats and/or mice for 117 of 150 xenobiotics. Comparative studies showed that these results are highly reproducible.

Animal Testing Alternatives↗
PubMed2004

A novel continuous toxicity test system using a luminously modified freshwater bacterium.

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.

Biological Assay↗
PubMed2001

Partition controlled delivery of hydrophobic substances in toxicity tests using poly(dimethylsiloxane) (PDMS) films.

Interpretation of toxicity test results may be hampered when doubt exists about the actual exposure concentration. Processes that are responsible for differences between the nominal and the actual concentration in aqueous test systems may include sorption, precipitation, volatilization, chemical and biological degradation, and uptake into biological or test tissue. In this study, the use of a poly(dimethylsiloxane) (PDMS) film containing the test compound is introduced as a versatile technique for partition controlled delivery of hydrophobic compounds to aqueous toxicity tests. Two methods developed produced preloaded films, having toxicant added to the PDMS prepolymer solution before film deposition and curing, and postloaded films, which are created by the addition of toxicant in a solvent to an already-polymerized PDMS film. Preloaded films were generally more easily prepared, may better accommodate larger molecules, and have a higher capacity than postloaded films. Postloaded films provided film-solution partition coefficients with higher precision and allowed for the use of films from stock and thus for a more portable technique. Chemical analysis showed that equilibrium between films and the aqueous solution was established within 7-10 min and was maintained for a suite of aromatic compounds (log Kow ranging from 2.8 to 6.1). The reliability of the film technique was demonstrated by application to the Microtox bacterial toxicity tests of solutions of polycyclic aromatic hydrocarbons (PAHs).

Absorption↗
PubMed1993

Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing. Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.

Animal Testing Alternatives↗