Species Toxicity Tests Research Articles

Temperature and food quantity effects on the harpacticoid copepod Nitocra spinipes: Combining in vivo bioassays with population modeling.

The harpacticoid copepod Nitocra spinipes has become a popular model species for toxicity testing over the past few decades. However, the combined influence of temperature and food shortage, two climate change-related stressors, has never been assessed in this species. Consequently, effects of three temperatures (15, 20 and 25°C) and six food regimes (between 0 and 5 × 105 algal cells/mL) on the life cycle of N. spinipes were examined in this study. Similarly to other copepod species, development times and brood sizes decreased with rising temperatures. Mortality was lowest in the 20°C temperature setup, indicating a close-by temperature optimum for this species. Decreasing food concentrations led to increased development times, higher mortality and a reduction in brood size. A sex ratio shift toward more females per male was observed for increasing temperatures, while no significant relationship with food concentration was found. Temperature and food functions for each endpoint were integrated into an existing individual-based population model for N. spinipes which in the future may serve as an extrapolation tool in environmental risk assessment. The model was able to accurately reproduce the experimental data in subsequent verification simulations. We suggest that temperature, food shortage, and potentially other climate change-related stressors should be considered in environmental risk assessment of chemicals to account for non-optimal exposure conditions that may occur in the field. Furthermore, we advocate combining in vivo bioassays with population modeling as a cost effective higher tier approach to assess such considerations.

Weatherfish (Misgurnus fossilis) as a new species for toxicity testing?

Selection of appropriate test species is a critical issue when assessing effects of environmental contamination on fish because the ecological relevance of commonly used test species might be restricted due to their exotic origin. In the present study, a European freshwater fish with frequent occurrence in agricultural areas is suggested as a potential alternative: the European weatherfish (Misgurnus fossilis). Its suitability for acute embryo toxicity tests (FET) was investigated with regard to practical implementation, sensitivity to contaminants and tolerance against environmental conditions of concern. For this purpose, weatherfish embryos were exposed (72h) to the reference substance 3,4-dichloroaniline (DCA) in three independent tests. Furthermore, the effects of dissolved oxygen (DO) deficiency on weatherfish embryos were studied to evaluate their suitability e.g. for sediment bioassays. Obtained results revealed that the sensitivity of weatherfish embryos towards DCA (72 h-EC50=0.52mg/l; 72 h-LC50=0.71mg/l) was highest compared to other species and three times higher than that reported for the commonly used zebrafish (Danio rerio). Even though knowledge of DO requirements during the embryonic period of European fish species is scarce, weatherfish can be stated as one of the most tolerant native species (LC90 for DO=0.53mg/l after 48h exposure plus 72h post-exposure). Its high ecological relevance for Europe, the particular sensitivity towards DCA and high tolerance against DO depletion highlight the potential of weatherfish as additional species for toxicity testing.

Spatially explicit assessment of estuarine fish after Deepwater Horizon oil spill: trade-off in complexity and parsimony.

Evaluating long-term contaminant effects on wildlife populations depends on spatial information about habitat quality, heterogeneity in contaminant exposure, and sensitivities and distributions of species integrated into a systems modeling approach. Rarely is this information readily available, making it difficult to determine the applicability of realistic models to quantify population-level risks. To evaluate the trade-offs between data demands and increased specificity of spatially explicit models for population-level risk assessments, we developed a model for a standard toxicity test species, the sheepshead minnow (Cyprinodon variegatus), exposed to oil contamination following the Deepwater Horizon oil spill and compared the output with various levels of model complexity to a standard risk quotient approach. The model uses habitat and fish occupancy data collected over five sampling periods throughout 2008-2010 in Pensacola and Choctawhatchee Bays, Florida, USA, to predict species distribution, field-collected and publically available data on oil distribution and concentration, and chronic toxicity data from laboratory assays applied to a matrix population model. The habitat suitability model established distribution of fish within Barataria Bay, Louisiana, USA, and the population model projected the dynamics of the species in the study area over a 5-yr period (October 2009-September 2014). Vital rates were modified according to estimated contaminant concentrations to simulate oil exposure effects. To evaluate the differences in levels of model complexity, simulations varied from temporally and spatially explicit, including seasonal variation and location-specific oiling, to simple interpretations of a risk quotient derived for the study area. The results of this study indicate that species distribution, as well as spatially and temporally variable contaminant concentrations, can provide a more ecologically relevant evaluation of species recovery from catastrophic environmental impacts but might not be cost-effective or efficient for rapid assessment needs.

Adsorption, uptake and distribution of gold nanoparticles in Daphnia magna following long term exposure

Gold nanoparticles (nAu) have recently been studied and developed within the biological and photothermal therapeutic contexts. The major clinical interest is within the application of novel drug delivery systems. Environmental exposure to nanoparticles can occur in different stages of the lifecycle of the product; from their synthesis, applications, product weathering and their disposal. Freshwater Daphnids, specifically Daphnia magna, have been used since the 1960s as a standard species in acute and chronic aquatic toxicity testing. Visualization of the interactions and uptake of nAu by D. magna was related to reproduction and molting patterns. Exposure to nAu was done using a chronic reproduction test performed for 14 days at six concentrations (0.5mg/L, 2mg/L, 5mg/L, 10mg/L, 15mg/L and 20mg/L). Microscopy was used to determine whether there was any uptake or interaction of nAu with daphnia. However the concentration of nAu in the media and the charge of particles played a role in the uptake and surface adsorption. As exposure concentrations of nAu increased it appeared that the nAu aggregated onto the surface and in the gut of the organisms in higher concentrations. There was no evidence of nAu internalization into the body cavity of the daphnia. Aquatic exposure to nAu resulted in increased adhesion of the particles to the carapace of daphnia, ingestion and uptake into the gut of daphnia and had no significant effect on reproduction and molting patterns.

Toxicity of Vertimec® 18 EC (active ingredient abamectin) to the neotropical cladoceran Ceriodaphnia silvestrii

The aim of the present study was to evaluate the toxicity of abamectin to the neotropical cladoceran Ceriodaphnia silvestrii. To this end, acute and chronic bioassays were conducted with the commercial formulation Vertimec® 18 EC. In addition, the toxicity of water samples taken from a microcosm experiment evaluating the effects of a single application (144μga.i./L) and two applications (2×36μga.i./L) of Vertimec® 18 EC, in the presence or absence of a tadpole species (Lithobates catesbeianus), was also assessed. The acute LC50-48h for immobilization was 1.47μga.i./L and chronic NOEC-8d for survival and fertility (number of neonates per female) were 169 and 84nga.i./L, respectively. Irrespective of the presence of tadpoles, water samples from the microcosms applied with the single concentration of 144μga.i./L remained toxic until the end of the experiment, even when samples were diluted 32 times with culture medium. Water in the repeated pesticide treatment showed a similar toxic response after both applications. Toxicity of water samples from the microcosms was lower than that expected based on the generated LC50 values, which is explained by a potential reduced bioavailability of the test compound resulting from absorbance to organic material. Potential side-effects on C. silvestrii related with the use of Vertimec® 18 EC in Brazil and the suitability of this species for tropical toxicity testing are discussed.

Comparative sensitivity of field and laboratory populations of Hyalella azteca to the pyrethroid insecticides bifenthrin and cypermethrin

Hyalella azteca are epibenthic invertebrates that are widely used for toxicity studies. They are reported to be more sensitive to pyrethroid insecticides than most other test species, which has prompted considerable use of this species in toxicity testing of ambient surface waters where the presence of pyrethroids is suspected. However, resident H. azteca have been found in some ambient water bodies reported to contain surface water and/or sediment pyrethroid concentrations that are toxic to laboratory reared H. azteca. This observation suggests differences in the sensitivities of laboratory reared and field populations of H. azteca to pyrethroids. The goal of the present study was to determine the sensitivities of laboratory reared and field populations of H. azteca to the pyrethroids bifenthrin and cypermethrin. Specimens of H. azteca were collected from resident populations at field sites that are subject to varied land-use activities as well as from laboratory populations. These organisms were exposed to bifenthrin- or cypermethrin-spiked water in 96-h water-only toxicity tests. The resulting data demonstrated that: 1) field-collected populations in urban and agricultural settings can be >2 orders of magnitude less sensitive to the pyrethroids than laboratory reared organisms; 2) field-collected organisms varied in their sensitivity (possibly based on land-use activities), with organisms collected from undeveloped sites exhibiting sensitivities similar to laboratory reared organisms; and 3) the sensitivity of field-collected "tolerant" organisms increased in subsequent generations reared under laboratory conditions. Potential mechanisms for these differences are discussed.

Determining the sensitivity of the Antarctic amphipod Orchomenella pinguides to metals using a joint model of survival response to exposure concentration and duration.

Developing water quality guidelines for Antarctic marine environments requires understanding the sensitivity of local biota to contaminant exposure. Antarctic invertebrates have shown slower contaminant responses in previous experiments compared to temperate and tropical species in standard toxicity tests. Consequently, test methods which take into account environmental conditions and biological characteristics of cold climate species need to be developed. This study investigated the effects of five metals on the survival of a common Antarctic amphipod, Orchomenella pinguides. Multiple observations assessing mortality to metal exposure were made over the 30days exposure period. Traditional toxicity tests with quantal data sets are analysed using methods such as maximum likelihood regression (probit analysis) and Spearman-Kärber which treat individual time period endpoints independently. A new statistical model was developed to integrate the time-series concentration-response data obtained in this study. Grouped survival data were modelled using a generalized additive mixed model (GAMM) which incorporates all the data obtained from multiple observation times to derive time integrated point estimates. The sensitivity of the amphipod, O. pinguides, to metals increased with increasing exposure time. Response times varied for different metals with amphipods responding faster to copper than to cadmium, lead or zinc. As indicated by 30days lethal concentration (LC50) estimates, copper was the most toxic metal (31µg/L), followed by cadmium (168µg/L), lead (256µg/L) and zinc (822µg/L). Nickel exposure (up to 1.12mg/L) did not affect amphipod survival. Using longer exposure durations and utilising the GAMM model provides an improved methodology for assessing sensitivities of slow responding Antarctic marine invertebrates to contaminants.

The Role of Biotransformation in Drug Discovery and Development

Biotransformation is a process by which organic compounds are transformed from one form to another, aided by organisms such as bacteria, fungi and enzymes. It plays a major role and determines the fate of the prospective drugs. Biotransformation must take place only after the drugs reach their specific target site and produce the desired effects. In addition, the nature of the metabolites produced from the drug, must be thoroughly studied; otherwise, the drugs would be rejected during the screening process. Hence, drug metabolism is a major criterion in the highthroughput screening of prospective drugs. Biotransformation has an important role in the determination of the pharmacokinetic parameters like oral bioavailability, drug-drug interaction, clearance and the half-life of the entity within the cell. It is very essential in the toxicity studies too. Biotransformation is used as a valuable strategy to build molecules, similar to parent drug in the drug discovery programme. It can play an important role in identifying factors underlying the problems, facilitate the optimal selection of compounds for further development, provide information on metabolites for possible improvement in drug design, and contribute to the identification of the appropriate animal species for subsequent toxicity testing. Hence, so

Chemical comparison and acute toxicity of water accommodated fraction (WAF) of source and field collected Macondo oils from the Deepwater Horizon spill

Two Source oils and five field collected oil residues from the Deepwater Horizon incident were chemically characterized. Water accommodated fractions (WAFs) of the Source oils and two of the field-weathered oils were prepared to evaluate the impact of natural weathering on the chemical composition and the acute toxicity of the WAFs. Toxicity test species representing different tropic levels were used (the primary producer Skeletonema costatum (algae) and the herbivorous copepod Acartia tonsa). The results suggest that the potential for acute toxicity is higher in WAFs from non-weathered oils than WAFs from the field weathered oils. The Source oils contained a large fraction of soluble and bioavailable components (such as BTEX (benzene, toluene, ethyl benzene, xylenes) and naphthalene), whereas in the surface collected oils these components were depleted by dissolution into the water column as the oil rose to the surface and by evaporative loss after reaching the sea surface.

Part 1: Laboratory culture of Centroptilum triangulifer (Ephemeroptera: Baetidae) using a defined diet of three diatoms

Development of methods for assessing exposure and effects of waterborne toxicants on stream invertebrate species is important to elucidate environmentally relevant information. Current protocols for freshwater invertebrate toxicity testing almost exclusively utilize cladocerans, amphipods or chironomids rather than the more typical aquatic insect taxa found in lotic systems. Centroptilum triangulifer is a parthenogenetic mayfly occurring in depositional habitats of streams and rivers of the Eastern U.S. and Canada. C. triangulifer is an ideal stream insect for toxicity testing under field and laboratory conditions because of its short life cycle, parthenogenetic mode of reproduction, and it represents a group considered sensitive to environmental stressors. In this study, a colony of C. triangulifer was reared using a defined diet of three diatoms, Mayamaea atomus var. permitis, Nitzschia cf. pusilla, and Achnanthidium minutissimum. Percent survival (⩾80%), fecundity measurements (⩾1000 eggs) and pre-egg laying weights were used as indicators of overall colony health and fitness in our laboratory water (Lab-line) and in Moderately Hard Reconstituted Water (MHRW). Lab-line reared C. triangulifer had average survival rate of 92.69% for eleven generations and 82.99% over thirteen generations. MHRW reared C. triangulifer had an average survival rate of 80.65% for four generations and three generations of fecundities greater than 1000 eggs per individual. Pre-egg laying weight and fecundity were highly correlated and a best-fit model equation was derived to estimate egg counts for future generations. Establishment of this culturing protocol provides a more ecologically relevant species for toxicity testing and aids in further stressor identification for stream bioassessments.

Effect of test duration and feeding on relative sensitivity of genetically distinct clades of Hyalella azteca

The amphipod Hyalella azteca is widely used in ecotoxicology laboratories for the assessment of chemical risks to aquatic environments, and it is a cryptic species complex with a number of genetically distinct strains found in wild populations. While it would be valuable to note differences in contaminant sensitivity among different strains collected from various field sites, those findings would be influenced by acclimation of the populations to local conditions. In addition, potential differences in metabolism or lipid storage among different strains may confound assessment of sensitivity in unfed acute toxicity tests. In the present study, our aim was to assess whether there are genetic differences in contaminant sensitivity among three cryptic provisional species of H. azteca. Therefore, we used organisms cultured under the same conditions, assessed their ability to survive for extended periods without food, and conducted fed and unfed acute toxicity tests with two anions (nitrate and chloride) whose toxicities are not expected to be altered by the addition of food. We found that the three genetically distinct clades of H. azteca had substantially different responses to starvation, and the presence/absence of food during acute toxicity tests had a strong role in determining the relative sensitivity of the three clades. In fed tests, where starvation was no longer a potential stressor, significant differences in sensitivity were still observed among the three clades. In light of these differences in sensitivity, we suggest that ecotoxicology laboratories consider using a provisional species in toxicity tests that is a regionally appropriate surrogate.

A review of recent non-target toxicity testing of vertebrate pesticides: establishing generic guidelines

An analysis of the range, extent and importance of New Zealand-based ecotoxicology studies has been completed to better understand the hazards and non-target risks associated with new toxins and baits. This review focuses on compounds that have been recently developed for incorporation into bait for terrestrial vertebrate pest control, namely cholecalciferol, para-aminopropiophenone (PAPP) and zinc phosphide and the effects of these toxins on non-target species. Testing of these compounds has included cage and pen toxicity and bait acceptance studies. Locally conducted acute toxicity studies clarify overseas data and enhance risk assessments. Consistency in approach and selection of surrogate species, and careful selection of native non-target species for acute toxicity testing in cage and pen trials are recommended as important steps before field trials. We propose a systematic approach to cage and pen trials and offer guidelines on species selection.

The Use of the Cyanobacteria, Cyanobium sp., as a Suitable Organism for Toxicity Testing by Flow Cytometry

Cyanobacteria are commonly found in a number of temperate and tropical bioregions, and provide important roles in fuelling many nutrient poor freshwater and marine ecosystems. Although cyanobacteria commonly occur in these environments, little is known about the use of cyanobacteria as suitable organisms for toxicity studies. Here, we propose the use of the unicellular cyanobacteria Cyanobium sp., as a potential species for tropical toxicity testing using flow cytometry. Cyanobium sp. was isolated from a composite sample of sea water in Halifax Bay, North Queensland, Australia. After careful isolation, cleaning and purification, Cyanobium sp. was used to determine the toxicity of copper, cobalt, and nickel at pH 8, and ammonia at pH 7 and 8. EC₁₀/₅₀ values were calculated using growth inhibition data determined via flow cytometry, which was found to provide rapid, accurate results, with the ability to define multiple endpoints. Cyanobium sp. was particularly sensitive to copper, cobalt and nickel, however, thrived at elevated concentrations of ammonia, irrespective of pH value. The results indicate that Cyanobium sp. is a useful test organism for tropical marine metal toxicity studies, however, is unsuitable for nutrient studies, particularly ammonia.

Target organ toxicities in studies conducted to support first time in man dosing: An analysis across species and therapy areas

An analysis of target organ toxicities in first time in man (FTiM) toxicity studies for 77 AstraZeneca candidate drugs (CDs) was conducted across a range of therapy areas. In the rodent, the most frequently affected organ was the liver followed by adrenal glands, kidney, spleen, bone marrow and thymus. In non-rodent, liver and thymus were the most frequently affected organs, followed closely by the testis and GI tract. The profile of affected organs was largely similar across the therapy areas of respiratory and inflammation, cardiovascular/gastrointestinal and CNS/pain. The oncology/infection therapy area differed with a larger range of organs affected. For the 75 CDs for which both rodent and non-rodent studies were conducted, new target organs were identified in non-rodents for 43 of the CDs. Notably, the changes seen only in non-rodents included organ systems of high relevance for human risk assessment such as the liver, male reproductive tissues and CNS. Additionally, profiles were similar for those CDs that progressed into human trials and those that did not. Overall, our data provide new insights into drug toxicity profiles in pre-clinical species and additionally confirm the value of using non-rodents as a second species in toxicity testing to support human safety.

Evaluating the suitability of Hydrobia ulvae as a test species for sediment metal toxicity testing applying a tissue residue approach to metal mixtures in laboratory and field exposures

A major weakness in evaluating the suitability of a biomonitor organism is the poor ability to predict the variability of the bioavailability of metals from measured environmental concentrations. In this study, the intertidal gastropod Hydrobia ulvae was used to evaluate its suitability as a test organism for assessing sediment metal toxicity. Toxicity tests were run with sediments spiked with copper, cadmium and zinc applied both as single metal and as a mixture to investigate toxicological interactions evaluating different lethal and sublethal effects. Dose–response relationships were constructed based both on tissue residue approach and particulate metal concentrations. Because metal-spiked sediments used in routine toxicity tests often do not exhibit the same adsorption/desorption kinetics as the natural sediments, the laboratory results were compared to 10-d bioassays conducted with natural field sediments collected from the Guadalete estuary (SW Spain). Highly significant correlations between tissue residue concentrations and particulate metal concentrations were found for all metal-spiked or field-collected and demonstrated that: (i) H. ulvae readily accumulated copper and cadmium in response to contamination and (ii) dietary uptake was determined to be the most significant route of metal exposure. The comparison of the modeled tissue residue-response curve developed from the mixture tests was in good agreement with the results from the bioassay conducted with field sediments and strongly demonstrated that H. ulvae is also a suitable test organism for assessing copper sediment toxicity. In contrast, the dose–response curve expressed as a function of total particulate metal concentrations would fail in predicting effect, erroneously assessing higher metal toxicity.

Aiptasia pulchella: a tropical cnidarian representative for laboratory ecotoxicological research

An urgent need exists to identify suitable tropical marine species for use in the development of sensitive and reliable test methods for routine laboratory ecotoxicological testing. Corals are a group of organisms not represented in routine ecotoxicology due to inherent difficulties in laboratory husbandry, and sea anemones from the same phylum (cnidaria) may be useful proxies. Aiptasia pulchella is a tropical symbiotic sea anemone with a wide geographic range. It is well suited to laboratory conditions and has been used extensively in research. However, its suitability as a toxicity test species has not been investigated. Assessment of juvenile recruitment in laboratory and semi-outdoor conditions showed higher production in semi-outdoor conditions; however, laboratory rearing produced enough recruits to run routine toxicity tests. In investigations of the sensitivity of A. pulchella to contaminants, acute tests were conducted on 1- to 2-mm juveniles using copper. Lethal concentration, 50% (LC50) values at 96 h estimated from tests using five and 10 replicates ranged from 30 to 83 and 60 to 90 µg/L, respectively, and a 28-d LC50 of 26 µg/L was estimated. During the present study, sublethal endpoints were investigated; chronic assessment of inhibited asexual reproduction looks promising (12-d effective concentration, 50% [EC50] 15 µg/L) and should be assessed further. Aiptasia pulchella is a species worthy of investigation as a cnidarian representative, and will be an invaluable contribution to tropical marine ecotoxicologists.

Identification of oestrogen-responsive transcripts in Potamopyrgus antipodarum

The freshwater caenogastropod Potamopyrgus antipodarum is a promising test species for partial lifecycle toxicity testing and also for studies of endocrine disruptors. However, endocrine signalling pathways and the genes/transcripts involved are barely identified and characterized in this snail. We therefore performed whole transcriptome sequencing (454 pyrosequencing), selected transcripts that are known to be hormone-responsive in vertebrates and determined their expression via quantitative real-time PCR after 24 h exposure to the oestrogens 17a-ethinyloestradiol (EE2) and 17b-oestradiol (E2). Examined mRNAs were: nuclear receptors [oestrogen receptor (ER), chicken ovalbumin upstream promoter-transcription factor (COUP-TF), ecdysone-induced protein (E75)], repressors of oestrogen-induced activity [prohibitin-2 (PHB-2), striatin (STRN)] and the egg yolk precursor vitellogenin (VTG). Significantly increased expression by EE2 and E2 treatment was detected for ER and VTG. E2 induced significant upregulation of E75, PHB-2 and STRN, while EE2 did not. COUP-TF expression was unaffected by EE2 or by E2. Variations between replicates were higher in snails exposed to EE2 than to E2. The identification of these oestrogen-responsive transcripts contributes to a better characterization of the endocrine system of P. antipodarum and provides possible biomarkers for the detection of endocrine disruption.

Preclinical development of an anti-CD30/anti-CD16A bispecific tetravalent TandAb antibody for the treatment of Hodgkin lymphoma.

e18532 Background: CD30 is a validated tumor target that is expressed on both Hodgkin’s Lymphoma cells and some activated immune cells. To improve antibody-based therapies targeting CD30, Affimed developed an NK cell-recruiting bispecific antibody. Methods: To enhance the recruitment and activation of NK cells for tumor cell lysis, we isolated a human antibody that is highly specific for the A isoform of FcgRIII (CD16A). Its variable domain (Fv) was used to construct a tetravalent bispecific TandAb, consisting of four Fvs linked in tandem, with two binding sites for both the CD16A on NK cell and the CD30 target cell antigens. The resulting anti-CD30/anti-CD16A bispecific tetravalent TandAb AFM13 was compared to both an anti-CD30/anti-CD16A bispecific diabody and an Fc-enhanced anti-CD30 IgG in binding and cytotoxicity assays in vitro. Pharmacokinetics and safety of the TandAb were investigated in Cynomolgus monkeys. Results: AFM13 is retained longer on NK cells than either the anti-CD30 IgG or the bispecific diabody due to its bivalency for CD16A; it binds both CD16A alleles (158F, 158V) equally well indicating the potential for broader patient response than that achieved with current therapeutic mAbs. AFM13 did not induce systemic activation of NK cells. It displays higher potency (EC50) and higher efficacy (maximal lysis) than either the anti-VD30 IgGs or the bispecific diabody in a cytotoxicity assay. The cytotoxic activity of AFM13 is unaffected by physiological concentrations of polyclonal IgG, whereas that of anti-CD30 IgGs is dramatically reduced. Cynomolgus monkeys were identified as the only relevant species for toxicity testing. Pharmacokinetics studies reveal AFM13 half-lives of up to 22.9 h after repeated dosing. MTD was not reached in a single dose escalation study ranging to 100 mg/kg maximum. A 28 days toxicity study, with dosing up to 10 mg/kg every other day, revealed an excellent safety profile. No signs of immunotoxicity or systemic cytokine release were observed. Conclusions: AFM13 may be a more effective treatment for Hodgkin’s Lymphoma that is refractory to treatment with anti-CD30 IgGs and is under evaluation in a Phase I clinical trial.

Development of a harpacticoid copepod bioassay: Selection of species and relative sensitivity to zinc, atrazine and phenanthrene

Worldwide, estuaries are under increasing pressure from numerous contaminants. This study aimed to identify a suitable marine harpacticoid copepod species for toxicity testing of New Zealand estuaries. Multiple aspects were considered for species selection and included: a broad regional distribution, ease of culture, reproductive rate under laboratory conditions, sexual dimorphism, and sensitivity to contaminants. Five species were evaluated and two (Robertsonia propinqua and Quinquelaophonte sp.) were able to be cultured. The relative sensitivity of these copepods to three reference toxicants was assessed by determining the medial lethal values following a 96h exposure (96h LC50) to these toxicants in the aquatic phase. LC50 values for zinc, phenanthrene, and atrazine respectively were 2.0, 0.89, and 7.58mg/L in R. propinqua and 0.64, 0.75, and 20.8mg/L in Quinquelaophonte sp. After evaluating all factors involved in choosing a bioassay species for New Zealand, Quinquelaophonte sp. was selected as the most suitable bioassay species.

Abstract 3522: Preclinical development of an anti-CD30/anti-CD16A bispecific tetravalent TandAb antibody for the treatment of Hodgkin lymphoma

Abstract NK cells are professional cytotoxic immune effector cells and, unlike cytotoxic T cells, exist in a constitutively activated state not requiring pre- or co-stimulation. Thus, NK cells are attractive effectors to employ for cancer immunotherapy with bispecific antibodies. To improve the recruitment and activation of NK cells for lysis of tumor cells, we isolated a human antibody that was highly specific for the A isoform of FcαRIII (CD16A) and displayed substantially lower dissociation constant than native or Fc-optimized mAbs. The variable domains of this antibody were used to construct a tetravalent bispecific antibody, termed TandAb, consisting solely of antibody variable domains, with two valencies for CD16A (on effector cells) and two for CD30 (on target cells). CD30 represents a validated tumor target which is expressed on Hodgkin's Reed-Sternberg lymphoma cells, and on some activated immune cells. The binding properties of the TandAb and its efficacy for inducing tumor cell lysis were compared to those of a native anti-CD30 mAb, a mAb with enhanced Fc receptor binding properties, and an anti-CD30 bispecific diabody, all of which comprised the same anti-CD30 variable domains as the TandAb. Due to its bivalency for CD16A, the TandAb was retained longer on NK cells than the IgGs or the bispecific diabody, and relatively high concentrations of IgG were necessary to displace it from NK cells. The TandAb bound equally well to both CD16A alleles (158F, 158V) indicating a potentially broader patient response than that achieved with current therapeutic mAbs. The TandAb did not induce any systemic activation of NK cells even though it's binding was bivalent to CD16A. In a cytotoxicity assay, the TandAb displayed higher potency (EC50) and higher efficacy (maximal lysis) than the IgGs or the bispecific diabody. Cynomolgus monkeys were identified as the only relevant species for toxicity testing. Pharmacokinetics studies revealed half-lives for the TandAb up to 22.9 h after repeated dosing. No MTD was reached in a single dose escalation study to a maximum dose of 100 mg/kg. A pivotal 28 days toxicity study in which animals were dosed repeatedly up to 10 mg/kg every other day revealed an excellent safety profile. Several immunological parameters were assessed indicating no signs of immunotoxicity or systemic cytokine release. In conclusion, TandAbs may provide a more effective treatment for tumors that are refractory to treatment with IgGs and is currently being evaluated in the Phase I clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3522. doi:1538-7445.AM2012-3522