AdmetAnimal StudiesADMET GROUP provides a variety of acute and sub-chronic toxicity, pharmacokinetics, toxicokinetics and biocompatibility studies with rodents and non-rodent animal species. ADMET GROUP conducts the following studies either as investigative pilot studies or as GLP studies in compliance with regulatory guidelines and requirements.
ADMET GROUP subcontracts dog, Pig and Primate studies
to other qualified laboratories. Acute Oral Toxicity Study in Rodents
Animals receive a single treatment and are observed daily for 14 days for clinical signs of toxicity, including, change in skin, fur, eyes, mucous membranes, and somatomotor activity. Three animals per sex are treated with a fixed dose that is based on prior knowledge of toxicity. If no toxicity information is available, the 200 mg/kg of body weight is used for the initial trial. Guideline: 1. OECD Guideline 423: OECD Guideline for the testing of chemicals adopted 22-03-96. Repeated Dose 28-Day Oral Toxicity Study in Rodents Animals are treated with three concentrations of the test article using a minimum of 10 animals (5 males and 5 females) per group. Concurrent control animals are also included in the study. The dose levels are selected based on previous toxicity and toxicokinetic data available for the test compound. Animals are dosed daily for a period of 28 days. Clinical observations are performed daily. Changes in body weight and food and water consumption are recorded weekly. At the end of the study, complete hematology and clinical biochemistry studies are performed for each animal. Pathology studies are made on vital organs and gross lesions, if present, as specified by the study protocol. Guideline: 1. OECD Guideline 407: OECD Guideline for the testing of chemicals adopted 27-07-95. Repeated Dose 90-Day Oral Toxicity Study in Rodents Animals are treated with three concentrations of the test article using a minimum of 20 animals (10 males and 10 females) per group. Concurrent control animals are also included in the study. The dose levels are selected based on toxicity data from a 28-day study. Animals are dosed daily for a period of 90 days. Clinical observations are performed daily. Changes in body weight and food and water consumption are recorded weekly. At the end of the study, complete hematology and clinical biochemical studies are performed for each animal. Pathology studies are made on vital organs and gross lesions, if present, as specified by the study protocol. Guideline: 1. OECD Guideline 408: OECD Guideline for the testing of chemicals adopted 21-09-98. Δ BACK TO TOP | < CAPABILITIES INDEX DMPK StudiesADMET GROUP performs pharmacokinetic (PK) studies in animal models for its pharmaceutical clients. Typically, a pilot PK study is done prior to a full GLP PK study. Once the results of an initial Pilot PK study indicate sufficient absorption and bio-availability and half-life of the compound in the plasma, a study specific protocol is generated and submitted to the sponsor. Measurements of the drug and its metabolites in the plasma are determined after acute or repeat dosing of animals at multiple time intervals. The time course distribution of the drug and its metabolites in the plasma samples is determined to characterize the plasma kinetics. The concentrations of both bound and free drug and its metabolites in the plasma are measured using appropriate analytical methods. A pharmacokinetic data analysis of plasma concentrations of the drug uses a non-compartmental approach to derive the following for the drug.
Oral Bio-availability Studies
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Bio-availability studies of pharmaceutical finished products in animals are designed and conducted by ADMET GROUP. Animal ADME / DM-PK / In-Vitro Metabolism ADMET GROUP performs a series of integrated in-vitro and in-vivo pre-clinical animal studies in mice, rats, guinea pigs and rabbits onabsorption, distribution, metabolism and elimination (ADME) in support of drug candidate screening and lead compound selection. Animal ADME Characterization ADME studies are conducted using LC/MS/MS measurement for rapid development of an assay method in support of low-level drug measurement.
All DM-PK studies are conducted using LC/MS/MS measurement with a LOQ typically from 1 to 5 ng/ml assaying 50 µl of plasma or urine.
Drug Candidate and Toxicity Screening TestsADMET GROUP uses the following approaches in assisting its clients in their drug discovery efforts and in the selection of lead drug candidates for further development. Many of these tests are also used for screening other compounds. Please click on the following sections for more information. In Vitro Absorption
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ADMET GROUP performs the Caco-2 Assay. Biologically active, orally bioavailable compounds are desirable because they are easier to administer, have better patient compliance and have a larger overall commercial market. Since In Vivo assays for determining bioavailability are expensive, time consuming, and are not geared to high throughput, the Caco-2 Assay was developed. Caco-2 cells are of human origin, and membranes formed by these cells exhibit morphological and biochemical characteristics similar to human intestinal mucosa. The membranes are relatively easy and inexpensive to grow and the assays provide a good indication of potential human absorption. Cells are seeded on polycarbonate or polyester membranes coated with collagen and are allowed to grow until monolayers form and the cells form tight junctions. The membranes are comprised of both apical and basolateral sides. Caco-2 Assays can be used to screen compounds for further testing, compare analogs for differences in absorption, determine the mechanism of absorption (passive or active transport, paracellular or transcellular), and determine if excipients enhance absorption. The Caco-2 Cell Assay is a valuable tool in the ever-challenging search for new and unique compounds beneficial to the health of mankind. It is well suited for the demands of high throughput screening, requires minimal compound, and is relatively fast and easy to perform. Genetic Toxicology: Non-GLP Screening TestAmes Screening Test
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Designed to evaluate developmental materials at a low cost with a quick turn-around time, volume discounts are provided for samples tested concurrently. Mutagenicity tests are performed with and without activation at 6 concentrations. Two tester strains (TA98 and TA100) duplicate plating with concurrent positive and solvent controls. Results are reported in tables and a short summary. In Vitro Chromosome Aberration Tests Chromosome Aberration Assay in CHO Cells
Same experimental design as above for the CHO cells In Vitro Micronucleus Tests Human Lymphoblastoid Cell Line TK6 Cells This assay evaluates the test article for its potential to induce genetic damage manifested by induced frequencies of micronuclei in the dividing cell population. This assay is increasingly gaining attention in genetic toxicology for its simplicity and rapidness and is commonly known as CBMN (Cytokinesis-blocked micronucleus assay). An additional advantage is that a large sample size can be evaluated in this assay. Five logarithmic doses will be screened with concurrent positive and negative controls. CHO Cells This assay evaluates the test article for its potential to induce genetic damage manifested by induced frequencies of micronuclei in the dividing cell population. This assay is increasingly gaining attention in genetic toxicology for its simplicity and rapidness and commonly known as CBMN (Cytokinesis-blocked micronucleus assay). The additional advantage is that a large sample size can be evaluated in this assay. Five logarithmic doses will be screened with concurrent positive and negative controls In Vitro Aneuploidy Tests Human Blood Lymphocytes This assay detects chemically induced aneuploidy in vitro by evaluating treated human blood lymphocytes in cultures. Generally, the exponentially growing lymphocytes are treated with various doses of test article, solvent control and known positive compound. The aneuploidy inducing potency of test article is characterized using the CREST-staining/ FISH using a pancentromeric DNA probe. Human TK-6 Cell This assay detects chemically induced aneuploidy in vitro by evaluating treated human TK-6 cells in cultures. Generally, the exponentially growing cells are treated with various doses of test article, solvent control and known positive compound. The aneuploidy inducing potency of test article is characterized using the CREST-staining/ FISH using pancentromeric DNA probe. In Vivo Micronucleus Test Micronucleus Assay in Mouse Bone Marrow
Human Blood Lymphocytes This assay evaluates the test article for its potential to induce genetic damage manifested by induced frequencies of micronuclei in the dividing cell population. This assay is increasingly gaining attention in the genetic toxicology for its simplicity and rapidness and commonly known as CBMN (Cytokinesis-blocked micronucleus assay). The additional advantage is that a large sample size can be evaluated in this assay. Five logarithmic doses will be screened with concurrent positive and negative controls. CBMN Assay This assay detects chemically induced micronucleus in vitro by evaluating treated chinese hamster ovary (CHO) cells and human peripheral blood lymphocytes (HPBL) in cultures. Generally the exponentially growing CHO cells are treated with various doses of test article, solvent control and known positive compound. The range finding assay uses 8-10 doses with duplicate cultures per dose. Nuclear division index (NDI), apoptosis and necrosis determine the cytotoxicity. The genotoxicity is determined by induction of micronuclei in the binucleated cells. The definitive assay is performed with 4-5 doses in duplicate. Guidelines: OECD Draft Guideline 487, June 14, 2004 (1st Version). Blood Brain Barrier StudiesADMET GROUP performs studies using animal models to evaluate a chemicalís ability to pass through the Blood Brain Barrier (BBB). These studies are usually performed to evaluate orally administered small molecules. If the results of an initial pilot PK study indicate sufficient absorption and bio-availability of the compound in the plasma, the concentration of the drug and metabolites in the plasma and brain are determined at multiple time points after acute or repeat dosing of animals. The time course distribution of the drug and its metabolites in the central nervous system (CNS) and the plasma are determined to characterize the plasma kinetics and CNS / plasma ratios. The concentrations of both bound and free drug and drug metabolites are measured using appropriate analytical methods. A pharmacokinetic data analysis of plasma and brain concentrations of the drug using a non-compartmental approach is used to derive the following:
In Vitro CytotoxicityADMET GROUP provides a panel of In Vitro Cytotoxicity Tests, which are used to screen potential drugs to select lead compounds for further development. A variety of primary cells and cell lines from human and laboratory animals are used for cytotoxicity screening. These tests can be performed with as little as 1mg of test material depending on the types of tests. Each test includes concurrent positive and negative controls. These assays are used for testing drug candidates, formulations, coating materials for medical devices such as stents and industrial chemicals. In vitro hepatotoxicity screening: Liver toxicity is a major adverse drug effect. We provide hepatotoxicity screening using cryopreserved human and/or animal hepatocytes. Results are available 3- 5 business days after sample receipt in our laboratories.In vitro organ toxicity screening: Screening for liver, kidney, CNS, and vascular toxicity using primary hepatocytes, renal tubule cells, neurons / astrocytes, and endothelial cells, respectively. In vitro anticancer activity screening: We screen drug candidates for anticancer activities using the NCI cancer cell panel. Proprietary drug-resistant cell lines are also available for specific applications. Multiple endpoint studies: Cytotoxicity screening with multiple endpoints offers useful, mechanistic information. The endpoints developed in our laboratory includes the following: Cell viability: Cell viability can be evaluated via the measurement of cytoplasmic enzyme release (e.g. LDH), cellular ATP content, lysozomal activities such as neutral-red uptake and mitochondrial metabolism such as MTT metabolism. These endpoints are applicable to proliferating and nonproliferating cells.Specialized cytotoxicity studies: We also perform nonstandard cytotoxicity assays to determine specific mechanisms of toxicity. Examples of specialized assays are as follows: Steatosis: Measurement of lipid accumulation in hepatocytes. Steatosis is a common effect of hepatotoxic drugs.Δ BACK TO TOP | < CAPABILITIES INDEX In Vitro MetabolismEvaluation of Metabolic Stability of Test Article in Liver Microsomes or Hepatocytes from Man, Rat, and Dog: Metabolic stability is a key drug property, which is important for both drug administration regiment design as well as toxicity. Species comparison in metabolic stability allows the determination of which animal species is the most appropriate model for the estimation of human metabolic stability of the test article. The use of liver microsomes allows the evaluation of metabolic stability as results of phase I oxidation. The use of hepatocytes allows the evaluation of both phase I oxidation and phase II conjugation. Species Comparison of Metabolite Profiles of Test Article: Studies with Human, Rat, Mouse, Dog, and Monkey Hepatocytes/Liver Microsomes Metabolite profile comparison allows the selection of the most relevant animal species for drug properties related to human drug metabolism (e.g. safety studies). The animal species that produce a metabolite profile similar to human would be considered a relevant animal species. The use of liver microsomes allows the evaluation of metabolites formed by phase I oxidation (mainly P450 oxidation). The use of hepatocytes allows the evaluation of metabolites formed by all hepatic drug metabolizing enzyme pathways, including both phase I and phase II pathways. Drug-Drug Interaction Study: Determination of Metabolite Profile of Test Article in Human Hepatocytes The FDA recommends the evaluation of metabolites generated by a drug for the evaluation of drug-drug interactions. The identity of the metabolites allows the assignment of major metabolic pathways, which will facilitate experimental design for the evaluation of drug-drug interaction potential. For instance, if a drug forms phase 1 oxidation metabolites, then P450 pathways may be involved in its metabolism. Experiments concerning P450 inhibition, induction, and P450 pathway identification (see below) are studies that would need to be performed. Drug-Drug Interaction Study: Metabolic pathway identification of test article using cDNA-expressed P450 Isoforms / Liver Microsomes If a test article is metabolized by phase I oxidation, then it is necessary to assign P450 isoforms that are involved in its metabolism. This is performed using cDNA-expressed isoforms (using microsomes containing only one specific isoform) or via the evaluation of isoform-specific inhibitors of the metabolism of the test article using liver microsomes. This approach is recommended by the U. S. FDA. Drug-Drug Interaction Study: Evaluation of P450 inhibition potential of test articles in cDNA-expressed P450 Isoforms / liver microsomes / hepatocytes A major mechanism of drug-drug interaction is the inhibition of drug metabolizing enzymes by a drug, thereby inhibiting the metabolism of co-administered drugs, which are substrates of the inhibited pathways. This can be performed using cDNA microsomes or liver microsomes. The FDA recommends using two independent methods for the assessment of P450 inhibitory potential. Drug-Drug Interactions: Evaluation of P450 Induction Potential of Test Article in Human Hepatocytes Enzyme induction is a major mechanism of pharmacokinetic drug-drug interactions. A drug that induces a specific drug metabolizing enzyme (e.g. a specific P450 isoform) would have the potential to enhance the metabolism of a co-administered drug that is a substrate of the induced pathway. Enzyme induction studies are generally performed using human hepatocytes. This approach is recommended by the U.S. FDA. In Vitro Toxicity Screening and Mechanistic Toxicity Studies Drug toxicity is a major challenge for the drug industry. Using in vitro primary cells, especially those derived from human tissues (e.g. human hepatocytes), one can select against chemicals or chemical moieties that may have serious toxicology consequences. In vitro cytotoxicity assays using necrotic and apoptotic endpoints have been developed in our laboratory using human primary cells such as hepatocytes (hepatotoxicity), kidney proximal tubule epithelial cells (nephrotoxicity), astrocytes (neurotoxicity), endothelial cells (vascular toxicity), and airway epithelial cells (pulmonary toxicity). In Vitro Hepatocyte Toxicogenomics Toxicogenomics, the evaluation of the effect of a drug on gene expression profiles of the human genome, is considered a valuable technology for the definition of toxicological potential. Our laboratory has developed collaborative relationships with gene array companies to allow the provision of contract research service in this area. The combination of hepatocytes and toxicogenomics allows the definition of hepatotoxicity, for instance, possible mechanism of action, and potential to cause idiosyncratic hepatotoxicity. Δ BACK TO TOP | < CAPABILITIES INDEXGenetic Toxicology TestingThe genetic toxicology assays are performed in support of regulatory submissions throughout the world. Our protocols comply with ICH and OECD guidelines. We offer the following categories of assays:
Microbial MutationBacterial Cells ñ Salmonella / E. coli Reverse Mutation Tests (Ames Assay) This assay evaluates the test article for its potential to cause mutations in the histidine operon of Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and the tryptophan operon of Escheria coli strain WP2uvrA. The Dose Range Finding test with and without activation, at 7-8 dose levels, uses tester strains TA100 and WP2uvrA. The Mutagenicity test with and without activation at five dose levels uses all five tester strains. This also includes concurrent positive and solvent controls. Mammalian Cell Mutation TestsMammalian Cells - L5178Y TK-/+ Mouse Lymphoma Assay This assay is used to evaluate the test article for its potential to cause mutations at the thymidine kinase locus of L5178Y TK-/+ mouse lymphoma cells. This assay has been effective in detecting the mutagenic activity of chemicals from a wide range of classes. The Dose Range Finding test, with and without metabolic activation, is performed using 10 dose levels. The Definitive test is performed with and without activation at 8 dose levels, and at least 5 test article-treated cultures are monitored for suspension and clonal growth. Each test includes concurrent positive and negative controls. The treatment period is 4 hours, except for ICH Guideline protocols that include an additional 24-hour treatment period without activation. CHO/HGPRT and AS52/XPRT Locus Mutation Tests The CHO/HGPRT assay is used to test for the chemical induction of gene mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in cultured Chinese hamster ovary (CHO) cells. The AS52/XPRT assay evaluates the test article for its potential to cause genetic damage as manifested by induced gene mutation at the XPRT locus in cultured AS52 cells. Both of these assays have been effective in detecting the mutagenic activity of chemicals from a wide range of classes. The Dose Range Finding test, with and without metabolic activation, is performed at 10 dose levels. The Definitive test with and without activation is performed at 5 dose levels, with duplicate cultures and a parallel toxicity test. Up to 5 test article-treated duplicate cultures from each Group are cloned in the definitive and confirmative test. In Vitro and In Vivo Chromosome Aberration and Micronucleus Tests In Vitro Chromosome Aberration and In Vitro / In Vivo Micronucleus Assays Δ BACK TO TOP | < CAPABILITIES INDEX Mammalian Cell CytogeneticsChromosome Aberrations Assay in CHO / CHL Cells (In Vitro Cytogenetics)
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The Chromosome Aberration Assay in either Chinese hamster ovary (CHO) cells or Chinese hamster lung (CHL) cells is used to evaluate the test article for its potential to cause genetic damage as manifested by induced chromosome aberrations in cultured CHO cells. The assay is performed with and without metabolic activation. The Range Finding Test uses 8 to 10 dose levels in duplicate and the cells are treated for 3 hours, and cells are harvested 18 hours after treatment. Cytotoxicity is determined by the Relative Mitotic Index (RMI). The Definitive Tests are performed using 4 or more dose levels in duplicate. Treatments are for three hours and cells are collected 18 hours after treatment. A Confirmatory Test is necessary if the first trial is negative. The Confirmatory test (a second independent trial) is conducted without activation and with an 18-hour treatment and harvest. A full Confirmatory, with and without activation, is used when the first trial is equivocal. If the first trial is positive, then no Confirmatory test is necessary. Concurrent positive and solvent controls are used. Parallel toxicity is assessed by RMI. 200 metaphases are scored at least 3 doses and the controls. Guidelines: 1. OECD Guideline for the Testing of Chemicals, Nƒ. 473. In Vitro Mammalian Chromosome Aberration Test. Adopted July 21, 1997. 2. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Federal Register 61 (80):18198-18202,1996. Chromosome Aberration Assay in Human Peripheral Blood Lymphocytes (in vitro Cytogenetics) The Chromosome Aberrations assay in Human Peripheral Blood Lymphocytes (HPBL) is used to evaluate the test article for its potential to cause genetic damage as manifested by induced chromosome aberrations in human blood lymphocytes. The assay is performed with and without metabolic activation. The Range Finding Test uses 8 to 10 dose levels in duplicate. The cells are treated for 3 hours and harvested 21 hours after treatment. Cytotoxicity is determined by the Relative Mitotic Index (RMI). The Definitive test is performed using 4 or more dose levels in duplicate. Treatments are for three hours and cells are collected 21 hours after treatment. The Confirmatory (a second independent trial) is conducted without activation only for a 21 hour treatment and harvest. Concurrent positive and solvent controls are included with each trial. Parallel toxicity is assessed by RMI. 200 metaphases are scored from at least 3 doses and the controls. Guidelines: 1. OECD Guideline for the Testing of Chemicals, Nƒ. 473. In Vitro Mammalian Chromosome Aberration Test. Adopted July 21,1997. 2. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Federal Register 61 (80):18198-18202,1996. Micronucleus Assay The Micronucleus Assay uses either rats or mice to evaluate the test article for its potential to cause genetic damage as manifested by induced micronucleated polychromatic erythrocytes (MPCE) in bone marrow cells of the animal used. The Micronucleus assay has been effective in detecting the clastogenic activity of chemicals from a wide range of classes. The assay is performed with at least 3 dose levels, 5 animals per sex per dose level, and 2 or 3 collection time points. Concurrent positive and vehicle controls are included and 2000 polychromatic erythrocytes (PCE) scored per animal. Guidelines: 1. OECD Guideline for the Testing of Chemicals, Nƒ. 474. Mammalian Erythrocyte Micronucleus Test. Adopted July 21,1997. 2. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Federal Register 61(80):18198-18202,1996. Mouse Bone Marrow Aneuploidy / Micronucleus Assay This Assay uses male mice to evaluate the test article for its potential to cause genetic damage as manifested by induced aneuploidy and micronuclei formation in mouse bone marrow cells. The Micronucleus Assay in rodents has been widely used to determine the clastogenic potential of a variety of chemicals. This Assay has the capability to distinguish micronuclei-containing centric chromosomes (micronuclei with kinetochores) from those containing acentric fragments (micronuclei without kinetochores), thereby differentiating between chemicals that are aneuploidy-inducing agents and clastogens. The Range Finding Test includes 6 dose levels and a vehicle control. The Assay includes two treatments 24 hours apart at 3 dose levels and harvest 24 hours after the last treatment. Two positive controls and vehicle control are also included in the assay. 2000 polychromatic erythrocytes (PCE) are scored for micronucleated polychromatic erythrocytes (MPCE) per animal, and the micronuclei are scored for the presence or absence of kinotochores, which are made visible using immunofluoresent staining techniques. 200 erythrocytes are scored for the percentage of PCE per animal as an indication of toxicity. Guidelines: 1. OECD Guideline for the Testing of Chemicals, Nƒ 474. Mammalian Erythrocyte Micronucleus Test. Adopted July 21, 1997. 2. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Federal Register 61 (80): 18198-18202,1996. Chromosome Aberration Assays in Mouse / Rat Bone Marrow Cells The Chromosome Aberration Assay uses either rats or mice to evaluate the test article for its potential to cause genetic damage as manifested by induced chromosome aberrations in bone marrow cells of the animal used. This assay has been effective in detecting the clastogenic activity of chemicals from a wide range of classes. Performed with at least 3 dose levels, 5 animals per sex per treatment Group, multiple harvest times and concurrent positive and vehicle controls. One hundred metaphases are scored per animal for chromosome aberrations and to determine the Relative Mitotic Index (RMI). Guidelines: 1. OECD Guideline for the Testing of Chemicals, Nƒ. 475. Adopted July 21, 1997. Mammalian Bone Marrow Chromosome Aberration Test. 2. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Federal Register 61 (80):18198-18202,1996. Sister Chromatid Exchange Tests In vitro The Sister Chromatid Exchange (SCE) Assay is used to evaluate the test article for its potential to cause genetic damage as manifested by sister chromatid exchange. This test uses Chinese hamster ovary (CHO) cells or human peripheral blood lymphocytes (HPBL). It is performed with and without metabolic activation. The Range Finding Test uses 8 to 10 dose levels with duplicate cultures per dose. Cytotoxicity is determined by the Relative Mitotic Index (RMI). The Definitive test is performed with 4 or more dose levels in duplicate. Concurrent positive and solvent controls are used. Parallel toxicity is assessed by RMI. Fifty (50) metaphases are scored from at least 4 doses and the controls. OECD Guidelines for the Testing of Chemicals, Nƒ 479. Adopted October 23, 1986. In Vitro Sister Chromatid Exchange Assay in Mammalian Cells. In vivo SCE This SCE test is performed with mouse bone marrow (50 animals). Each test includes 3 dose levels with concurrent positive and vehicle controls. Five males and five females are treated per Group and harvested 24 hours after treatment. Fifty (50) metaphases are scored per animal for SCEs. One hundred (100) metaphases are scored per animal for all doses to determine the Relative Mitotic Index. Unscheduled DNA Synthesis (DNA Repair) TestsThe Unscheduled DNA Synthesis (UDS) Assay is performed with rat hepatocytes. It is used to evaluate the test article for its potential to damage DNA as manifested by induced DNA synthesis to repair the damage. The UDS Assay can be performed either in vitro or in vivo / in vitro. In the first case, cells are exposed to the test article in culture. In the second case, rats are exposed to the test article and after four-sixteen hours hepatocytes are harvested from their livers and placed in culture. In both cases the cells are grown in the presence of tritiated thymidine and DNA synthesis is subsequently measured by the presence of tritiated thymidine in the nuclear DNA. In Vitro UDS
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The Range Finding Test uses 10 dose levels. Cells are treated for 18 hours, after which toxicity is measured by viable cell counts. Toxicity is expressed as Relative Cell Survival by comparing the treated Groups with the negative control cultures. The Definitive Test uses 5 or more dose levels in triplicate. In addition, three replicate cultures per dose are treated to monitor toxicity. Treatments are for 18 hours in the presence of tritiated thymidine. After exposure the Relative Cell Survival of each culture used to assess toxicity is determined. The cells from cultures used to determine UDS are fixed and processed for scoring for UDS. One hundred fifty nuclei per Group are scored for UDS. In Vivo / In Vitro UDS The Range Finding Test uses 5 to 6 dose levels; three animals per dose. The animals are treated according to the method and regimen elected by the Sponsor. The animals are observed for three days and toxicity is evaluated based on the number of deaths, loss of body weight, and other clinical symptoms that may be evident. Each assay consists of three test article doses, one vehicle control and one positive control. Three animals per dose per harvest time are used for the assay. At harvest time, hepatocytes are prepared and three cultures per animal are incubated in the presence of tritiated thymidine for 18 hours. The cells from cultures used to determine UDS are fixed and processed for scoring. One hundred fifty nuclei per animal are scored for UDS. Cell Transformation TestBALB/c-3T3 Cells
This assay evaluates the test article for its potential to cause genetic damage as manifested by induced morphological cell transformation in cultures of BALB/c-3T3 cells. These cells have been used extensively and have been effective in detecting carcinogenic chemicals from a wide range of classes. The assays are performed without activation or with and without activation using rat liver S9. A Range Finding Test is performed to select test article concentrations for the Cell Transformation Assay using ten doses. Three or four test article concentrations are evaluated in the assay. A minimum of 20 plates per test article concentration are seeded with cells and exposed to the test article for eighteen hours. The cells are cultured for a period of 5 to 6 weeks after which the cells are fixed and scored for Type III foci. Type III foci are composed of highly polar, fibroblastic cells that form colonies that are multilayered and contain crisscrossed arrays of densely stained cells. Analytical and Bioanalytical ServicesAnalytical method development and method validation in support of dosing samples label content confirmation, bioanalytical analysis and metabolism studies utilizing the following analytical instrumentation:
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