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Safety Studies - Toxicology Protocols

The following are standard sets of protocols for a number of studies performed by SRI Biosciences. Other routes of administration and study designs are available upon request.

Mammalian Toxicology
Acute toxicity studies
Repeat-dose toxicity studies
Other studies

Molecular and Genetic Toxicology
Microbial mutagenesis (Ames Test)
Gene mutation in mammalian cells in vitro (Mouse lymphoma assay)

In vitro cytogenetics with CHO or human lymphocytes
In vivo cytogenetics

Gene Mutation Assays
Transgenic rodent mutagenesis assay

Rodent Micronucleus Assay
Bone marrow erythrocyte assays
Mouse peripheral blood assay

Primary DNA Damage Assays
Unscheduled DNA synthesis

Recommended genetic toxicology battery for ICH guidelines

Immunotoxicology Studies

Mammalian Toxicology

Acute Toxicity Studies

M101 Acute Limit Test (Rodents)

  • One group of 5 male and 5 female rats (or mice)

  • Single administration of test substance at 5 g/kg by gavage or i.p. injection (other routes available)

  • Animals are weighed weekly and observed for clinical signs of toxicity and death for 2 weeks after treatment

  • Necropsy is performed on all animals that die and on all survivors at the end of the 2-week study

  • No tissues are preserved; necropsy findings are recorded

M102 Acute Toxicity Study (Rats or Mice)

  • Groups of 5 male and 5 female rats
  • Single administration of test substance at 3 dose levels by gavage or i.p. injection (other routes available)
  • Animals are weighed weekly and observed for clinical signs of toxicity and death for 2 weeks after treatment
  • Necropsy is performed on all animals that die and on all survivors at the end of the 2-week study
  • No tissues are preserved; necropsy findings are recorded

M120 Dose Escalation Study (Dog)

  • Single group of 1 male and 1 female beagle dog
  • Single administration of test substance by gavage (other routes available)
  • Repeat with same dogs, escalating dose every 4 days; total of 4 escalations
  • Animals are weighed daily during escalation cycle, and observed for clinical signs of toxicity and death daily for 4 days after treatment
  • Non-terminal study; no necropsy
  • Non-GLP

M103 Acute Dermal Limit Test

  • One group of 5 male and 5 female rabbits
  • A single 24-hr dermal exposure to 2 g/kg
  • Animals are weighed weekly and observed for clinical signs of toxicity and death for 2 weeks after treatment
  • Necropsy is performed on all animals that die and on all survivors at the end of the 2-week study
  • No tissues are preserved; necropsy findings are recorded

M104 Acute Dermal (LD50) Study

  • Groups of 5 male and 5 female rabbits
  • A single 24-hr dermal exposure to 3 dose levels of the test substance
  • Animals are weighed weekly and observed for clinical signs of toxicity and death for 2 weeks after treatment
  • Necropsy is performed on all animals that die and on all survivors at the end of the 2-week study
  • No tissues are preserved; necropsy findings are recorded

M105 Primary Dermal Irritation Study

  • The shaved intact skin of 6 rabbits is exposed to the test substance for 4 hr
  • After the exposure period, the skin is graded for irritation at 1, 24, 48, and 72 hr
  • Observation period may be extended for up to 21 days to evaluate the reversibility of the effects observed.

M106 Primary Eye Irritation Study

  • Test substance is instilled into one eye of each of 6 rabbits (unwashed eyes)
  • The cornea, iris, and conjunctival tissue of the treated eyes are graded for irritation effects at 1, 24, 48, and 72 hr after instillation
  • Observation period may be extended for up to 21 days to evaluate the reversibility of the effects observed.

M106-A

  • Same as M106 but with 3 additional rabbits with washed eyes
  • Observation period may be extended for up to 21 days to evaluate the reversibility of the effects observed.

M110 Local Lymph Node Assay in Mouse

  • Groups of 5 female CBA/CA mice are administered the test article at 4 dose levels on the dorsum of each ear for 3 consecutive days; a vehicle and positive control are included
  • On Day 6, mice are injected with 3H-thymidine and euthanized 5 hr later; lymph nodes draining the ears are removed and analyzed for incorporation of 3H-thymidine into proliferating T cells that entered the lymph nodes in response to the test article
  • The degree of 3H-thymidine incorporation is a measure of the ability of the test article to elicit an immune response

M115 Rabbit Vaginal Irritation Assay

  • Five groups of 5 female rabbits; control, positive control, 3 dose levels
  • Daily vaginal administration for 10 days
  • Daily clinical observations and visual scoring of genital region
  • Gross necropsy and preservation of reproductive organs and bladder
  • Histological evaluation of all preserved tissues

  • Calculation of mean irritation score (MIS) based on histopathologic evaluation of vagina

Repeat-Dose Toxicity Studies

M204 14-Day Oral Toxicity Study (Rodents)

  • Groups of 5 males and 5 females are treated with the test substance by gavage at 3 dose levels for 14 days; a vehicle control group is included (40 animals total)

  • Study measurements include daily clinical observations, weekly body weights, and feed consumption

  • Hematology and serum clinical chemistry evaluations at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

M204-H

  • Same as M204, with the following changes:

  • Full histopathological examinations are conducted on the control and high-dose groups and on any unscheduled deaths; target organs are examined in the intermediate groups

M204-RH

  • Same as M204-H, with the following changes:

  • Additional recovery group with 5 males and 5 females per dose level (80 rats total)

  • Recovery group evaluated on Day 28

M205 14-Day Oral Toxicity Study (Dogs)

  • Groups of 2 males and 2 females are treated with the test substance by capsule or gavage at 3 dose levels for 14 days; a vehicle control group is included (16 dogs total)

  • Study measurements include daily clinical observations, weekly body weights, and feed consumption

  • Hematology and serum clinical chemistry evaluations prestudy and at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

M205-H

  • Same as M205, with the following changes

  • Full histopathological examinations are conducted on high dose and control animals; target organs are examined in the intermediate groups

M205-RH

  • Same as M205-H, with the following changes:

  • 2 males and 2 females evaluated after 14 days; recovery group of 1 male and 1 female evaluated on Day 28 (24 dogs total)

M201 28-Day Oral Toxicity Study (Rodents)

  • Groups of 10 males and 10 females are treated with the test substance by gavage at 3 dose levels for 28 days; a vehicle control group is included (80 rats total)

  • Study measurements include daily clinical observations, weekly body weights and feed consumption, and ophthalmological examination pretest and before study termination

  • Hematology, serum clinical chemistry and urinalysis evaluations at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

  • Full histopathological examinations are conducted on the control and high-dose groups and on any unscheduled deaths

  • Target organs are examined in the intermediate groups

M201-R

  • Same as M201, with the following changes:

  • Additional recovery group with 5 males and 5 females per dose level (120 rats total)

  • Recovery group evaluated on Day 42

M202 28-Day Oral Toxicity Study (Dog)

  • Groups of 4 males and 4 females are treated with the test substance via capsule at 3 dose levels for 28 days; a vehicle control group is included

  • Study measurements include daily clinical observations, weekly body weights and feed consumption; ophthalmological examination pretest and before study termination

  • Hematology, serum clinical chemistry, and urinalysis at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

  • Full histopathological examinations are conducted on all animals

M202-R

  • Same as M202, with the following changes:

  • 3 males and 3 females evaluated after 28 days; recovery group of 2 males and 2 females evaluated on Day 42 (40 dogs total)

M211 90-Day Oral Toxicity Study (Rodent)

  • Groups of 10 males and 10 females are treated with the test substance by gavage at 3 dose levels for 90 days; a vehicle control group is included

  • Study measurements include daily clinical observations, weekly body weights and feed consumption, and ophthalmological examination pretest and before study termination

  • Hematology and serum clinical chemistry at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

  • Full histopathological examinations are conducted on the control and high-dose groups and on any unscheduled deaths

  • Target organs are examined in the intermediate groups

M211-20

  • Same as M211 except 20 males and 20 females per treatment group

M212 90-Day Oral Toxicity Study (Dog)

  • Groups of 4 males and 4 females are treated with the test substance via capsule at 3 dose levels daily for 90 days; a vehicle control group is included

  • Study measurements include daily clinical observations, weekly body weights and feed consumption, and ophthalmological examination pretest and before study termination

  • Hematology, serum clinical chemistry, and urinalysis at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

  • Full histopathological examinations are conducted on all animals

M215 90-Day Dermal Toxicity Study (Mouse)

  • Groups of 20 males and 20 females are treated with the test substance by dermal application at 3 dose levels for 90 days; a vehicle control group is included; a satellite toxicokinetics group of 6 males and 6 females is included for plasma drug evaluations

  • Study measurements include daily clinical observations, daily Draize irritation scores at application site, weekly body weights and food consumption

  • Plasma drug evaluations at approximately 4 time points on Days 1 and 28

  • Hematology and serum clinical chemistry at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

  • Full histopathological examinations are conducted on the control and high-dose groups and on any unscheduled deaths

  • Target organs are examined in the intermediate groups

M213 Two-Year Chronic Toxicity/Carcinogenicity Study (Rats; Dosed Food or Water)

  • Groups of 50 males and 50 females are treated with the test substance by dosed feed or water at 3 dose levels for 104 weeks; an untreated feed/water control group is also included

  • A satellite group of 20 males and 20 females at the high dose and 10 males and 10 females in the control group are also included for evaluation of non-neoplastic toxicity for 52 weeks

  • Study measurements include weekly clinical observations; body weights weekly for 13 weeks and monthly thereafter; weekly food and water consumption consumption, and ophthalmological examination pretest and before study termination

  • Hematology and serum clinical chemistry, on all animals at termination and in 10/sex/dose at 3, 6, 12, and 18 months; urinalysis in 10/sex/dose at 12 months and at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

  • Full histopathological examinations are conducted on the control and high-dose groups (main study and satellite groups) and on any unscheduled deaths

  • Target organs are examined in the intermediate groups

M214 6-Month Carcinogenicity Study in p53+/- Transgenic Mice

  • Groups of 15 males and 15 females are treated with the test substance by dosed feed or water at 3 dose levels for 104 weeks; an untreated feed/water control group is also included

  • Study measurements include weekly clinical observations; body weights weekly; weekly food and water consumption consumption

  • Hematology and serum clinical chemistry on all animals at termination

  • Complete necropsy is performed on any animal that dies or upon termination, and weights are recorded for major organs

  • Full histopathological examinations are conducted on the control and high-dose groups and on any unscheduled deaths

  • Target organs are examined in the intermediate groups

Other Studies
SRI also has the capability to conduct the following studies or additional evaluations:

  • Toxicokinetics

  • Electrocardiographic Evaluations

  • Continuous Intravenous Infusion

  • Medical Device Evaluations

  • Mini- and Micro-pig Studies

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Molecular and Genetic Toxicology Studies

Microbial Mutagenesis

G101 Salmonella/Microsome Plate Incorporation Assay (Ames Test)

  • Preliminary dose range finding experiment with and without metabolic activation
  • Mutagenicity experiment conducted with 4 strains at 6 dose levels, with and without metabolic activation
  • Three plates per dose; positive, negative, and sterility controls

G101-R

  • Same design as G101 with independent repeat experiment performed

G102 Salmonella/Microsome Preincubation Assay (Ames Test)

  • Same design as G101
  • Includes a 20-min preincubation step

G102-R

  • Same design as G102 with independent repeat experiment performed

G103 Salmonella/E. coli Reverse Mutation Assay (using four Salmonella and one E. coli strains)

  • Preliminary dose range finding experiment with and without metabolic activation
  • Six dose levels, with and without metabolic activation
  • Three plates per dose; positive, negative, and sterility controls
  • Use of plate incorporation or preincubation procedures
  • Independent repeat experiment performed

G104 Salmonella/E. coli Reverse Mutation Assay (using four Salmonella and two E. coli strains)

  • Preliminary dose range finding experiment with and without metabolic activation
  • Six dose levels, with and without metabolic activation
  • Three plates per dose; positive, negative, and sterility controls
  • Use of plate incorporation procedure for first assay and preincubation for repeat experiment

G105

  • Same design as G104 but with five Salmonella strains

G106

  • Salmonella assay with volatile liquids and gases, reductive conditions, or microsuspension assay (Kado method)

Gene Mutation in Mammalian Cells In Vitro

G201 Mouse Lymphoma L5178Y/tk+/- Cell Gene Mutation Assay

  • Analysis with and without metabolic activation
  • Preliminary dose range finding experiment (4 hour exposure) to determine toxicity
  • Mutagenesis experiment (4 hour exposure) quantitating the frequency of triflurothymidine-resistant cells
  • Selection of mutants from at least five test article concentrations, 3 x 106 cells per culture
  • Two independent cultures per concentration
  • Solvent and positive control cultures
  • Analysis of colony size distribution of selected cultures, including controls

G201-R

  • Same as G201, with the following changes:
  • Range finding experiment conducted with 4 and 24 hour (without metabolic activation) exposures
  • 24-hour exposure without metabolic activation for test articles evaluated as negative in the initial mutagenesis experiment or repeated with 4 hour exposure for test articles evaluated as being positive in the initial mutagenesis experiment
  • With replicate mutagenesis experiments

G202 Mouse Lymphoma L5178Y/tk+/- Cell Gene Mutation Screening Assay

  • Mutagenesis experiment with and without metabolic activation
  • Up to 15 test article concentrations, single cultures per concentration, with replicate solvent and positive controls
  • Selection of mutants from at least six test article concentrations
  • Optional in situ protocol for expression and selection of mutants

G203 Chinese Hamster Ovary or V79 Cell Mutation Assay at the hprt Locus

  • Analysis with and without metabolic activation
  • Preliminary dose range finding experiment to determine toxicity
  • Mutagenesis experiment quantitating the frequency of thioguanine-resistant cells
  • Selection of mutants from at least five test article concentrations, 2-3 x 106 cells per culture

G203R

  • Same as G203, with the following changes:
  • With replicate mutagenesis experiment with and without metabolic activation

In Vitro Cytogenetics with CHO or human lymphocytes

G401 Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells

  • Preliminary cytotoxicity experiment using 5 concentrations and solvent control
  • Definitive experiment with at least 3 concentrations and positive and solvent controls
  • 100 metaphase cells scored per concentration
  • Experiments conducted with and without metabolic activation using duplicate cultures

G401-2

  • Same as G401 but with 200 metaphases per concentration

G401-R

  • Same as G401 but with repeated experiment (100 metaphases)

G401-R2

  • Same as G401 but with repeated experiment and 200 metaphases per concentration

G401-R2H

  • Same as G401-R2 but with an additional harvest time in the repeated experiment

G402 Chromosomal Aberrations in Human Lymphocytes

  • Preliminary cytotoxicity experiment with 5 concentrations and solvent control
  • Definitive experiment with at least 3 concentrations and positive and solvent controls
  • 100 metaphase cells scored per concentration
  • Experiments conducted with and without metabolic activation using duplicate cultures

G402-2

  • Same as G402 but with 200 metaphases per concentration

G402-R

  • Same as G402 but with repeated experiment (100 metaphases)

G402-R2

  • Same as G402 but with repeated experiment and 200 metaphases per concentration

G402-R2H

  • Same as G402-R2 but with an additional harvest time in the repeated experiment

G403 Sister Chromatid Exchanges in Chinese Hamster Ovary (CHO) Cells

  • Preliminary cell-cycle kinetics experiment with 5 concentrations and solvent control
  • Definitive experiment with at least 3 concentrations and positive and solvent controls
  • 50 cells scored per concentration for SCE
  • Cell harvest delay if indicated
  • Experiments conducted with and without metabolic activation using duplicate cultures

In Vivo Cytogenetics

G501 Chromosomal Aberrations in Rodent Bone Marrow Cells

  • Single-exposure protocol
  • Three dose levels and positive and vehicle controls
  • Three time points (6, 12, and 24 hr)
  • Five males and 5 females per dose level per time point
  • Fifty cells in metaphase scored per animal (110 animals)

Other Endpoints

G502 Single-Exposure Bone Marrow Cytotoxicity Assay

  • Five dose levels and vehicle controls, 24-hr time point
  • Three males and 3 females per dose level
  • Mitotic index based on 1000 cells per animal

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Gene Mutation Assays

Transgenic Rodent Mutagenesis Assay

G301 Big Blue® Rodent Mutagenesis Assay (Somatic Tissues)

  • Available in B6C3F1 mouse, C57BL/6 mouse or F-344 rat (male or female)
  • 28 daily doses followed by 3-day expression (other dosing or expression periods optional)
  • Three dose levels plus vehicle control
  • Five animals per dose group (20 animals total)
  • Includes preliminary range-finding experiment in nontransgenic animals using 3 animals/dose at 5 doses
  • Available for liver, kidney, spleen, brain, lung; inquire about availability of other tissues
  • 200,000 plaques/tissue, 20 tissues total
  • Available in Lac I or CII gene

G303 Processing of Tissues for Big Blue® Rodent Assay

  • Mutagenesis analysis including DNA isolation, packaging, plating, counting 100,000 plaques
    • Lac I
    • CII

Other Services Available

  • DNA Sequencing of Mutants
  • Other Transgenic Mutation Assays

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Rodent Micronucleus Assay

Bone Marrow Erythrocyte Assays

G601 Three-Dose Administrations, One-Sample Protocol

  • Available in rat or mouse
  • Includes preliminary range-finding experiment using 3 animals/dose at 5 dose levels
  • Acridine orange stain used to unequivocally differentiate DNA from RNA and to eliminate basophil-derived artifacts
  • Ratio of PCE/NCE (cytotoxicity index) determined based on 200 RBC
  • Three daily doses at 24-hr intervals
  • Definitive experiment using three dose levels plus vehicle and positive controls
  • Sample 18 to 24 hr after last dose administration
  • Five males and 5 females per dose group; positive control in one sex
  • 1,000 PCE per animal scored for micronuclei

G601-2

  • Same as G601, but scoring 2,000 PCE per animal

G602 Two-Dose Administrations, Two-Sample Protocol

  • Same design as G601 but 2 daily doses at a 24-hr interval, followed by 2 samples at 24 and 48 hr after the last dose administration
  • Positive control (single sex) at one sampling time

G602-2

  • Same as G602, but scoring 2,000 PCE per animal

G603 One-Dose Administration, Three-Sample Protocol

  • Same design as G601 but 1-dose administration, followed by 3 samples at 24, 48, and 72 hr after the dose administration
  • Positive control (single sex) at one sampling time

G603-2

  • Same as G603, but scoring 2,000 PCE per animal

G604-2 One-Dose Administration, Two-Sample Protocol

  • Same design as G601 but 1-dose administration, followed by 2 samples at 24 and 48 hours
  • Positive control (single sex) at one sampling time
  • 2,000 PCE per amount scored for micronuclei

Mouse Peripheral Blood Assay

G605 Three-Dose Administrations, One-Sample Protocol

  • Three daily doses at 24-hr intervals
  • Three dose levels plus vehicle and positive controls
  • Sample 24 hr after last dose administration
  • 5 males and 5 females per dose group; positive control in one sex
  • Includes preliminary range-finding experiment at 5 dose levels
  • Acridine orange stain used to unequivocally differentiate DNA from RNA and to eliminate basophil-derived artifacts
  • Ratio PCE/NCE (cytotoxicity index) based on 5,000 RBC
  • 1,000 PCE per animal scored for micronuclei

Sample Evaluation of Prepared Micronucleus Slides

  • Unstained smears of blood or bone marrow may be prepared by the client from animals exposed during toxicity assays. SRI will advise on appropriate tissue and sampling time and will stain and score samples. Slides already stained with Giemsa or other stains may also be acceptable. Free evaluation of acceptability of preparation. Minimum of 10 slides.

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Primary DNA Damage Assays

Unscheduled DNA Synthesis (UDS)

G701 In Vitro Hepatocyte UDS Assay (Rodent)

  • Ten concentrations tested with positive, solvent, and media controls
  • 30 cells scored per slide, 3 slides per concentration
  • Concurrent toxicity evaluation

G701-R

  • Same as G701 but including replicate experiment with at least 5 concentrations

G702 In Vitro Hepatocyte UDS Assay (Human)

  • Ten concentrations tested with positive, solvent, and media controls
  • 35 cells scored per slide, 3 slides per concentration
  • Concurrent toxicity evaluation

G702-R

  • Same as G702 but including replicate experiment from a different human liver with at least 5 concentrations

G703 In Vivo-In Vitro Hepatocyte UDS Assay

  • Available in rat or mouse of either sex
  • Three doses, single time point (16 hr)
  • Positive and negative control groups
  • Minimum of 3 animals per group
  • 105 cells scored per animal

G703-A

  • Same as G703 but including 3 doses at additional time point (e.g., 2 hr)

Other Studies Available

In vitro UDS analysis in hepatocytes from non-rodent species

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Recommended Genetic Toxicology Battery for ICH Guidelines

Microbial Mutagenesis

G103 Salmonella/E. coli Reverse Mutation Assay (using four Salmonella and one E. coli strains) - Ames Test

  • Preliminary dose range finding experiment with and without metabolic activation
  • Six dose levels, with and without metabolic activation
  • Three plates per dose; positive, negative, and sterility controls
  • Use of plate incorporation or preincubation procedures
  • Independent repeat experiment performed

Gene Mutation in Mammalian Cells In Vitro

G201-R Mouse Lymphoma L5178Y/tk+/- Cell Gene Mutation Assay

  • Analysis with and without metabolic activation
  • Range finding experiment conducted with 4 and 24 hour (without metabolic activation) exposures
  • Mutagenesis experiment (4 hour exposure) quantitating the frequency of triflurothymidine-resistant cells
  • Selection of mutants from at least five test article concentrations, 3 x 106 cells per culture
  • Two independent cultures per concentration
  • Solvent and positive control cultures
  • Analysis of colony size distribution of selected cultures, including controls
  • 24-hour exposure without metabolic activation for test articles evaluated as negative in the initial mutagenesis experiment or repeated with 4-hour exposure for test articles evaluated as being positive in the initial mutagenesis experiment
In Vitro Cytogenetics (choose one of the following)

G401-R2 Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells

  • Preliminary cytotoxicity experiment using 5 concentrations and solvent control
  • Experiments performed on duplicate cultures with at least 3 concentrations and positive and solvent controls
  • Initial experiment performed with 3-hour exposure with and without metabolic activation and harvest time of 1.5 cell cycle (20 hour)
  • If negative results are obtained in the initial experiment, then an experiment is performed with 21-hour treatment without metabolic activation and 3-hour treatment with metabolic activation. If positive results are obtained in the initial experiment, then results are confirmed using the conditions of the initial experiment
  • 200 metaphase cells scored per concentration are analyzed for structural aberrations and polyploidy

G402-R2 Chromosomal Aberrations in Human Lymphocytes

  • Preliminary cytotoxicity assay with 5 concentrations and solvent control
  • Experiments performed on duplicate cultures with at least 3 concentrations and positive and solvent controls
  • Initial experiment performed with 3-hour exposure with and without metabolic activation and harvest time of 1.5 cell cycle (22-24 hour)
  • If negative results are obtained in the initial experiment, then an experiment is performed with 24-hour treatment without metabolic activation and 3-hour treatment with metabolic activation. If positive results are obtained in the initial experiment, then results are confirmed using the conditions of the initial experiment
  • 200 metaphase cells scored per concentration are analyzed for structural aberrations and polyploidy
Bone Marrow Erythrocyte Assays

G604-2

  • Available in rat or mouse
  • Includes preliminary range-finding experiment using 3 animals/sex/dose at 5 dose levels
  • Acridine orange stain used to unequivocally differentiate DNA from RNA and to eliminate basophile-derived artifacts
  • Ratio of PCE/NCE (cytotoxicity index) determined based on 200 RBC
  • Definitive experiment using three dose levels with concurrent vehicle and positive controls
  • Single dosing regimen with sampling times at 24 and 48 hour after last administration of dose
  • Five males and five females per dose group; positive controls in one sex
  • 2000 PCE per animal scored for micronuclei

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Immunotoxicology Studies

M302 30-Day Immunotoxicology Study in BALB/c Mice

  • Groups of 12 female mice treated with test substance at 3 dose levels for 30 days; one positive control group and one vehicle control group is included

  • Daily clinical observations and weekly body weights

  • Gross necropsy on any animal that dies or at termination. This will include examination of all tissues and weights of spleen and thymus

  • Six mice from each group will be immunized with KLH at day 27 to induce antibody production

  • Humoral immune response in immunized mice - ELISA to measure anti-KLH IgM and IgG

  • Hematology and splenic lymphocyte immunophenotyping in non-immunized mice at termination

  • Cellular immune response in non-immunized mice - T and B cell proliferation in response to stimulation with Con A and LPS - Mixed lymphocyte reaction (MLR) on spleen cells

Options:

  • Addition of in vitro cell-mediated cytotoxicity

  • Addition of in vivo cell-mediated cytotoxicity

M303 Host Resistance Assays in Mouse

  • Listeria monocytogenes

  • Streptococcus pneumoniae

  • Both assays listed above include:

    • 3 weeks of dosing prior to infection

    • 15 mice per group

    • 2 doses of test substance + controls

    • Monitor for mortality

Customized Immunological Studies

  • Histopathology

  • ELISA or bioassays for cytokine production

  • MHC expression

  • Macrophage phagocytosis

  • Apoptosis

 

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     Last Updated Jul 17, 2008

 

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