Abstract for TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data

Integration of Multiple Endpoints in Toxicology Studies: NTP Perspective

Rajendra S. Chhabra
National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

NTP Commitment

Develop and validate mechanistically-based alternative test methods such as computerized modeling and prediction systems, genetically-engineered cell systems, tissue cultures, transgenic animals, and non-mammalian species.
Refine existing test methods to utilize new scientific information and techniques that improve their usefulness in risk assessment.

NTP Toxicity and Carcinogenicity Studies

Designed to seek maximum information by using reduced (optimum) numbers of animals, replacement of animal testing with non-animal methods whenever possible, and refinement of animal procedures to minimize pain and distress.
In general, the chemicals selected for carcinogenic evaluation are studied in a sequence of acute (single exposure), subacute (14-day exposure), subchronic (90-day exposure) and chronic (2-year exposure) studies.
Most of the studies are performed in Fischer 344 rats and B6C3F1 mice.

Acute Toxicity Studies

NTP has discontinued conduct of acute toxicity studies since 1980.
The dose levels for 14-day toxicity studies are estimated from information available in the literature.
If information does not exist in the literature, then dose levels are extrapolated from the data on structurally related chemicals.
In rare situations when an acute toxicity study is essential, the acute toxic class method, a stepwise procedure with the use of 3 animals per step is used rather than the traditional LD₅₀ test method.

14-Day Toxicity Studies

If sufficient information is available in the literature on the toxicity of the chemicals being studied, the 14-day studies are not performed.
In such instances, the dose levels for 90-day studies are selected from the information available in the literature.

Cost and Animal Use Reduction Analysis: Review of NTP 14-Day Toxicity Study Database

The objective was to evaluate if the number of dose groups can be reduced from five to three. A sample of 30 14-day studies was randomly selected from the recently reported toxicology reports. Each study had five treated groups plus control. The studies were done in rats and mice of both sexes. The following parameters were analyzed from each study:

Major Findings:

Dose Groups 1-5 (lowest to highest)
Mortality	0	1	1	5	10
BW decreases	0	1	3	5	9
Toxicity	1	2	5	8	10

Recommendations

Use three dose groups plus control
Use four fold difference in dose levels instead of two

Benefits

Saving of 40 animals per study
Saving of about 20% cost

90-Day Toxicity Study Endpoints

Sperm Morphology and Vaginal Cytology Examination (SMVCE)
Micronucleated Erythrocyte Frequencies
Clinical Pathology
Immunotoxicity
Neurotoxiciy Evaluations
Biochemical/Mechanistic Aspects
Molecular Toxicology

Sperm Morphology and Vaginal Cytology Examination (SMVCE)

These evaluations are used as a screen for potential reproductive toxicants and are performed in core animals of all 90-day studies.
SMVCE endpoints include epididymal sperm concentration, motility, and morphology, vaginal cytology, and male reproductive organ weights.
Analysis of the database suggests these endpoints can provide a valuable preliminary indication of the likely reproductive toxicity.

Micronucleated Erythrocyte Frequencies

The frequencies of micronucleated erythrocytes in peripheral blood samples from mice are measured routinely as an indicator of chromosomal damage in all 90-day studies.
Eleven chemicals out of about 100 chemicals positive for micronucleus assay in the 90-day studies were also positive in 2-year carcinogenicity tests, suggesting a strong predictive value of this test.

Clinical Pathology

Hematology and clinical chemistry analysis in rats are performed on day 3, 21 and 90 in all 90-day toxicity studies. Additional animals are incorporated for day 3 and day 21 evaluations.
For day 90 evaluations, blood samples from the core animals are collected at the termination of the studies.
Evaluation of the database suggests that these endpoints are useful in identification of target organ toxicity, especially for liver and kidney.

Immunotoxicity

Each 90-day study includes histology of thymus, lymph node, and spleen; spleen and thymus weights; complete blood count and differential; and splenic cellularity.
Several alternate tests are under development such as in vitro Mishell Dutton assay, basal serum immunoglobulin levels as predictor of immunotoxicity in short term studies, and a combined assay for detecting irritation and sensitization potential.

Neurotoxiciy Evaluations

The functional endpoints such as behavior are favored in the initial assessment of chemicals because they are widely regarded as an indicator of the net sensory, motor and integrative process occurring in the central and peripheral nervous system.
Clinical signs of toxicity (alteration in behavior) are observed and recorded routinely in toxicity studies.
For selected chemicals, the core animals in 90-day studies are subjected to a Functional Observational Battery at least three time point intervals.

Biochemical Indices

Incorporation of biochemical parameters depends on the background information such as known structural-toxicity relationship and on the target organ(s) of toxicity.
Examples of biochemical determinations include mixed function oxidase activity; cholinesterase activity; thyroid and gonadal hormone levels; cell proliferation rates in suspected cancer target tissues; and analysis of a number of cell cycle parameters.

Molecular Toxicology

The National Toxicology Program has initiated collection of tissue samples at the terminal sacrifice of 90-day rat and mouse toxicity studies. The intended objective for the samples is to use existing and evolving molecular arrays and related technologies to screen the samples for gene alterations that might provide mechanistic information related to toxicity. The following tissues are proposed to be frozen quickly when they appear normal grossly:

Spleen
Liver
Kidney
Lung
Forestomach
Thymus
Skin
Testis
Femur
Glandular stomach

Use of 90-Day Toxicity Data in Setting Priorities

Testing Priority	Triggers in 90-Day Toxicity Studies
Reproductive	SMVCE, hormonal levels, histology, organ weights
Neurobehavioral	clinical signs of toxicity, FOB, histology
Immunotoxicity	histology of thymus, lymph node, and spleen; spleen and thymus weights; complete blood count and differential; and splenic cellularity
Carcinogenesis/Mechanistic	MN frequencies, cell proliferation, biochemical markers, metabolic profiles

Conclusions

Data from 90-day NTP studies are the primary information source used for non-cancer risk assessment and selection of dose levels for 2-year studies, if performed. A number of endpoints that serve as screens for system toxicity are incorporated into most 90-day studies, depending on the information needed for an individual chemical. These endpoints are evaluated in core animals when feasible or extra animals may be incorporated. This approach helps in prioritizing the need for systemic toxicity studies, and in reduction of cost and animal use.

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