Mammalian toxicity testing: in vivo, in vitro, multigeneration; C 2, 8, 9, 10, 20; Molecular methods, high throughput testing; C 9, 31

In running assays how do we account for alternative causes and background? This requires being able to distinguish a signal due to the causative agent from a signal due to background noise. This is best accomplished by use of an assay that is well characterized and validated. For both bioassays or analytical assays this means the assay must operate within well-characterized ranges for a series of quality control parameters. These are criteria for valid assays:

1. Precision -- reproducibility (A given input results in a predictable output.)

2. Accuracy -- evaluation of reference standards yields expected results (as determined by an independent method or from a consensus of others running similar tests on the same standard materials)

3. Specificity -- measures the intended analyte or parameter even in the presence of potentially interfering substances (lack of specificity = nonspecificity or cross-reactivity)

4. Sensitivity (Limit of Detection) -- lowest measurable, nonzero dose (e.g., LOAEL); also, in a chemical analytical sense, the slope of the response curve, dY/dX, the ability to differentiate one dose from the next.

Bias is found when a result curve is obtained that is different from that seen in another assay. The idea combines sensitivity and specificity dimensions.

Note that a high signal to noise ration, S/N, implies a high analytical sensitivity. It will do well in discriminating the presence of signal from background.

Comparisons among assays are often evaluated using a Chi-square approach where the distinct possible outcomes of the reference assay or evaluation (for example, development of disease or the known content of a compound) are placed on the Y-axis and compared to the outcomes of the test assay (or drug trial). For a 2x2 evaluation where each test has yes or no answers (i.e., they are qualitative or semiquantitative) each of the cells can be readily described.

1. When both tests give positive assays, the result is entered in a square termed "true positives," TP.

2. When both are negative, the result is entered in a square termed "true negatives," TN.

3. When the reference test is positive, but the test assay is negative, the results are "false negatives," FN.

4. When the reference test is negative, but the test assay is positive, the results are "false positives," FP.

With this information, the test assay can be evaluated (relative to the reference) by five qualitative features:

1. Sensitivity = (TP/(TP + FN)); note, this is not the same as lowest measurable dose or slope of the curve for a single assay.

2. Specificity = (TN/(TN + FP)); this is related to, but not the same as measuring the intended parameter.

3. Predictive Value for a Positive Test = (TP/(TP + FP))

4. Predictive Value for a Negative Test = (TN/(TN + FN))

5. Efficiency = ((TP + TN)/(TP + TN + FP + FN)); this is the overall ability of the test to correctly predict or of a compound or drug to have the expected result.

ASTM testing methods:

chronic oral toxicity: http://www.astm.org/DATABASE.CART/PAGES/E1619.htm

ASTM Bioassay Table:

http://www.cwo.com/~herd1/bioassay/Table.htm

LOAEL, NOAEL, Zero dose

Note how these appear differently in a sigmoid model of toxicity in which there is no difference between the beginning of the dose-response curve and zero dose, i.e., the response curve is monotonically increasing beginning at zero dose, and in the threshhold model of toxicity in which there is an actual point on the dose axis above zero dose at which the response falls to zero.

In the monotonically increasing case, repeated measuremens of the dose response curve provide an error estimate around the points of the dose curve including the zero dose. The mean of the zero dose + 2 (or 3) standard deviations of the zero dose, define a line through the dose response curve below which a response cannot be distinguished from the impact of a zero dose. Since doses are chosen for testing, any dose falling below the line just described would yield no effect. The highest dose below that line should be the NOAEL dose. The first dose above that line should be the LOAEL. Note the line defined could also be refered to as the maximal tolerated dose, MTD.

The threshhold model is variant of the linear extrapolation model. The latter assumes there is no dose that does not have some effect. If our methods are sensitive enough, this model would, in fact, have no NOAEL or MTD. If, more realistically, there are physiological mechanisms that pose barriers for toxicity, e.g., detoxification systems, sequestration mechanisms, or repair processes, there will be a dose that does not generate a response in the organism. In this situation, there will be no response at a finite, nonzero, dose which can be termed the NOAEL or the MTD. The first dose above that level would be the LOAEL. Operationally, it is normally impossible to distinguish between a true threshhold model and a sigmoid model given the limits of response measurement technologies and the finite number of dosages that can be tested. Thus, the determination of NOAEL, LOAEL, and MTD fall back to the approach outlined above under the sigmoid model.

What happens with delayed effects? Cancer, neurodegeneration, immunocompromise (where opportunistic infections by secondary agents may actually be the ultimate causes of the observed toxic effect). In all these situations the phase delay between exposure and toxic response may be difficult or impossible to detect above background "noise." But the obvious places to begin exploring these situations are in the tissues most potentially affected. In the case of cancer this would probably be most prominent in the tissues demonstrating the most active proliferation: testicular germinal epithelium, bone marrow, lining of the small intestine, placenta, or embryonic tissues. Note how the clearance and repair processes make the connection of cause and effect even harder because the causal agent may be cleared long before the impacts of its effects are expressed by the biological systems triggered to set in motion processes or cascades that are proximally responsible for observed effects.

Practically, these delays have important impacts on the process of testing drugs and food additives since they may take 10-20 years or more to become apparent. Current patents are only good for 17-20 years including much of the time needed for the later phases of testing. There is little wonder companies try to optimize profits through the course of the initial patent as they need to address the costs not only of development, but also of liability litigation that may arise as a result of delayed untoward effects that may not be made apparent during the time of testing. Animal models are not perfect substitutes for humans and not all humans can be adequately modeled even with other humans.

So the question arises as to whether the potential for delayed deleterious toxic effects might be grounds for delaying the granting of a patent, prolongation of the testing phase, governmentalization of the responsibility for prolonged testing, or automatic patent renewal if no such deleterious effects are reported during the initial patent.

Genetic Toxicology: What is covered?

1. Anything that changes DNA [Joanne]

2. Others?

It includes discussions of:

1. some forms of neoplasia/cancer

2. inherited disorders

3. teratology (gestational losses)

4. ?somatic mutations (such as those responsible for immune system variation)

What are changes in DNA or mutations?

mutations leading to truncations

1. missense

2. deletions or insertions

1. point mutations

a. transitions (G<->A, C<->T)

Because wobble in the third base of many amino acid t-RNA codons allows transitions, transitions will often produce silent mutations.

b. transversions (G<->C, A<->T, G<->T, A<->C)

Because wobble in the third base of many amino acid t-RNA codons often allows transitions but not transversions, transversions are more likely to yield a nonsilent or missense change that will either change an amino acid in protein products of translation or will produce an inappropriate stop codon and a truncated gene product.

2. larger changes

a. frameshifts (1 base deletions or insertions)

These frequently arise during dimer or adduct formation and repair. They may give rise to truncations or gene fusions.

b. large deletions (often occur in repetitive sequences during synapsis in meiosis)

c. sequence duplications (often occurring in repetitive sequences during synapsis in meiosis)

d. inversions (often occur in repetitive or palindromic sequences during synapsis in meiosis)

All of the larger changes can give rise to more obvious chromosomal changes including: insertions, deletions, duplications, inversions, and translocations.

Additional chromosomal changes are possible due to problems arising at the cellular level during meiotic or mitotic division. Chromosome breakage or nondisjunctions fall in this class and often give rise to trisomies or aneuploidies. Note the mechanisms by which these may arise do not have to target DNA, they may come about by interference with the proteins and metabolic pathways responsible for the mechanics or regulation of cell division. They include spindle proteins, centrosomal proteins, centromeric proteins, telomeric proteins, aster proteins, cytokinesis proteins, nuclear membrane proteins and others.