Alternatives to Animal Use in Research, Testing, and Education

Chapter 8

Alternatives to

Animal Use in Testing

Queen:

I will try the forces

Of these compounds on such creatures as

We count not worth the hanging, but none human . . .

Cornelius:

Your Highness

Shall from this practice but make hard your heart.

Shakespeare,

Cymbeline

Act I, Scene VI

The experimental means to be used for safety evaluations is left open to suggestion. As

unorthodox as this might sound, leaving such means open for consideration is the best

solution.

Safety evaluations should not be based on standard, specified series of tests. They

are best approached by first raising all pertinent safety questions and then searching for

the experimental means to provide the best answers. Under such circumstances, even the

standard LD test might on occasion be the best experimental means to resolve outstanding

satfety questions.

Constantine Zervos

Food and Drug Administration

Safety Evacuation and Regulation of Chemicals 2,

D. Homburger (cd.) (Base]: Karger, 1985)

—.—

CONTENTS

Page‘

Continued, But Modified, Use of Animals in Testing . . . . . . . . . . . . . . . . . . . . . ..175

Avoiding Duplicative Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Reducing Pain and Distress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177

Use of Living Systems in Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

In Vitro Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177

Nonanimal Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179

Use of Nonliving Systems in Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

180

Chemical Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

180

Mathematical and Computer Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Epidemiologic Data on Humans . . . . . . . . . . . . . . . . . . . . . . . . . . ..:......

.181

The LD

Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...181

Using Fewer Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.182

The Limit Test and Other Refinements . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..182

In Vitro and Nonanimal Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182

Skin and Eye Irritation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ]83

In Vitro Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183

Chick Embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

Repeated-Dose Toxicity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

184

Hepatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......185

Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185

Microorganism Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....186

In Vitro Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Tests Using Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Carcinogenicity ...,.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..187

The Ames Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .187

Use of the Ames Test in a Battery of tests . . . . . . . . . . . . . . . . . . . . . . . . . .188

Current Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...188

Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

190

Chapter preferences.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

191

Table

Table No.

Page

8-1. The Response of Known Human Carcinogens to Rodent Carcinogenicity

and Bacterial Mutagenicity Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..188

Figure

Figure No.

Page

8-1. Chronological Sequence of Chick Embryo Chorioallantoic

Membrane Assay.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..184

Chapter 8

Alternatives to Animal Use in Testing

Alternatives to using animals in testing serve the

same purposes that using whole animals does—

protecting and improving human health and com-

fort. The technologies on which alternatives are

based result primarily from biomedical and bio-

chemical research. Several of them are reviewed

in this chapter, though they are discussed in

greater detail in chapter 6. Some alternatives that

might eventually replace the tests covered in chap-

ter 7 are also described here.

Notable progress in the move to alternatives has

been achieved in certain areas (78). For example,

biochemical tests to diagnose pregnancy have re-

placed those using rabbits, and the Limulus ame-

bocyte lysate test, which relies on the coagulation

of a small amount of blood from a horseshoe crab,

has replaced rabbits in testing for the presence

of bacterial endotoxins that would cause fever

(25,117). Many companies have modified the widely

used LD

test to use fewer animals (22) and have

otherwise refined the methods used to test for tox-

icity (100). Mammalian cell culture assays are used

extensively in industrial laboratories for safety test-

ing of medical devices (52,53) and pharmaceutical

CONTINUED, BUT MODIFIED,

It has been suggested that many more animals

are used for testing than are needed (90) and that

changes in experimental design or improved meth-

ods of data analysis could substantially reduce the

number of animals used. Each experiment has

unique requirements (see ch. 7), and the ways in

which the number of animals might be reduced

will vary accordingly.

Many of the methods discussed in chapter 6 for

the modified use of animals in research are also

applicable to testing, such as gathering more data

from each animal or improving the analysis of re-

sults by using random block design or covariance

analysis. In random block design, animals with a

particular characteristic, such as litter mates or

animals of a certain size, are randomly assigned

to different groups to balance whatever effect

substances (1,84) and as immune response assays

(97,98).

The development of alternatives to animals in

testing has accelerated in recent years with the

establishment of programs having development

and implementation of alternatives as their goal

(see ch. 12). However, the barriers to adoption of

these tests are more than the technical barrier of

developing and validating anew technology. Test-

ing is an integral part of many regulatory schemes

and product liability law, and validation ultimately

rests on acceptance by the scientific, regulatory,

and legal communities.

Public concern over animal

to be increasing in tandem

for product and drug safety

use in testing appears

with public concern

. Ironically, the pub-

lic’s-increasing concern for safety could lead to

more testing. Yet it also provides an incentive to

develop new techniques, particularly those that

promise to be cheaper and faster than current

whole-animal methods. A further irony is that de-

veloping alternatives, as well as validating them,

sometimes requires animal use.

USE OF ANIMALS IN TESTING

these variables might have. If the groups being dis-

tributed are sufficiently large, the results can also

be analyzed to determine the effect of the mask-

ing variable (47). Covariance can be used to ana-

lyze results when some of the experimental varia-

bles are uncontrolled but known, thus estimating

their effect on the results.

As in research, the number of animals needed

as controls can be reduced by using the same group

as a control for several simultaneous experiments.

A laboratory’s ability to do this will be limited by

its size and the amount of lead time available to

allow testing to be coordinated. Another difficulty

is that environmental conditions must be exactly

the same and the tests must start and finish at ex-

actly the same times. The reduction in animal use

that simultaneous experiments brings about is

175

176 ● Alternatives to Animal Use in Research, Testing, and Education

modest because the control group should be larger

if it is being used in several simultaneous experi-

ments (34),

The use of historical data for control groups is

constrained by the difficulty of exactly duplicat-

ing the conditions of a study. However, the size

of the groups and other controlled variables can

be better planned if historical data are used to dis-

cover the background incidence of specific tumors

or other diseases before testing begins. This use

of historic controls has been recognized by the

National Cancer Institute, the world Health Orga-

nization, the Canadian Government, and the now-

defunct Interagency Regulatory Liaison Group

(104). The Federation of American Societies for

Experimental Biology has developed a data book

containing such information based on the Labora-

tory Animal Data Bank (see ch. 10) (2).

Avoiding Duplicative Testing

Animal use in testing can and has been reduced

by industry and others through improved commu-

nication and cooperation in the planning and exe-

cution of testing, thereby avoiding unintentional

duplication. Trade groups such as the Chemical

Manufacturers Association, the Pharmaceutical

Manufacturers Association, and the Soap and De-

tergent Association play important roles in this co-

ordination.

The sharing of data after testing has occurred

is often done for pesticides (see chs. 10 and 11).

And in 1978, the Food and Drug Administration

implemented a policy of permitting approval of

new drug applications solely on the basis of pub-

lished scientific papers (113). The possibility of an

unintentional repetition of an experiment is also

avoided through the work of organizations such

as the Chemical Industry Institute of Toxicology

(CIIT) (Research Triangle Park, NC). Using contri-

butions from member companies, CIIT conducts

toxicological tests and distributes the results

widely.

Governments contribute greatly to information

sharing, which allows duplicative testing to be

avoided, by providing both access to test results

and information about their own planned and on-

going tests. The International Agency for Research

on Cancer makes it easy for duplicative carcinoge-

nicity testing to be avoided by informing testing

facilities and governments about planned and on-

going testing, Federal and international databases

and publications also contain information about

planned tests and those under way (see ch. 10).

Reducing Pain and Distress

As with research, testing can be modified to re-

duce animal pain or distress in two ways: by pro-

viding relief with drugs or by changing the proce-

dures so that less pain or distress is produced (see

ch. 6). A third alternative might be to use a less

sensitive species, but there is no method by which

relative distress among species can be discerned.

Relief from pain and distress is accomplished through

analgesics, anesthetics, tranquilizers, or sedatives

and modification of the test itself.

Few pain-relieving drugs have been developed

and marketed for animals. Little information is

available on recommended doses (122) or on the

likely effect on test results. Thus, before pain re-

lief could be incorporated into a test, it would be

necessary to determine the needed dose and the

effect on the toxic response, thus using additional

animals as well as subjecting them to pain.

Several small changes that do not interfere with

the experimental design can be made by an inves-

tigator. Small needles can be substituted for large.

Animals can be comforted by petting. Social ani-

mals can be caged in groups, although there are

often reasons that multiple housing cannot be used.

Smaller doses can be used and tests can be ended

at the earliest feasible time. Sometimes, smaller

doses will actually result in increased sensitivity

of the test (38). Making such changes sometimes

depends on the attitude and expertise of individ-

ual researchers rather than the contents of test-

ing guidelines, which may not be sufficiently

detailed.

Ch. 8—Alternative to Animal Use in Testing . 177

USE OF LIVING SYSTEMS IN TESTING

As detailed in chapter 6, two kinds of living sys-

tems can reduce whole-animal use—in vitro sys-

tems based on animal or human components (cell,

tissue, and organ cultures) and systems based on

organisms not considered animals for purposes

of this report (micro-organisms and invertebrates).

(Some people consider both of these in vitro

system s.)

In Vitro Systems

Cells, tissues, and organs can be kept alive out-

side a living organism and used for testing. Al-

though animals are still required as a source for

these in vitro systems, the animal would experi-

ence distress for a much shorter time, and per-

haps less distress overall, than occurs with whole-

animal testing because it would be killed before

any experimental manipulations were carried out.

Occasionally, different cells, tissues, or organs from

the same animals can be used for different inves-

tigations. In addition, many fewer animals would

be required for a given test, in part because varia-

bility in the toxic response is smaller than it is with

whole-animal tests and in part because one ani-

mal can be used for multiple data points, further

reducing variability. The fact that human tissues

sometimes can be used confers an additional ad-

vantage because the need for extrapolation from

animal data is obviated.

These isolated components also have disadvan-

tages. They are usually unable to produce the com-

plete physiologic responses of a whole organism.

The components often become undifferentiated

and lose their ability to perform their special func-

tions when isolated from the organism, particu-

larly when the sample is broken up into its con-

stituent cells, and even more so when the cells

replicate. Another disadvantage is that the effect

of the route of exposure, a variable that can have

profound effects on test results, is often impossi-

ble to determine.

There are many measures of damage to differen-

tiated or undifferentiated cells—the rate of repro-

duction, the rate of synthesis of certain substances,

Microscopic View of Cell Culture From Rabbit Corneal Epitheliums

Photo credit: Kwan Y. Chan, University of Washington

178 ● Alternatives to Animal Use in Research, Testing, and Education

changes in membrane permeability, and damage

to some part of the cell structure. Those functions

having to do with viability and growth are most

frequently measured because they require an in-

tegration of many physiologic events within the

cell, are sensitive, and lend themselves to automa-

tion (73).

Quantifiable tests are preferred over subjective

ones, and a wide variety of quantitative approaches

are available to measure irritation, including the

release of prostaglandins (35); the production of

enzymes (46), proteins (57), antigens, antibodies,

or hormones (73); and the migration of certain

white blood cells (macrophages) to the area of ir-

ritation (12,101). Irritation can also be measured

by the extent to which cells exfoliate from the sur-

face of the tissue. The extent of damage can be

determined by counting cells and by examining

the nuclei (1O2). Another indicator of irritation,

the integrity of cell membranes, can be monitored

through the uptake of nutrients through the cell

wall. Where the nutrient uptake is active (that is,

when the cell is required to expend energy for

transport), uptake can also be used to indicate

changes in metabolism (86,102).

Liver cells have been the subject of considerable

research, in part because they play such an im-

portant role in an organism’s removal of toxic sub-

stances and in part because they retain most of

their special functions when cultured. The re-

sponse of liver ceils to toxic substances may be

measured in many ways: the use of sugar as an

indication of metabolic activity; the production of

proteins or other substances that have been cor-

related with toxicity; uptake of amino acids as an

indication of protein synthesis; changes in appear-

ance that parallel those observed in livers of whole

animals (106); and morphological changes and re-

ductions in viability (75). Other promising tech-

niques in this rapidly expanding field include cul-

turing:

●

beating heart cells to detect the effect of cer-

tain vapors on irregularities in heartbeat (68);

rabbit kidney tubules to detect substances that

can cause acute renal failure, and rat vaginal

tissue to test vaginal irritancy of contracep-

tives (27);

various kinds of cells to test for biocompati-

bility of implants (15,52,53); and

nerve cells to test for the synthesis of neuro-

Dispensing Apparatus for Deiivery of Cuiture Medium to Ceiis Within a Piastic Cuiture Piate

Photo credit: The Johns Hopkins University

Ch. 8—Alternative to Animal Use in Testing ● 179

transmitter chemicals, the formation of syn-

apses, and the conduction of impulses (7).

Although tissue and organ cultures may approx-

imate more closely the physiology of the human

or whole-animal model, they are more difficult to

manipulate than cell cultures (see ch. 6). Sophisti-

cated equipment must be used to monitor and con-

trol the environment and to perfuse the sample

with nutrients. Where the sample is more than

a few cell layers thick, uniform delivery of the test

substance, nutrients, and oxygen is difficult, as is

the removal of waste products. Cell differentia-

tion can usually be maintained in tissue and or-

gan cultures, albeit with some difficulty (50).

Human placentas have proved quite useful in

testing the ability of a drug to cross the placenta

from mother to fetus. There are certain logistical

problems with this method, however. The placenta

must be transferred to the perfusion apparatus

within 5 minutes after it is eliminated from the

uterus, and it is only useful for about 3 hours af-

terward (77).

Nonanimal Organisms

There are a variety of nonanimal organisms that

can replace some animals in testing, ranging from

plants to single-celled organisms to invertebrates.

All of these can respond to certain noxious stimuli,

and some may experience pain. However, many

commentators believe that they do not experience

pain or suffering in the same way that animals do,

particularly in those cases where there is no brain

or neural tissue (90). The use of such organisms,

which has never been controlled under any Fed-

eral or State law, is regarded as a replacement for

animals in this report.

Micro-organisms

In recent years, increased emphasis has been

placed on the use of bacteria and fungi to meas-

ure certain genotoxic effects. A major advantage

of these organisms is that they can be cultivated

much more easily and quickly than most animal

or human cells. Their genetic makeup is simple

compared with that of animals and humans and

the fact that a great deal is known about it facili-

tates their use, particularly in toxicological re-

search leading to new methods (74). A change in

genetic material is relatively easy to detect and

characterize. Fungal systems have been shown to

be especially useful in mutagenicity testing and

seem to be more sensitive than bacteria (126), per-

haps at the expense of falsely indicating a hazard.

Other species that have proved useful include slime

molds, algae, and protozoa (74).

Protozoa, although rather primitive overall, fre-

quently have specialized functions that mimic those

of humans. For example, the cilia of protozoa re-

spond to smoke or phenols as do the cilia in the

human bronchial tube (5). Various protozoans have

been used in toxicity testing of cigarette smoke.

protozoans are currently being evaluated for use

in screening tests for carcinogenesis, mutagene-

sis, and reproductive toxicity (93).

Invertebrates

Invertebrates have made major contributions in

biomedical research because certain aspects of

their physiology are sufficiently similar to that of

mammals (74). Although models for toxicity test-

ing require greater similarity to animals or more

thorough characterization of differences than

models for research, invertebrates offer exciting

possibilities.

Of the invertebrates, insects offer the greatest

selection of models, there being over 2 million spe-

cies from which to choose (74). Among them, the

fruit fly, Drosophila rnelanogaster, is the best un-

derstood. procedures have been developed for de-

tecting mutagenicity (18), as well as teratogenic-

ity (11) and reproductive toxicity (93).

The sea urchin has long been a favored test

organism for basic reproductive research (74). Con-

sequently, the mechanisms and procedures of

testing this invertebrate can easily be developed

and performed. The sea urchin model for fertili-

zation and development can be used in screening

for reproductive toxicity, teratogenicity, and muta-

genicity. Nematodes, annelids, and mollusks are

also used for alternative mutagenesis testing re-

gimes and, additionally, mollusks are used in the

area of reproductive toxicology. Sponges, mollusks,

crustaceans, and echinoderms are being used in

metabolism studies, as understanding metabolize

formation in nonmammalian species can lend in-

sight to interspecies variation (93).

180 ● Alternatives to Animal Use in Research, Testing, and Education

USE OF NONLIVING SYSTEMS IN TESTING

Animal use can sometimes be avoided altogether

with nonliving biochemical or physiochemical sys-

tems, although most such systems currently re-

quire animal derived components. Computer simu-

lation can also be used when there are sufficient

data available for substances related to the one

of interest and when the mechanisms of toxicity

are at least partially understood.

Chemical Systems

Whole animals have been replaced with analyti-

cal chemistry for tests involving detection of a sub-

stance or measurement of potency or concentra-

tion, such as for vaccines, anticancer drugs, and

vitamins (10). However, toxicity testing in nonliv-

ing systems is quite limited at this time.

Recently developed methods of detection or

measurement are based on the selective binding

that occurs between a particular substance and

the antibodies to it. In an assay for botulism toxin

(which traditionally required up to 200 mice), an-

tibodies obtained from rabbits are modified so that

the binding of the toxin can be detected easily. The

rabbits are initially injected with a small, harm-

less dose of the botulism toxin. Small amounts of

blood are then removed from the rabbits at regu-

lar intervals. In 4 weeks, a rabbit can produce

enough antibody, with little discomfort, to perform

tests that would otherwise require thousands of

mice (32).

Chemical systems that test for toxicity are based

on determining whether a substance undergoes

a specific reaction. For example, it is well known

that carbon monoxide binds to hemoglobin in the

blood, thus greatly reducing the blood’s ability to

carry oxygen. The extent to which a substance

would displace oxygen in hemoglobin can be a

measure of its ability to produce asphyxiation. Sub-

stances can also be tested in isolation for their ef-

fects on enzymes crucial to certain bodily functions.

An important limit of chemical systems is that

they do not indicate the extent to which an organ-

ism can recover from or prevent these reactions.

For example, a substance that binds strongly to

hemoglobin may not be a problem because it is

not absorbed. A substance will not have a signifi-

cant effect on an enzyme of interest if it is excreted

before it has an effect.

Physiochemical systems have some ability to

determine whether a substance will be absorbed

and what will happen to it. The tendency of a sub-

stance to accumulate in a biological system can

be roughly estimated by the relative proportions

that dissolve in equal volumes of water and the

organic solvent octanol (34,55). Artificial skin made

with filter paper and fats is being tried as a means

of mimicking absorption of cosmetics and drugs

(45). Reactivity and other toxicity-related proper-

ties can be deduced from chemical structure alone

(109).

Mathematical and Computer Models

Advances in computer technology during the

past 20 years have contributed to the development

of sophisticated mathematical models of quantita-

tive structure activity relationships (QSAR). These

models are used to predict biological responses

on the basis of physical and chemical properties,

structure, and available toxicological data. The limi-

tations of such models are due in part to a lack

of understanding of the mechanisms by which

toxic effects occur.

In applying QSAR, the biological effects of chem-

icals are expressed in quantitative terms. These

effects can be correlated with physiochemical

properties, composition, and/or structure. Fre-

quently used properties include an affinity for fats

versus water (octanol/water partition coefficient),

the presence of certain reactive groups, the size

and shape of molecules, and the way reactive frag-

ments are linked together.

The simplest extrapolation is for a series of

closely related chemicals. The several character-

istics they have in common need not be incorpo-

rated into the model as variables. This type of

analysis has been performed for several hundred

families of chemicals and has established that rela-

tionships within a series are fairly predictable (64).

Another approach, more broadly applicable, is

to examine the contributions of various portions

of a molecule. In more elaborate computer pro-

Ch. 8—Alternative to Animal Use in Testing . 181

grams, it is possible to identify likely reactions and

cascading physiological events in various species,

techniques first developed for pharmacology (54).

A similar approach is the use of multitiered clas-

sification schemes that use large databases to draw

semiempirical conclusions (36).

Epidemiologic Data on Humans

Perhaps the most useful alternative to animal

testing is epidemiologic studies on humans. Such

studies were used to detect carcinogenicity in hu-

mans as early as the 18th century (49,85,87). The

most well known study detected scrotal cancer

in chimney sweeps (85). A more recent example

in which epidemiologic evidence was used to de-

tect a human carcinogen was the finding that vi-

nyl chloride causes a rare liver cancer in humans

(26). A major disadvantage of epidemiologic studies

is that considerable human exposure can take place

before a toxic effect is detectable, particularly in

the case of diseases that take many years to de-

velop. Another disadvantage is that they can be

quite expensive to conduct. Privacy must also be

considered (112), preventing many data that would

be useful from being collected or analyzed.

Epidemiologic studies may be divided into three

general types: experimental, descriptive, and ob-

servational. Experimental epidemiology is the hu-

man equivalent of animal testing—providing or

withholding a substance to determine its toxic or

beneficial effects. Such studies are greatly limited

by ethical and legal considerations, as well as the

difficulties involved in securing the cooperation

of a large number of people.

Descriptive epidemiology analyzes data on the

distribution and extent of health problems or other

conditions in various populations, trying to find

correlations among characteristics such as diet,

air quality, and occupation. Such comparisons are

frequently done between countries or smaller geo-

graphic regions, as is the case for cancer statistics

collected and analyzed by the National Cancer In-

stitute (9).

observational epidemiology uses data derived

from individuals or small groups. Data would be

evaluated statistically to determine the strength

of the association between the variable of interest

and the disease. In cohort studies, a well-charac-

terized and homogeneous group is studied over

time. In case-control studies, a control group is

selected retrospectively based on variables thought

to be relevant to the effect, Both methods rely on

an accurate prediction of the variables that are

important and are subject to various selection

biases (62)112).

THE LD

TEST

The LD

testis one of the most widely used tox-

icity tests, and the development of alternatives to

it is regarded by many as a high priority. As de-

scribed in chapter 7, this acute toxicity test meas-

ures the amount of a substance needed to kill half

the population of the test species. The LD

used as a rough indicator of the acute toxicity of

a chemical,

The LD

is useful for testing biological thera-

peutics, although there remain few such sub-

stances for which the LD

is the only available

means of standardization (13)90). Other applica-

tions, perhaps not so well justified (90), are deter-

mining doses for other toxicological tests and set -

ting regulatory priorities.

There has been political pressure to abolish the

and it has been criticized by many toxicolo-

gists on scientific grounds. It has poor reproduci-

bility and the results are difficult to extrapolate

to humans because there are so many mechanisms

by which death could occur (70,90,125).

Despite the many criticisms of the LD

, most

toxicologists agree that acute toxicity information

has valid uses, and that measurements of lethality

also are important. Nevertheless, the precision with

which the LD

is measured is often unjustified

for several reasons. First, most applications of the

information do not require precision. Second, even

if the information were precise for a given spe-

cies, the LD

varies so much from species to spe-

182 Ž Alternatives to Animal Use in Research, Testing, and Education

cies that extrapolation to humans is only rough.

Third, the LD

of a given substance varies signifi-

cantly from laboratory to laboratory, and even in

the same laboratory.

Various regulatory classification schemes make

distinctions between levels of toxicity (“highly toxic”

versus “toxic, ” versus “moderately toxic, ” versus

“nontoxic”). The LD

for two neighboring levels

typically differs by a factor of 4 to 10. Yet, the

reproducibility of test results does not justify even

these distinctions. A recent study, though not nec-

essarily typical, indicates the magnitude of the

problem. A series of LD

tests were performed

in 60 European laboratories for five substances

on one species. The LD

for one substance ranged

from 46 mg/kg body weight to 522 mg/kg, possi-

bly ranging over three toxicity levels in some clas-

sification schemes. Although the variations were

not this large for the four other chemicals tested,

the smallest variation was 350 to 1,280 mg/kg. Each

test was done with 50 or more animals so that the

results would be precise (61).

Using Fewer Animals

The standard LD

requires at least three groups

of 10 animals or more each. An alternative proce-

dure for dete

rmining the Approximate Lethal Dose

(ALD) was developed as early as the

1940s (29),

in which individual animals are administered doses

that increase by 50 percent over the previous dose.

Depending on the initial dose level, the total num-

ber of animals needed is usually 4 to 10. Because

the test substance might not be cleared between

doses or because there maybe cumulative effects,

the ALD can be lower than the LD

, perhaps by

70 percent, though more typically by less than 20

percent (29).

Many other acute toxicity tests that require fewer

animals than the LD

have been developed (14,

17,33,61,69,71,94,105,107). Most require that the

doses increase sequentially, thereby allowing the

experiment to stop when a certain limit is reached.

Thus, fewer animals die in the conduct of a test,

but its duration could increase from 2 weeks to

a month or more. Although many investigators

are moving to less precise LD

tests, no generally

accepted alternative seems to have emerged.

The Limit Test and

Other Refinements

If a substance is not lethal at high doses, its pre-

cise LD

is not very important. In the limit test

(80), a small number of animals is given a single

oral dose, e.g., 5 g/kg body weight. If no animals

die and no major ill effects occur, no further test-

ing is needed. However, this limit is so high that

this approach may have little practical value in re-

ducing animal use (24).

Rather than determining the dose that is lethal,

studies can also be done to detect toxic effects at

doses that are not lethal. As with the LD

, increas-

ing doses can be administered to a small number

of animals, perhaps stopping when some limit is

reached. This approach can be further refined so

that animals that are in distress could be sacrificed

without affecting the outcome of the test (14).

In Vitro and Nonanimal Methods

Cell toxicity—changes in cell function or death

of cells-can sometimes be used to detect acute

toxicity. However, cell toxicity cannot be expected

to function as a replacement for the LD

because

lethality can occur by so many mechanisms that

are supercellular. Cell toxicity is particularly use-

ful in comparing members of chemical families,

such as alcohols and alkaloids (79).

At present, mathematical modeling has limita-

tions, although it may have some utility in range-

finding and in screening substances for testing

(109). Modeling of acute toxicity fails to meet one

of the criteria suggested by a working party on

quantitative structure activity relationships, namely

that the mechanism by which the response occurs

should involve a common rate determining step

(88). Nonetheless, in a large study involving thou-

sands of substances, a computer program was de-

veloped that predicted LD

values within a fac-

tor of 2.5 for 50 percent of the substances and

within a factor of 6 for 80 percent. Considering

Ch. 8—Alternative to Animal Use in Testing Ž 183

the reproducibility of the test itself, this might be

satisfactory for some purposes, and it certainly

warrants further investigation. Furthermore, many

of the larger deviations in this study, upon fur-

ther examination, were found to involve report-

ing errors. This program relied on a multi-tiered

classification scheme based on chemical structure

(36).

SKIN AND EYE IRRITATION

The widely used Draize eye irritation test and,

to a somewhat lesser extent, the skin irritation test

have been criticized because of the amount of pain

inflicted and because they are unsatisfactory mod-

els for human irritation (91,95). First, the rabbit

eye has structural differences, such as a thinner

cornea and differing tearing apparatus (103), and

animal skin is much less sensitive and discriminat-

ing than human skin (56,63). Second, both of these

tests are sensitive to too many variables, making

reproducibility poor (83,118).

As with most tests, the number of animals used

can sometimes be reduced. Several refinements

have also been proposed. For example, screening

tests based on pH or skin irritancy might also serve

as alternatives to eye irritancy tests in limited cir-

cumstances, although preliminary studies indicate

that this approach is frequently misleading (119).

Other refinements involve local anesthetics (51,65,

110), applying smaller (43) or more dilute (120)

doses, and testing whole eyes in vitro (20). The lat-

ter method has particular appeal when cow eyes

are used because they are so readily available from

slaughterhouses. In the case of smaller doses, a

recent comparison with over 500 accidental human

exposures showed that doses smaller than those

now in use yielded results more predictive of the

human response while causing less severe irrita-

tion (38).

Skin and eye irritation are similar in many re-

spects. Thus, even though little work has been done

to develop alternatives to skin irritation tests, the

many approaches just summarized for eye irrita-

tion may eventually be applied to skin testing as

well (91).

In Vitro Tests

Several in vitro alternatives have been examined,

and it appears to some commentators that no sin-

gle alternative will be adequate, but that a battery

of in vitro tests might be a useful replacement (67).

Several types of cell cultures have been used in

developing an in vitro test for eye irritation. The

cells used are rabbit and human corneal cells (72),

mouse and hamster fibroblasts, human hepatoma

cells, and mouse macrophages (96).

A variety of effects have been used as surrogates

for eye irritation, such as the rate of uptake of uri-

dine as an indication of cell functioning and re-

covery, visible changes in cell structure, decreases

in the concentration of cell protein (96), and re-

lease of plasminogen activator from the injured

cells (21). Some techniques appear promising, par-

ticularly in their ability to rank substances based

on irritancy, Rapid progress is being made in the

development of techniques, but none can be con-

sidered validated at this time (91).

To date, little work has been done on in vitro

replacements for skin irritancy testing. However,

the growth of skin in tissue culture is of interest

for treating burn victims, and it is expected that

culture techniques currently being developed for

that purpose can be used in testing methods. In

addition, it has also been suggested that suitable

specimens can be obtained from cadavers and

surgery and from judicious use of human volun-

teers (63).

Chick Embryo

One test system receiving considerable attention

is the fertilized chicken egg. A part of the eggshell

is removed and the test substance applied to the

chorioallantoic membrane surrounding the devel-

oping embryo (see fig. 8-l). This test has the po-

tential for assessing both eye and skin irritancy.

The chorioallantoic membrane of the chick em-

bryo is a complete tissue, including arteries, capil-

184 ● Alternatives to Animal Use in Research, Testing, and Education

Figure 8-1.—Chronological Sequence of Chick

laries, and veins, and is technically easy to study.

Embryo Chorioallantoic Membrane Assay

An embryonic membrane tested after 14 days of

incubation responds to injury with a complete in-

flammatory reaction, a process similar to that in-

Day O

duced in the conjunctival tissue of the rabbit eye.

The embryonic membrane can show a variety of

signs of irritation and has capabilities for recov-

ery (59,60).

Assessment of toxicity is made and the embryo

●

Day 14

size,

contours and surface,

color,

retraction of surrounding chorioallantoic

membrane,

spokewheel pattern of vessels,

● overall grade of severity, and

● necrosis (confirmed microscopically).

Although this is, strictly speaking, an in vivo test,

the chorioallantoic membrane does not have nerve

cells, and thus it is unlikely that the organism ex-

periences any discomfort. In addition, fertile eggs

are inexpensive and do not require elaborate ani-

mal room facilities.

Day O. Fertile eggs are incubated at 37” C. Day 3. The shell is

penetrated in two places: A window is cut at the top, and 1.5 to 2

milliliters of albumin is removed with a needle and discarded. The

chorioallantoic membrane forms on the floor of the air space, on

top of the embryo. The window is taped. Day 14. A test sample is

placed on the embryonic membrane and contained within a plastic

ring. Day 17. The chorioallantoic membrane is evaluated for its

response to the test substance, and the embryo is discarded.

SOURCE: J. Leighton, J. Nassauer, and R. Tchao, “The Chick Embryo in Toxicol-

ogy: An Alternative to the Rabbit Eye,”

Food Cherry.

Tox/co/.

23:293-298.

REPEATED-DOSE TOXICITY TESTS

Repeated-dose toxicity testing involves the re- peated-dose testing, the long-term effects of

peated application of a substance to a biological

repeated, sublethal exposure to a substance are

assay system and subsequent measurement of

of interest, rather than acute, lethal effects. Cell

many different effects of the substance. In re-

cultures may be useful adjuncts for suspected tar-

Ch. 8—Alternative to Animal Use in Testing ● 185

Chick Embryo Chorioallantoic Membrane Assay

Photo credit: Joseph Leighton, Medical College of Pennsylvania

Typical react ion seen 3 days after certain concentrations

of household products have been placed on the 14-day-

old chorioallantoic membrane. The thin white plastic ring

has an internal diameter of 10 millimeters. The area of

injury within the ring is well defined with a distinct edge.

All of the cells in the injured area are degenerating or

dead. The severity of this positive lesion is quantified by

measuring its diameter.

get organs or tissues, but they are not a replace-

ment for whole-animal testing. The most promising

alternatives in the near future involve modifica-

tions of animal use (for example, by combining

tests), and the use of screening tests and computer

simulation for improved experimental design. The

screening tests with the greatest promise are for

hepatotoxicity and neurotoxicity.

Hepatotoxicity

Several in vitro alternatives for hepatotoxicity

have been developed, including perfused liver

(108), liver cell suspensions (39), and liver cell cul-

tures (39)44). Liver perfusions can only be main-

tained for a few hours, and with some difficulty.

Cell cultures can retain the special functions of

liver cells with specially prepared culture media

(76,81). However, the cells are viable for only a

limited period of time and do not replicate in a

reproducible manner. Although these techniques

have been used to study mechanisms of liver tox-

icity, only limited attention has been given to their

use in screening or as alternatives (91).

Neurotoxicity

The development of alternatives for neurotox-

icity is more difficult than for hepatotoxicity. The

nervous system is the most complex organ in the

body, both in terms of structure and its function.

Because many neurotoxins affect only one kind

of cell, a battery of in vitro tests would probably

be required to replace whole-animal testing–if

anything could. Substances can also affect vari-

ous areas differently, partly because of distribu-

tion factors, For example, very few substances are

able to enter the brain because of the ‘(blood-brain

barrier.” Thus, pharmacokinetic studies will con-

tinue to be very important.

Some in vitro tests (41) and tests using inverte-

brates (8) seem useful, at least for screening. As

yet, however, the primary use of in vitro tech-

niques has been the elucidation of mechanisms

of known toxic effects (31). Many toxic effects to

neural tissue have been correlated with concen-

trations of specific chemicals in or around the cells,

thus offering the means for developing in vitro

tests (31).

MUTAGENICITY

Mutation, the change in the DNA sequence of

is passed from the mutated cell to its descendants.

genes, is a mechanism by which toxic effects may

Mutation can lead to cell death or the gain or loss

be initiated. If the DNA replicates, the mutation

of certain functions. When it occurs in germ cells,

38-750 0 - 86 - 7

186 ● Alternatives to Animal Use in Research, Testing, and Education

the gene pool is affected, even if the mutation is

not expressed in the progeny. The mutations that

occur in somatic cells that are of greatest concern

are those that lead to cancer (18).

Recent advances in the techniques of cell biology

have led to an increase in the types and sophisti-

cation of mutagenicity tests available. Mutations

can be detected by analyzing DNA or its fragments

or by observing changes in the size, shape, or num-

ber of the chromosomes (which contain DNA), as

well as by observing changes in a whole organism

(34). Mutation can also be detected by measuring

the amount of DNA repair.

Micro-organism Tests

The most commonly used test for mutagenicity

is the Ames test for “reverse mutation” in Salno-

nella typhimurium

(3). Mutagenicity is detected

by exposing an already mutated strain to poten-

tial mutagens. If the mutation is reversed, the bac-

teria regain their ability to produce the amino acid

histidine and will proliferate in a histidine-deficient

culture medium.

The Ames test, as well as most other mutagenic-

ity tests involving micro-organisms, does not avoid

animal use entirely, To determine whether the meta-

bolic products of a substance might be mutagenic

even if the substance itself is not, liver prepara-

tions from rats or other rodents are used to pro-

duce at least some of the likely metabolic products.

Microorganism systems may fail to detector may

overpredict mutagenic changes that could occur

in whole animals or humans. For example, the sys-

tem provided for metabolism may not be capable

of reproducing conditions in vivo, or in the case

of screening for carcinogenicity, mutation may not

be the initiating event. on the other hand, such

systems may indicate mutagenicity when the DNA

repair system of mammals would reverse the mu-

tation.

Other bacterial tests have been developed using

S. typhimurium, Escherichia coli, and Bacillus sub-

tilis. These systems do not seem to offer any par-

ticular advantage over the Ames test, although

thorough evaluation is hampered by lack of a com-

parable database of results (28). Tests have also

been developed for molds (3

)

fungi (16), and

yeasts (18,82).

In Vitro Tests

In vitro mutagenicity tests maybe done with cul-

tured mammalian cells that are exposed to toxic

substances, although many mammalian in vitro

tests also have an in vivo variant. Such tests typi-

cally measure acquired resistance or lost resistance

to the effects of the toxic substance. Most com-

monly used are a mouse lymphoma ceil line or ham-

ster ovary cells, but almost any well-characterized

cell can be used. ovary cells are often used be-

cause, as germ cells, they have half the number

of chromosomes to be evaluated (18).

A test known as the specific locus test can be

done with Chinese hamster ovary cells. They are

exposed to a test substance and their response to

the normally lethal 8-azaguanine or 6-thioguanine

in cell culture determined. The cell’s ability to sur-

vive, requiring the ability to metabolize the 8-

azaguanine or 6-thioguanine, is an indication of

the occurrence of mutation as a result of exposure

to the test substance. This test can also be done

with mouse lymphoma cells exposed to 5-bromo-

deoxyuridine or trifluorothymidine (23).

The sister chromatid exchange test relies on the

fact that certain substances will cause DNA break-

age and reunion. This damage can be observed

by staining the original chromosomes so that any

segments exchanged during replication can be ob-

served. Commonly used cells include human lym-

phocyte cells and rodent and human fibroblasts

(37). Both the specific locus test and the sister chro-

matid exchange can also be performed as in vivo

procedures (see ch. 7).

Although the cells are usually derived from ani-

mals, there is a considerable net savings in animal

lives when in vitro mutagenicity tests are per-

formed. For example, the rat mast cell assay can

be used to screen severe irritants, and one rat can

supply enough tissue to replace the use of 48 ani-

mals in in-vivo procedures (103).

Tests Using Insects

The most widely used insect for genetic studies

is the fruit fly, Drosophila melanogaster (114, 115).

The fruit fly has well-characterized genetics and

is similar to mammals in many key reactions, A

variety of end points can be detected. The most

common, and probably most sensitive, test is the

Ch. 8—Alternative to Animal Use in Testing • 187

sex-linked recessive lethal assay (18). Treated males

ured include the loss, gain, or breakage of chro-

are mated with untreated females, and the progeny mosomes detected by examining germ cells. With

are mated

to each other. The number and charac- the availability of mutant strains, the measurement

teristics of the male progeny are evaluated to de-

of reverse mutations can be a valuable tool. Eye

termine if lethal mutations (that is, mutations that

color is a popular method of following genetic ef-

prevent viability) have occurred.

fects in the fruit fly (18).

Other

tests involving fruit flies also exist or are

likely to be developed. End points that can be meas -

CARCINOGENICITY

Many assays meant to replace carcinogenicity

testing

are designed to detect the initiation of can-

cer rather than the formation of tumors. First, de-

tecting initiation is faster and easier than detect-

ing cancer. Second, although not all initiation leads

to cancer, certain kinds are considered reliable

surrogates for the disease.

A major problem with evaluating the predictive-

ness of alternatives to whole animals for carcinoge-

nicity testing is that very few human carcinogens

have been positively identified. Most substances

treated as human carcinogens, although docu-

mented to be known animal carcinogens, must be

viewed as probable or suspected human carcino-

gens. The development of alternatives is somewhat

hampered by a lack of epidemiologic data on hu-

mans.

Various molecular and physiochemical prop-

erties of substances have been correlated to car-

cinogenicity. Some structure-activity models de-

veloped for families of chemicals have predicted

the carcinogenic properties for 75 to 97 percent

of them. The chemicals modeled include polycyclic

aromatic hydrocarbons (123), nitrosamines (89,99,

121), and aromatic amines (124).

The Ames Test

Because mutation is often the first step in car-

cinogenesis, the Ames

test has been suggested as

possible screen or replacement for carcinoge-

nicity testing. It has been evaluated for this pur-

pose, both alone and as one in a battery of tests.

Alone, it is less predictive than whole-animal tests.

In a battery, it has been shown to be about as pre-

dictive as animal testing for certain families of

chemicals and substantially less predictive for

others for the substances tested. Table 8-1 shows

the predictiveness of mouse and rat bioassays and

the Ames test for some known human carcinogens.

The Ames test has been performed thousands

of times in over 2,000 laboratories throughout the

world and has provided results on over 1,000

chemical substances since it was developed less

than two decades ago. Portions of this large body

of analytical data have been reviewed in over a

dozen evaluation studies with the intent of deter-

mining the test ability to predict carcinogenicity

(6,19,66). These evaluations show that the percent-

age of human carcinogens that are also mutagens

(mutagenic carcinogens) ranges from 50 to 93 per-

cent and is most likely about 80 percent (48). About

20 percent of the human carcinogens were not

mutagens (nonmutagenic carcinogens) in the Ames

test, and it is believed that cancer associated with

these carcinogens is initiated by a mechanism other

than mutation.

A critical analysis of several studies (19) identi-

fied several sources of variation. These include

methods of chemical selection, sample coding, use

of a high proportion of chemicals known to work

well or poorly with Ames testing, and differences

in metabolic activation during the test procedure.

The conclusion was that a reasonably careful ap-

plication of the Ames technique to a nonbiased

group of chemicals would be expected to yield a

predictive accuracy of approximately 80 percent

for mouse and rat carcinogens.

The Ames test tends to be positive for a large

proportion (about 40 percent) of substances that

have not been identified as carcinogens in rodent

bioassays. It should be noted, however, that these

188 ● Alternatives to Animal Use in Research, Testing, and Education

Table 8.1.—The Response of Known Human Carcinogens to

Rodent Carcinogenicity and Bacterial Mutagenicity Assays

Rat

Mouse

Ames

Chemical

bioassay bioassay

test

4-Aminobiphenyl . . . . . . . . . . . . . . . . . . . . . . . . .

Arsenic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Asbestos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Benzene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Benzidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Bis(chloromethyl)ether . . . . . . . . . . . . . . . . . . . .

Chromium; some chromium compounds . . . . .

Cyclophosphamide . . . . . . . . . . . . . . . . . . . . . . .

Diethylstilbestrol . . . . . . . . . . . . . . . . . . . . . . . . .

Melphalan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Mustard gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2-Naphthylamine . . . . . . . . . . . . . . . . . . . . . . . . .

Soot, tars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Vinyl chloride.. . . . . . . . . . . . . . . . . . . . . . . . . . .

—

n.d.

—

KEY:+ = Positive results (carcinogenic to rodents or mutagenic to bacteria)

– = Negative results (not carcinogenic

ornot mutagenic)

n.d.

= No data.

SOURCES: From H.

Bartsch, L. Tomatis, and C. Malaveille, ’’Mutagenicity and Carcinogenicityof Environmental Chemicals:’

Regu/ Tox/co/. Pharmacoi. 2:94-105, 1982;D. Brusick, devaluation of Chronic Rodent Bioaasays andAmes Assay

Tests as Accurate Modeis for Predicting Human Carcinogens,”

Application

Bioiogicai

Markets to Carcinogen

Testing,

Milman and S. Sell (ads.) (New York: Plenum Press, 1963);

B.D.

Goldstein,

C.A.

Snyder, S. Laskin et

al.,

“Myelogenous Leukemia in Rodents Inhaling Benzene,”

TcJx/coi.

f.ett.

13:169-173,

1962; and

J.V.

Soderman (cd.),

Handbook of identified Carcinogens and

Noncarcinogens, Vols.

and

(Boca Raton, FL: CRC Press, 1982),

substances have not been shown to be noncarcino-

genic, and many authorities maintain that the in-

formation is insufficient to make any statement

about the proportion of noncarcinogens that are

also nonmutagens in the Ames test (4,116).

Use of the Ames Test in

a Battery of Tests

The predictive value of the Ames test, or other

mutagenicity tests, can be improved by combin-

ing it with additional short-term assays to form

a test battery. Although no US. regulatory agency

has yet recommended a specific combination, most

authorities recommend that an appropriate bat-

tery should include information from a minimum

of three types of tests:

●

gene mutation (Ames test, mouse Iymphoma

test);

chromosomal mutation (in vivo Chinese ham-

ster ovary cell cytogenetics); and

DNA damage (sister chromatid exchange,

—

unscheduled DNA repair).

At least one test should include a mammalian in

vitro cell, tissue, or organ culture assay (4).

In a recent study, 18 Ames tests averaged 66 per-

cent “accuracy” (number of chemicals correctly

identified/number of chemicals tested). Compara-

tive results from six batteries of short-term tests

that included the Ames test increased the accuracy

to 82 to 90 percent (58,111).

CURRENT TRENDS

As long as toxicological data continue to be re- thermore, there are several impediments to devel-

quired by regulators and by the courts to protect opment and implementation:

human health, animal testing will continue for the

foreseeable future. Even major progress in the de-

. A large number of scientists have been trained

velopment and implementation of alternatives will

to solve health problems and to invent new

not necessarily eliminate whole-animal tests. Fur-

products using animal models.

Ch. 8—Alternative to Animal Use in Testing ● 189

●

Regulatory schemes, product liability law, and

patent law also incorporate notions of animal

models.

A large body of animal testing information al-

ready exists that is useful in interpreting new

testing data.

There are substantial costs and delays associ-

ated with the development and adoption of

alternatives. One study indicated that it takes

about 20 years for an in vitro test to be devel-

oped, validated, adopted, and implemented

(92).

At the same time, there are several factors

facilitating the development and implementation

of alternatives:

●

Rapid progress is being made in techniques

for culturing mammalian cells and organs, in

instruments for detecting and quantifying

cellular and molecular changes, and in the

understanding of the cellular and molecular

processes underlying toxicity. Improved un-

derstanding is leading to the ability to predict

long-term effects and carcinogenicity from

short-term biochemical and morphological

changes.

As such advances are made, the research lab-

oratories that have developed the expertise

are often willing to apply it to the develop-

ment of new testing methods, and can do so

efficiently (42).

Organizations such as The Johns Hopkins Cen-

ter for Alternatives to Animal Testing and the

Rockefeller University laboratory have been

set up to facilitate and coordinate research

on alternatives (see ch. 12).

Many organizations have been established to

pressure those who conduct animal testing

or use data based on it to adopt alternatives

or conduct research that will lead to alter-

natives.

Strategies to speed the development and adop-

tion of alternatives will depend on the needs and

resources of the organization involved. The fol-

lowing recommendations encompass a variety of

perspectives. They were promulgated by the Tox-

icity Committee of the Fund for the Replacement

of Animals in Medical Experiments, which met

from 1979 through 1982 (40). Some involve re-

assessment of testing needs and priorities; others

involve technical strategies thought to be likely to

lead to better methods, both in testing and in evalu-

ating results:

●

Provide a mechanism for reviewing the need

for a given test.

Investigate the consequences of not requir-

ing or possessing testing data other than what

already exists. Particular attention should be

given to widely used tests such as the LD

and skin and eye irritation tests with a view

toward eliminating unnecessary requirements.

Encourage flexible use of testing guidelines

and frequent reappraisal of them in light of

new knowledge.

Strive for broader-based international har-

monization and mutual recognition of data

from other countries so that duplicative test-

ing can be avoided.

Encourage detailed publication of all testing

results, particularly for costly or painful tests

or those requiring many animals.

Investigate the possibility of time limits on the

confidentiality of test results.

Make greater use of studies on absorption,

distribution, biotransformation, and excretion

in humans, as well as in test animals, to select

the most relevant exposure conditions, to aid

in extrapolation of results, and to improve the

reliability of test results.

Perform preliminary studies before undertak-

ing long-term studies so that results can be

as useful as possible.

Make greater use of the structural and con-

formational computer models used in devel-

oping drugs for the prediction of toxicity.

Standardize screening tests based on in vitro

and nonanimal tests, both to promote efficient

use of testing resources and to evaluate the

predictiveness of these tests.

Try to predict toxic reactions before testing,

both as a means for improving prediction tech-

niques and to avoid testing highly irritating

substances, particularly in the eye, if possible.

Conduct research on the mechanisms by which

toxic effects occur to facilitate the develop-

ment of new testing methods.

Develop more accurate, reproducible instru-

mentation for measuring toxic effects, avoid-

190 ● Alternatives to Animal Use in Research, Testing, and Education

●

ing subjective measurements and reducing

measurement errors.

Make greater use of depositories in standard-

izing cell lines or strains of micro-organisms

used for testing.

Study the relationship between physiochemi-

cal properties and pharmacokinetic proper-

ties, as well as between physiochemical and

toxicologic properties.

Develop techniques for detecting nonmuta-

genic carcinogens.

Develop systematic methods for objectively

evaluating new techniques.

Conduct postmarketing surveillance for ad-

verse effects, noting any discrepancies with

test results from animals.

Substitute very specific tests for the LD

and

other general toxicity tests, particularly for

substances having specialized uses, such as

drugs.

Use skin irritation testing as a rough screen-

ing tool for eye irritation.

Attempt to describe specific effects in eye ir-

ritation studies, rather than reporting only

the magnitude of the response.

Investigate specific effects such as neurotox-

icity to the extent possible when conducting

general toxicity tests.

●

Search for cell lines that retain their special

functions upon replication and develop tech-

niques for culturing them.

Evaluate the statistical precision needed in

various circumstances with a view toward

using the smallest number of animals likely

to be adequate.

Use statistics to maximize the utility of results.

Techniques such as blocking, covariance anal-

ysis, and factorial design should be used rou-

tinely.

Improve standards of care and diet to reduce

background effects.

Take care that those conducting tests are qual-

ified to do so, including having been trained

in humane handling of animals.

Combine tests wherever possible and keep

them as short as possible, compatible with the

nature of the test,

Place greater emphasis on “no observed effect

levels” than on lethal doses when they have

greater predictive value.

Use more than one species only to answer spe-

cific questions, and not for general safety as-

sessments.

SUMMARY AND CONCLUSIONS

There has been a small but significant shift away

from whole-animal testing to in vitro and non-

animal techniques in recent years, partly as a re-

sult of advances in biological techniques and partly

in response to political and economic pressures.

Many new methods are being developed for com-

monly used tests. Most of these are not yet vali-

dated, but they already have potential uses for

screening substances for the animal testing they

may eventually replace.

There are several kinds of alternatives. The first

entails the continued, but modified, use of ani-

mals-changes in experimental design or data anal-

ysis so that fewer animals are needed or changes

in protocols to reduce pain or distress. Living tis-

sues, organs, and cells derived from humans or

animals can sometimes be used instead of whole

animals. These systems require a larger investment

of time and money to develop than do modifica-

tions of whole-animal techniques, but their advan-

tages may also be greater. They are usually faster

and often cheaper than the corresponding whole-

animal test, and they have scientific advantages

as well. However, they almost always are less

predictive than whole-animal tests and often fail

to provide reliable dose-response data, informa-

tion that is critical in estimating potential toxicity

to humans.

Data, both anecdotal and epidemiologic, on toxic

effects in inadvertently exposed humans are some-

times useful. However, these data are often con-

founded by lifestyle and exposure to other toxic

Ch. 8—Alternative to Animal Use in Testing Ž 191

factors. Another drawback is that human exposure

can be great if there are long delays between ex-

posure and observable effects.

The LD

, probably the most common and most

criticized toxicity test, is well suited to the limited

use for which it was first developed. The biggest

obstacle to limiting or eliminating use of the LD

is institutional: Many regulatory schemes rely on

it for classifying substances. The most promising

alternatives in the short term are testing sequences

that require fewer animals. Cell culture techniques

and computer modeling show some promise, but

they have limited value at this time.

Another common and widely criticized test is

the Draize eye irritation test. Several promising

in vitro alternatives have been developed with cell

cultures. Another technique uses the outer (chorio-

allantoic) membrane of a 14-day-old chicken em-

bryo. This technique, although it uses a whole ani-

mal embryo, is thought to involve no pain because

the membrane has no nerves. These alternatives

may also apply to skin irritation.

Alternatives to carcinogenicity testing and re-

peated dose toxicity testing are of special interest,

in part because the potential savings in testing costs

and time are quite large, and in part because these

tests require large numbers of animals. The most

promising replacements are batteries of tests in-

volving cell cultures and living, nonanimal organ-

isms. Mutagenicity testing uses many in vitro or

nonanimal protocols. Mutagenicity is of particu-

lar interest because mutation can be the first event

in other kinds of toxicity, including carcinogenic-

ity, and because it can permanently affect the hu-

man gene pool. The most well known nonanimal

mutagenicity assay is the Ames test. When it is com-

bined with other tests, the Ames shows promise

as an alternative to carcinogenicity testing, but it

is not yet validated for this use.

In general, the development of alternatives is

being facilitated by the rapid development of bio-

logical techniques, which are being applied to the

search for

alternatives in many different labora-

tories. Major contributions to the coordination of

these developments in the United States are being

made by Rockefeller University and The Johns

Hopkins Center for Alternatives to Animal Testing.

The implementation of alternatives is hindered

by various forms of institutional inertia, such as

regulatory schemes (see ch. 7), product liability

law (see ch. 7), and general resistance to change.

Important impediments are the large body of ex-

isting information

—derived from animals—that is

relied on for the interpretation of new data and

the lack of sufficient information to support the

use of alternatives.

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