SAFE USE OF CHEMICALS: A Practical Guide - Chapter 3 pot

Toxicological studies are essential to understanding the possible adverse effects that a candidate chemical or combination of chemicals may cause to animals, humans, fauna, and flora, an

and Chemical Safety

Toxicology is the branch of science concerned with understanding the gross and intrinsic capabilities of a chemical substance on biological systems—that is, on plants, animals, and humans Toxicology is a multidisciplinary science and closely interrelated with many other branches of science Chemical substances are required for health, progress, and societal development In the very close linkage with an array of chemical substances and societal development, human health cannot be ignored Therefore, thinkers of the past and present around the world framed regula-tions about the manners and methods of use of chemical substances There are no safe chemical substances and all are toxic in one way or the other No chemical sub-stance is absolutely safe In fact, the safety of a chemical subsub-stance depends upon the concentration and manner of exposure and use This is important and should be very well understood and remembered by all students, industrial workers, and household users who handle, store, transport, and dispose of different chemical substances Improper and negligent use and management of chemical substances cause injury, death, and disaster The present chapter focuses on and briefly discusses the elements

of toxicology vis-à-vis effects of chemical substances and their use

Chemical substances as and when they are marketed for human use in the form

of drugs, food additives, cosmetics, and many others items require safety data and detailed quality evaluations To generate quality data about the candidate chemi-cal substance, different countries and international regulatory agencies have framed elaborate procedures By understanding the basics of toxicology and correctly adher-ing to regulations and observadher-ing precautions, the benefits of chemicals would enrich human society and free it from hunger and disease

Toxicological studies are essential to understanding the possible adverse effects that

a candidate chemical or combination of chemicals may cause to animals, humans, fauna, and flora, and to make relevant, reliable, reproducible predictions The gen-eration of toxicological data after conducting experiments with short- and long-term exposure in species of organisms and laboratory animals using different routes of exposure provides substantial and basic guidance to establish safe levels of chemi-cals Depending on route of exposure, the duration of exposure, and the quantity of the test chemical, the experimental animals develop signs and symptoms of toxicity The test provides information about

the nature of toxicity of the test chemical substance;

r

the dose and concentration of the chemical substance that cause adverse r

effects in the animal;

the toxicity profile in male and female test animals, oral, dermal, and respi-r

ratory routes;

the immediate and long-term health effects; and

r

the effects of two or more chemicals as additives or synergistic effects r

3.2.1 H ISTORY OF T OXICOLOGY

What is toxicology? What is the history of toxicology? What is the importance of toxicology to modern society? The answers to these questions can provide a better and more meaningful understanding of the management of chemical substances to protect health Toxicology is a scientific discipline many thousands of years old Reports trace the history of toxicology dating from 3000 bc to the Middle Ages (476–1453) to the periods of the Renaissance (1400–1600) and subsequent years The history of toxicology needs to be traced along with the global development To trace and document the history of toxicology to certain parts of the world alone is both incomplete and incorrect It is therefore necessary to know the origin and global development of the science of toxicology

The science of toxicology has a very solid and authentic historical base In fact, elementary knowledge about toxicology dates back to early times of human history and civilization India is well known as the birth place of ayurveda, the very ancient Indian system of medicine and human health care Although recent documents indi-cate ayurveda’s origin as ca 5000 bc, according to the Indian scriptures, which have stood the test of time, dates extend to much earlier periods of human history The ayurveda system of medicine and health care has valid links to the ancient books

of wisdom—the Vedas The word Veda in Sanskrit (Samskruta) means knowledge, and the language is Samskruta/Sanskrit, or Devanagari script The term ayurveda comes from two words: ayuh (meaning life) and veda (meaning knowledge—the

knowledge of longevity and life) Thus, ayurveda originated in India long ago in

the prevedic period—the Rigveda and Atharva-veda (5000 years bc) The texts of

ayurveda, such as Charak Samhita and Sushruta Samhita were documented about

1000 years bc As has been documented elsewhere, ayurveda is one of the oldest sys-tems of health care, describing both the preventive and curative aspects of different herbal medicines for improvement in the quality of life Ayurveda in a most compre-hensive way describes medication for human ailments and bears a close similarity

to the principles of health care of the modern era propounded by the World Health Organization

The ancient seers of India in the Astanga Hrudaya of Vagbhata and others have

paved the way for the understanding of the concept of human health Human

ail-ments, including poisoning, are the areas covered by ayurveda In brief, ayurveda

discusses the combination of four essential parts of the system—namely, human body, mind, senses, and the soul—and unravels the effect of toxic chemical sub-stances on the body and the manner of its elimination by adopting different pro-cesses Further, the history of indigenous Indian medical science along with the

Indus Valley civilization dates back to more than 3000 bc The most well planned cities of Harappa and Mohenjodaro exemplify not only the rich cultural heritage of India, but also its advanced systems of hygiene and human health care.1–4

For the people of the Indian subcontinent in particular the way of life and the association with food and drink was quite different and stringent as compared to the human populations in occidental regions of the world The elementary knowl-edge about the use and restricted use of certain substances and food items and drinks was the guiding principle for the maintenance of good health This is very evident in the dictum of the native language of India, Samskruta The dictum may be grouped under health and hygiene or the Yoga system of philosophy—a path to lead a life

of righteousness The dictum in Samskruta runs as follows: ati sarvatra varjayet,

meaning avoid excess in eating, drinking, and/or other activities, anywhere, anytime

Even nector (ambrosia), the drink of the angels, when consumed in excess can cause

adverse effects! There are many regulations well documented for human health care

The dictum langanam parmaushadham, meaning fasting or moderate food before bed at night, is the best medicine to maintain a proper and good health and madyam

na pibeyam means not to be alcoholic In fact, Rigveda, the ancient scriptures of India, clearly mention visha, a term in Sanskrit for poison Similar references are

also made in hymns to poison liquids that produce ecstasy In the Purana legends of India (ancient scriptures), mention of poison is made during the mythological

pro-cess of churning the cosmic ocean before the drink (amruta) of immortality is won.

Much later (1493–1541), Paracelsus, the father of modern toxicology, pronounced

a dictum of his own: Sola dosis facit veneum (“only the dose makes the poison”) “All

substances are poisons, there is none which is not a poison The right dose differenti-ates a poison from a remedy.” In other words, no substance is absolutely safe What a glorified commonness between ancient thinkers from India very much earlier in his-tory and of the West in later periods, without knowing each other during the periods

of world history This is the glorious saga of the global history of toxicology.4

In the Western world the ancient Greeks were probably the first to dissociate medicine from magic and religion Important and valuable contributions of several thinkers improved the quality of human health and our understanding in toxicology Some of the important ones include:

Shen Nung, 2696

r
bc: the father of Chinese medicine, noted for tasting 365

herbs He wrote the treatise On Herbal Medical Experiment Poisons and

died of a toxic dose

Ebers Papyrus, 1500

r
bc: the oldest well preserved medical document from ancient Egyptian records dated from approximately 1500 bc contains 110 papyrus pages on anatomy and physiology, toxicology, spells, and treatment Homer, 850

r
bc: wrote of the use of arrows poisoned with venom in the

epic tales of The Odyssey and The Iliad The Greek word toxikon is arrow

poison

Hippocrates, 460

r
bc: a Greek physician born on the island of Cos, Greece

He became known as the founder or father of modern medicine and was regarded as the greatest physician of his time A person of many talents, he

named cancer using the Greek word karkinos (crab) because of the creeping,

clutching, crab-claw appearance of cancerous tissue spreading into other tissue areas He moved medicine toward science and away from superstition

He was also noted for his oath of ethics still used today

Plato, 427–347

r
bc: reported the death of Socrates (470–399 bc) by hemlock

(Conium maculatum).

Socrates’ death by ingesting hemlock, 399

religious heresy and corrupting the morals of local youth The active chemi-cal used was the alkaloid coniine, which, when ingested, causes paralysis, convulsions, and potentially death

Aristotle, 384–322

r
bc: familiar with the venom of jellyfishes and scor-pion fishes

Mithridates VI, 131–63

r
bc: from a young age, fearful of being poisoned He went beyond the art of poisons to systematically study how to prevent and counteract poisons He used both himself and prisoners as “guinea pigs” to test his poisons and antidotes He consumed mixtures of poisons to protect

himself, which is the origin of the term “mithridatic.” The term Mithridatism

is well known in pharmacology It is named after Mithridates when he was king of Pontus (112–63 bc) and an enemy of the Roman Empire To avoid his assassination, he took small doses of poison to immunize himself against it

He was the first to develop antidotes in his quest of the universal antidote Sulla, 82

r
bc: Lex Cornelia de sicariis et veneficis—law against poisoning

people, including prisoners; it was forbidden to buy, sell, or possess poisons Aulus Cornelius Celsus (25

recom-mended the washing of wounds with an antiseptic such as vinegar He

published De Medicina, which contained information on diet, pharmacy,

surgery, and preparation of medical opiods

Pedanius Dioscorides, 40–90

the time of Nero who wrote De Materia Medica, the basis for the modern

pharmacopeia that was used until 1600 ce

Devonshire Colic, 1700s, Devonshire, England: High incidence of lead colic r

among those who drank contaminated cider The apple press was constructed partly of lead Discovered and described in the 1760s by Dr George Baker Ramazzini, 1700: documented the possible preventive measures to control r

industrial hazards among workers

John Jones, 1701: extensively researched the medical effects of opium r

Richard Meade, 1673–1754: wrote first English language book dedicated to r

poisonous snakes, animals, and plants

Percivall Pott, 1775: born in 1714 and apprenticed to Edward Nourse, made r

some groundbreaking discoveries in the fields of cancer research and sur-gery techniques He discovered the link between occupational carcinogens and scrotal cancer in chimney sweeps and wrote multiple scientific articles

in his lifetime

Friedrich Serturner, 1783–1841: first successful scientist in isolating mor-r

phene crystals from the poppy plant—in effect, creating a much stronger and more effective painkiller

Francois Magendie, 1783–1855: born in France, researched the different r

motor functions of the body in relation to the spine, as well as nerves within

it In addition, he researched the effects of morphine, quinine, strychnine, and

a multitude of alkaloids Noted as the father of experimental pharmacology Louis Lewin, 1854–1929: German scientist who took up the task of classify-r

ing drugs and plants in accordance with their psychological effects The clas-sifications were Inebriantia (inebriants), Exitantia (stimulants), Euphorica (euphoriants), Hypnotica (tranquilizers), and Phantastica (hallucinogens) Serhard Schrader, 1903–1990: Born in Germany, chemist Schrader acciden-r

tally developed the toxic nerve agents sarin, tabun, soman, and cyclosarin while attempting to develop new insecticides As a result, these highly toxic gases were utilized during World War II by the Nazis He is sometimes called the “father of the nerve agents.”

For more information, refer to the literature.5,5b

3.2.2 B RANCHES OF T OXICOLOGY

Chemicals are used extensively in industries, homes, and crop fields to meet growing challenges for healthy living It has been reported, however, that a vast majority of chemicals lack basic toxicity data and this has caused concern Generation of quality data on the toxicity and safety of chemical substances, proper evaluations, and mean-ingful interpretations to human health and environmental safety demand the support

of specialized branches of science In simple terms, the chemical substance under test has to pass through different branches for evaluation These are (1) analytical toxicology, (2) aquatic toxicology, (3) biochemical toxicology, (4) clinical toxicol-ogy, (5) ecotoxicoltoxicol-ogy, (6) environmental toxicoltoxicol-ogy, (7) epidemiological toxicoltoxicol-ogy, (8) genetic toxicology, (9) immunotoxicology, (10) nutritional toxicology, (11) mam-malian toxicology, and (12) regulatory toxicology and many other related branches Recent advances in toxicology and technology have now taken yet another impor-tant turn with the emerging discipline of nanotechnology and nanotoxicology.5a In fact, nanotechnology is one of the top research priorities of the U.S government Nanotechnology involves research and technology development at the atomic, molecular, or macromolecular level, in the length scale of approximately 1–100 nm This technology creates and uses structures, devices, and systems that have novel properties and functions because of their small and/or intermediate sizes and their novel ability to be controlled or manipulated on the atomic scale

The nanomaterials thus manufactured in different industries—particularly drugs and pharmaceuticals—might pose risks to human health and other organisms due to their composition, reactivity, and unique size Nanotechnology research and devel-opment, particularly in medical research, work at the micro- and nanoscale levels

to develop new drug delivery methods, therapeutics, and pharmaceuticals In such areas of research it is equally important to consider the potential interactions of nano-materials with the environment and the associated risks This involves studying the effects of natural nanoparticles in the air and soil, life cycle aspects of manufactured nanomaterials, and their fate and transport Risk assessment also includes studies

on the toxicity of natural and manufactured nanomaterials, as well as their routes of exposure to humans and other organisms and potential for bioaccumulation Also, the nanoscale colloidal particles thus produced are involved in the transformation and transport of metals, toxic organic compounds, viruses, and radionuclides in the environment because nanomaterials have been found to cause toxic responses in test animal systems In fact, data on the toxicology of nanoparticles and nanotubes (tiny carbon tubes) are very sketchy The nanoparticles perhaps have undesirable effects

on the lungs and other body systems Nanoparticles in food may cross into the gut lymphatic system Nanoparticles that are inhaled have been known to travel from nasal nerves to the brain and cause health disorders

The nanomaterials and the structures thus formulated with characteristic dimen-sions (approximately 1–100 nm) contain a variety of unique and tunable chemical and physical properties In fact, these properties have made the nanoparticles cen-tral components of the emerging global technologies The use of nanotechnology

is increasing Its potentially adverse effects on biological systems with particular reference to human health, however, have not been adequately understood In order

to accurately conduct hazard assessments, there is a need to know the concepts that apply to pathways of dermal, oral, and respiratory exposure with reference to nanomaterials This gains added importance in the study of biological systems that include but are not limited to membrane transfer, screening methods, and impact on major body organs and systems

While there are differences in the methods of data generation from one branch

to another, all branches are interrelated to provide complete data about the toxicity and safety of a candidate test chemical substance vis-à-vis human safety Toxicity

of a chemical is the result of several reactions and interactions between the candi-date chemical and its metabolites and the cellular receptors These include enzymes, glutathione, nucleic acids, hormone receptors, and the like The degree of toxicity of

a chemical could be explained as follows:

Toxicity C Ar (chemical) (receptor), where

Ar = the specific affinity of the receptor for the toxic chemical C The tox-icity of a chemical can also be expressed as toxtox-icity = k (C) (R) Ac, where toxicity is dependent upon C, R, and Ac

C = concentration of the candidate chemical in the tissue

R = concentration of the endogenous receptor of the tissue

Ac = affinity of the receptor for the chemical

The toxicological evaluations related to human safety of chemical substances are a very complex process involving the determination of the intrinsic toxicity and hazard of the test chemicals Subsequently, this evaluation leads to determin-ing and establishdetermin-ing a “no observed effect level” (NOEL): the highest dose level tested experimentally that did not produce any adverse effects This dose level then

is divided by a safety factor to establish an acceptable daily intake (ADI) of the can-didate chemical substance The ADI value is normally based on current research and

long-term studies on species of laboratory animals with several doses, including high doses, and observations of humans Subsequently, the NOEL is scaled by a safety factor based on judgment, experience, and international convention Typically, the safety factor ranges between 100 and 1000, depending on the biological relevance and severity of the observed effect and to extrapolate the differences between test animals and humans This provides a substantially lower level and thus a large mar-gin of safety for humans

ADI is a measure of a specific chemical substance—the pesticide residue or a food additive—in food, beverages, or drinking water that can be ingested over a lifetime period and without an appreciable health risk ADIs are expressed by body mass, usually in milligrams per kilogram of body mass per day The higher the value

of ADI is, the safer is the chemical substance in food or water and for regular inges-tion In fact the concept of ADI is a measure to indicate the toxicity from long-term exposure to repeated ingestion of chemical substances in foods This concept was first introduced in 1957 by the Council of Europe and later the Joint Expert Committee

on Food Additives (JECFA) of the U.N Food and Agricultural Organization (FAO) and the World Health Organization This internationally accepted concept is applied when estimating safe levels of food additives, pesticides, and veterinary drugs

3.2.3 T YPES OF T OXICOLOGICAL S TUDIES

All kinds of chemical substances have the intrinsic property of toxicity in one way

or another, depending on the quantities of the chemical substance involved, system conditions, and nature of the surroundings, to mention a few The purpose of the toxicological studies is to define the biological effects of the different chemical sub-stances commonly used by humans Further, the studies are also required to under-stand the intrinsic properties of chemical substances on children, animals, and the living environment The regulatory agencies of different countries require informa-tion on doses of the test chemical substance that produce adverse biological effects

in species of test animals as well as doses that cause no significant toxicological or pharmacological effects (NOEL) The spacing of the doses also provides an assess-ment of the dose–response relationship

3.2.3.1 Acute Toxicity

Acute toxicity tests in laboratory animals are conducted to generate data of the test chemical and its ability to cause systemic damage as a result of a one-time exposure

to relatively large amounts through a specified route of exposure The test substances

in specific amounts either as one oral dose or multiples within 24 hours are admin-istered to the animals Chemical substances that are acutely toxic cause damage in

a relatively short time (within minutes or hours) Exposure to a single concentrated test chemical substance induces irritation, burns, illness, and other signs and symp-toms of toxicity, including death (Appendix 3.1) Commonly used chemicals, such as ammonia and chlorine, cause severe inflammation, shock, collapse, or even sudden death when inhaled in high concentrations Corrosive materials such as acids and bases may cause irritation, burns, and serious tissue damage if splashed onto the skin

or eyes Exposure to chemical substances, development of symptoms of poisoning,

methods, standard procedures, and estimation of LD50 values are available in the literature.4,4a,4b,8–14

3.2.3.2 Chronic Toxicity

Chronic toxicity studies provide information on the long-term health effects of chem-ical substances Adverse health effects in exposed animals and subsequent severe damage are known to occur after repeated exposure to low doses over a period of time The slow accumulation of mercury or lead in the body or after a long latency period from exposure to chemical carcinogens is an example Chronic or prolonged periods of exposure to chemical substances may also cause adverse effects on the reproduction and behavior of animals and humans The symptoms caused after chronic exposure usually differ from those observed in acute poisoning from the same chemical In fact, when exposed to low concentrations of chemical substances,

as is the case with chronic toxicity studies, the industrial worker and common public are unaware of the exposure

Chronic toxicity also includes exposure to embryotoxins, teratogenic agents, and mutagenic agents The embryotoxins are substances that cause any adverse effects

on the fetus (death, malformations, retarded growth, functional problems) Terato-genic compounds specifically cause malformation of the fetus Examples of embryo-toxic compounds include mercury and lead compounds Mutagenic compounds can cause changes in the gene structure of the sex cells that can result in the occurrence

of a mutation in a future generation Approximately 90% of carcinogenic compounds are also mutagens

The regulatory agencies of different countries of the world require toxicity profiles

of candidate chemical substances It is mandatory that all such data (1) be generated through a battery of genetic toxicity tests about the chemical substances, (2) involve

a 90-day feeding study both in a rodent species (usually the rat) and in a nonrodent mammalian species (usually the dog), (3) show a two-generation reproduction study with a teratology component in rats, and (4) include other specialized testing studies

to define adequately the biological effect of the test chemical substance The special-ized studies include testing for (1) neurotoxicity, (2) immunotoxicity, and (3) effects following in utero exposure The regulatory agencies also advocate and require data

on toxicity tests performed for safety evaluation of direct food additives, as well as color additives used in food and food products

The Organization for Economic Co-Operation and Development (OECD) Guide-lines for the Testing of Chemicals are a collection of the most relevant internation-ally agreed-upon testing methods used by governments, industries, and independent laboratories to assess the safety of chemical products to man and animals These guidelines represent a basic set of important tools that are primarily for use in regu-latory safety testing and subsequent chemical product notification and chemical reg-istration in different governments around the world.5b

The details of several other toxicological tests (namely, repeated-dose toxic-ity, subchronic toxictoxic-ity, chronic toxictoxic-ity, genotoxictoxic-ity, mutagenictoxic-ity, teratogenictoxic-ity, carcinogenicity, neurotoxicity, and ecotoxicology) and the methods, purposes, and importance of safety evaluation studies to achieve human health have been discussed

in the literature.4,4a,9–14 Humans are exposed to chemical substances normally through contamination, food poisoning, accidental ingestion, skin absorption, and/or respira-tory route To generate toxicity data, species of laborarespira-tory animals are exposed to test chemicals through the three major routes However, more often than not, chemi-cal substances enter through more than one route (e.g., skin absorption, accidental ingestion, and inhalation) into the bodies of industrial workers who are negligent during work

To generate data on the toxicity profile of the test chemical substance and for further extrapolation of the data to human situations, other routes of exposure have also been used in laboratory animals These routes include (1) inhalation (breathing in), (2) absorption (through the skin or eyes), (3) oral ingestion (eating, swallowing), (4) transfer across the placenta to the unborn baby, (5) intravenous (injection into the vein), (6) intramuscular (injection into the muscle), (7) subcutaneous (injection under the skin), and (8) intraperitoneal (injection inside the membrane that lines the interior wall of the abdomen) These routes are advocated by the regulatory authori-ties of governments for the generation of quality data about chemical substances and drugs and subject to specific data requirements The laboratory animals used for testing should represent the species in which the drug will be used The most sensi-tive breed or class of test animal should be selected for testing The species of test animals should be free of disease and not exposed to environmental conditions and environmental pollutants

Additional experimental parameters should be included in the animal safety studies when they might reveal suspected adverse properties of the test chemical substance or product This is to know the species sensitivity to the test product or related drug product The test animals should be properly acclimated to the study environment Subsequent studies should be adequately designed, well controlled, and conducted by qualified investigators to generate meaningful data Further, the safety evaluation of the test chemical substances should be identical to the product intended to be marketed, meaning (1) the same chemical substance, (2) same particle size, and (3) the same formulation, if any Because the Center for Veterinary Medi-cine (CVM) regulates the manufacture and distribution of food additives and drugs that are given to animals, a discussion between the sponsor and CVM prior to use of

an alternative drug product is recommended

The routes of administration should be the same as proposed in the protocol as well as by labeling This, however, as in some of the studies, requires modifications (e.g., drench in lieu of medicated feeds) In order to minimize autolytic decomposi-tion, necropsy should be performed promptly after death on all animals that die during the study The necropsy should be performed by a qualified and experienced person A complete physical examination should be performed, and baseline data should be collected by a qualified and trained worker Data should be obtained prior

to the start of the trial and at reasonable, predetermined intervals thereafter in accor-dance with the study protocol

The clinical observations should be recorded twice daily, 7 days a week, dur-ing the entire study period, or accorddur-ing to the study protocol Appropriate clinical pathologic procedures should be conducted on all test groups This is required on all animals in each group or, when appropriate, on a representative number (usually

one half or a previously agreed upon number) of animals preselected at random from each group and at predetermined intervals and described in the study protocol After the completion of studies, tissues should be collected and preserved for his-tologic examination Again, all animals or a representative number (usually one half

or a previously agreed upon number) from each group is selected for further studies All or selected tissues of test animals exposed to the highest dose treatment and from control groups should be examined for possible histological changes Wherever microscopic lesions are observed, the corresponding tissues of the test group from the next lower treatment group should be examined until a NOEL is established Documentation of all studies should be made indicating the representative test conditions and the manner of use of the test chemical substance It is very important

to remember that more often than not, the toxicological effects observed in animals and humans caused by chemical substances involve various modulating factors Over the years, the potential health risks that might be caused by chemical substances act-ing in combination have been found to be important In fact, the interaction between chemical substances does take many forms Such interactions between chemical substances have become very relevant to determine the potential health risks vis-à-vis human safety Some of the known and common forms of interactions include the following four categories:

An additive effect is one in which the combined effect of two chemical

sub-stances is equal to the sum of the effects of each (2 + 2 = 4)

An antagonistic effect occurs when the toxic effect of the combination of

chemical substances is less than what would be predicted from the indi-vidual toxicities The antagonistic effect or antagonism is like adding 1 + 1 and getting 1 as the result

A synergistic effect occurs when the combined toxic effect of two chemical

substances is much greater or worse than the sum of the effects of each by itself Synergism is similar to adding 2 + 2 and getting 5 as the result

Potentiation is the ability of one chemical substance to enhance or increase the

simple summation of the two expected activities (1 + 0 = 1)

The toxicological interactions among chemical substances depend on the chemi-cals present, their mode of action, and their concentrations Of the four types of interactions, additive effects are the most plausible This requires that the chemicals act through similar mechanisms and affect the same target tissue For instance, the (combined) action of two or more chemicals causing irritation effects is often an added effect rather than attributable to any one candidate chemical substance

It is also important to remember that while tissue irritation studies in laboratory animals are conducted using different chemical substances including products of cosmetics or injectable drugs, the protocol should include data on the product vehicle and at least two times the use level concentration of the active ingredient The same volume of both preparations should be administered to all animals of the experi-mental groups Observation should be made about tissue inflammation, swelling, necrosis, and other reactions