Laboratory Guide in Toxicology

College of Veterinary Medicine University of Mosul, Iraq

Laboratory Guide in Toxicology A Technical Manual For Veterinary Medical and Related Health Sciences Students

Third Edition, 2013

Fouad Kasim Mohammad, BVMS, MS, PhD Professor, Pharmacology and Toxicology College of Veterinary Medicine, University of Mosul, Iraq

DEDICATED TO MY PARENTS, FAMILY AND FRIENDS

The first edition of Laboratory Guide in Toxicology was published in 2000 by F.K. Mohammad, Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, Iraq.

Laboratory Guide in Toxicology A Technical Manual for Veterinary Medical and Related Health Sciences Students Third Edition, Š 2013 by F.K. MOHAMMAD Published by F.K. MOHAMMAD Direct Publishing Cover designed by M.K. MOHAMMAD Copyrighted materials

ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED, STORED IN RETRIEVAL SYSTEMS OR TRANSMITTED, IN ANY FORMS OR MEANS, (DIGITAL, RECORDED OR PRINTED), WITHOUT PRIOR WRITTEN PERMISSION OF THE AUTHOR. For permissions, contact F.K. MOHAMMAD fkmohammad@vetmedmosul.org PHONE: + (964) 770-160-5334 Disclaimer The author and the publisher, F.K. Mohammad, make no warranties that the information contained herein is totally free from errors; therefore we disclaim all liability for direct, indirect, and/or consequential damages resulting from the use of material contained in the publication.

This publication is provided by F.K. Mohammad, (author and publisher), on an "as is" basis. F.K. Mohammad (author and publisher), makes no representations or warranties of any kind, express or implied, as to the usage of the guide, the information, content, materials, or products included in this publication. F.K. Mohammad (author and publisher), to the full extent permissible by applicable law, disclaims all warranties, express or implied, including but not limited to, implied warranties of merchantability and fitness for a particular purpose. F.K. Mohammad (author and publisher), will not be liable for any damages of any kind arising from the use of this publication, including but not limited to direct, indirect, incidental punitive, and consequential damages.

Laboratory Guide in Toxicology A Technical Manual For Veterinary Medical and Related Health Sciences Students

Third Edition, 2013 Fouad Kasim Mohammad, BVMS, MS, PhD

Laboratory Guide in Toxicology

Table of Contents TABLE OF FIGURES..................................................................................................... III THE AUTHOR ............................................................................................................ V PREFACE ................................................................................................................ VII INTRODUCTION.......................................................................................................... 1 SOME DEFINITIONS IN TOXICOLOGY ............................................................................... 3 COMMON LABORATORY ANIMALS USED IN TOXICOLOGY ................................................... 7 ESSENTIAL CALCULATIONS IN TOXICOLOGY .................................................................... 13 DRUG RESIDUES ...................................................................................................... 17 TOXICITY TESTING .................................................................................................... 21 NEUROBEHAVIORAL TOXICOLOGY................................................................................ 25 DETERMINATION OF THE MEDIAN LETHAL DOSE (LD50) .................................................. 33 DETERMINATION OF PLASMA CHOLINESTERASE ACTIVITY BY AN ELECTROMETRIC METHOD .... 37 ORGANOPHOSPHATE AND CARBAMATE INSECTICIDES POISONING...................................... 43 NEUROTOXICITY OF LINDANE ...................................................................................... 47 CADMIUM AND GENERAL ANESTHETICS........................................................................ 49 ASPIRIN – INDUCED GASTRIC IRRITATION ...................................................................... 51 SIDE EFFECTS OF KETAMINE ....................................................................................... 53 XYLAZINE–INDUCED DIURESIS .................................................................................... 55 ERYTHROCYTE FRAGILITY TEST FOR IRRITANT DRUGS ....................................................... 57 PHARMACOVIGILANCE .............................................................................................. 59 POLYPHARMACY ...................................................................................................... 63 PRACTICAL ASPECTS OF PHARMACOKINETICS ................................................................. 67 BASIC ANALYTIC TOXICOLOGY TESTS ............................................................................ 71 COMMON ANTIDOTES .............................................................................................. 87 ORAL MEDIAN LETHAL DOSES (LD50) OF SELECTED CHEMICALS IN RATS ............................. 89 APPENDIX ............................................................................................................... 95 REFERENCES............................................................................................................ 99 INDEX .................................................................................................................. 105 ATTACHMENTS ...................................................................................................... 109

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Laboratory Guide in Toxicology

Table of Figures FIGURE 1: OPEN-FIELD ACTIVITY ................................................................ 30 FIGURE 2: NEGATIVE GEOTAXIS.................................................................. 31 FIGURE 3: STEPS FOR THE ELECTROMETRIC DETERMINATION OF BLOOD CHOLINESTERASE ACTIVITY ........................................................................ 39

FIGURE 4: STEPS FOR THE ELECTROMETRIC DETERMINATION OF TISSUE CHOLINESTERASE ACTIVITY ........................................................................ 40

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Laboratory Guide in Toxicology

The Author

Fouad Kasim Mohammad, BVMS, MS, PhD Professor, Pharmacology and Toxicology Vice President for Administrative Affairs, University of Mosul, Mosul, Iraq BVMS, College of Veterinary Medicine, University of Baghdad, Iraq, 1976.

MS, Veterinary Pharmacology, University of Missouri-Columbia, MO, USA, 1981.

PhD, Pharmacology/Minor in Neuroscience, University of MissouriColumbia, MO, USA, 1984.

F. K. Mohammad, BVMS, MS, PhD

Dean, College of Veterinary Medicine, University of Mosul, Iraq (2003-2012).

Winner of the "New Investigator Research Award� 1987, Behavioral Teratology Society, USA. The Distinguished First Professor of the University of Mosul, Iraq, 2001/2002.

Honored and distinguished by the Ministry of Higher Studies and Scientific Research-Iraq, for scientific achievements, Science Day, 2009.

Honored by the Ministry of Higher Education and Scientific Research for patent achievement, Science Day April 6, 2010.

Appreciation by the Minister of Higher Education and Scientific Research for heading the Committee of Deans of the Colleges of Veterinary Medicine, June 3, 2010.

Honored and distinguished by the Ministry of Culture (Iraqi Cultural House) for scientific innovation, Innovation Festival, April 4, 2011.

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The Author

Fouad Kasim Mohammad, BVMS, MS, PhD Honored and distinguished by the Ministry of Higher Education and Scientific Research for scientific innovation and publications, Science Day, 2011 & 2012.

Honored and distinguished by the Ministry of Higher Education and Scientific Research for obtaining the College of Veterinary Medicine international recognition for one of its programs (as a Dean) and for recognition of the Iraqi Journal of Veterinary Sciences among the journals in the same field (as an Editor-inChief), Science Day April 6, 2011.

Honored and distinguished by the Ministry of Higher Education and Scientific Research for recognition of the Iraqi Journal of Veterinary Sciences among the journals in the same field (as an Editor-in-Chief), Science Day April 8, 2012.

Editor-in-Chief of the Iraqi Journal of Veterinary Sciences.

Associate Editor, BMC Research Notes, BMC Veterinary Research. Member of the editorial boards of Journal of Animal and Veterinary Advances and Human and Veterinary Medicine.

Honorary member of the National Society of Phi Zeta, Pi Chapter, Columbia, Missouri, USA. Member of International Neurotoxicology Association.

Member of Iraqi Veterinary Medical Association.

Member of the International Brain Research Organization.

Member of Turkish Toxicology Society.

Supervision of 35 postgraduate students (Diploma, MSc & PhD).

Publications: Total of 216: Journal publications-150; Books-3; Abstracts-37; Contract pharmaceutical studies-26.

Scientific meetings attended and reports delivered: 73.

Patents: 1

Supervision postgraduate students: 1 Diploma, 25 MSc and 9 doctoral students (Total 35).

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Laboratory Guide in Toxicology

Preface-3rd edition All the experiments should comply with institutional regulations addressing animal use; proper attention, humane care and ethical considerations should be given to animals used in the experiments.

The standards set forth in the following guide should also be followed:

Guide for the Care and Use of Laboratory Animals Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council National Academy Press Washington, D.C. 1996 ISBN: 0-309-05377-3; Copyright 1996, by the National Academy of Sciences.

The Guide’s rules (which every research facility is obliged to maintain) must be observed in addition to any local, institutional, and governmental codes and regulations; even when despite the fact, this Laboratory Guide Toxicology may or may not unreservedly advocate a different approach.

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Laboratory Guide in Toxicology

Introduction The Laboratory Guide in Toxicology represents a blend of study obtained from years of academic and practical experience in pharmacology and toxicology. It is intended to cover a major part of practical toxicology curriculum for the colleges of veterinary medicine and allied health sciences. The guide is designed to acquaint the students of colleges of veterinary medicine and allied health sciences with practical aspects of toxicology, specially with sections on animal experimentation, so that the side effects of chemicals or their signs of poisoning can be demonstrated within the laboratory period which usually extends to two or three hours.

The doses given in the experiments in this technical guide were found to produce the desired effects. However, they may have to be adjusted because of the expected variations between different experimental conditions.

The students, researchers, and users of this guide are required to carefully review each subject and study the relevant subject matters before attending the laboratory; in addition, they are encouraged to observe, document, and record the results for any follow-up discussion and/or case studies.

The 3rd edition of the Laboratory Guide in Toxicology includes new chapters; some chapters have been updated and expanded. I would like to thank all colleagues who have expressed interest in the guide and have made helpful suggestions and criticisms.

FKM

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Laboratory Guide in Toxicology

Some Definitions in Toxicology Antidote A substance which antagonizes or reduces the toxic effect(s) of a toxicant in the living organism; e.g. atropine vs. acetylcholine.

Bioactivation Production of reactive chemical species which are more active or toxic than the parent substance or its metabolite.

Bioactivation usually results from the oxidation reaction (phase I) by the liver microsomal enzymes (Cytochrome P450); e.g. bioactivation of malathion into malaoxon.

Chronicity factor CF = (LD50) single dose/(LD50) 90 doses.

If CF > 2, the toxicant is relatively cumulative in the body of the living organism.

If CF < 2, the toxicant is relatively non-cumulative.

If CF = 90, the toxicant is absolutely cumulative.

Clinical toxicology This science studies the diseases induced by or associated with toxicants in man (clinical toxicology) or animals (veterinary clinical toxicology). It is also concerned with diagnosing, treatment and management of cases of poisoning.

Drug side effect An undesirable effect that results from using therapeutic doses of drugs in man or animals.

Environmental toxicology Science of environmental contamination or pollution, chemical residues in animal tissues and their impact on public health, human life and other living organisms including wildlife, birds, insects, fishes and other aquatic animals.

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Forensic toxicology The science of diagnosing and interpreting cases of poisoning and their associations with legal aspects.

Hazard The intrinsic ability of a substance to cause adverse effect(s) under a specified condition of use.

Iatrogenic disease A disease condition caused by exposure to chemicals in man or animals.

Industrial toxicology Science of poisoning that results from exposure of the living organism to chemicals used in industries.

Maximum tolerable dose The highest dose that the living organism can tolerate without the appearance of detectable toxic effect(s).

Median lethal dose (LD50) The dose of a chemical that kills 50% of the test animals. It is usually determined within 1-3 days.

Minimum toxic dose (MTD) The lowest dose of a chemical that causes detectable toxic effect(s).

NOAEL The maximum dose of a substance that does not induce detectable adverse effects in the living organism (noobserved-adverse effect level). NOEL is no-observed-effect level.

Poison (toxicant) Any substance that can adversely affect the processes of life after gaining access into the body or when applied on it. It does not include thermal or mechanical effects.

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Laboratory Guide in Toxicology

Pollutant (contaminant) A substance which occurs in the environment possibly by human activity and adversely affects the living organisms and/or their environment.

Risk The likelihood of a hazard causing an adverse effect under specific conditions of exposure or use.

Risk = hazard X exposure.

Toxicity The intrinsic ability of a quantity of a toxicant to induce poisoning in the living organism or cause death under certain conditions of use or exposure.

Toxicology The science that deals with poisons and poisoning. It is concerned with adverse effects of chemicals (e.g. insecticides, herbicides, drugs) on various systems of the living organism.

Toxicosis (poisoning) The state of being poisoned or intoxicated.

Toxicovigilance The process of identification, investigation and assessment of toxic effects produced by chemicals in the living organism and taking measures to reduce contaminations and control exposures.

Toxin A poison of natural origin; e.g. from insects (bee venom), animals (snake venom), plants (hypericin) and fungi (mycotoxins).

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Study questions

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• 1. Differentiate between Toxicity and Toxicology. Support your answer with examples.

• 2. Differentiate between Hazard and Risk.

Support your answer with examples.

• 3. What is meant by Toxicovigilance and Pharmacovigilance?

• 4. Give examples of five toxins.

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Laboratory Guide in Toxicology

Common Laboratory Animals Used in Toxicology

The mouse (Mus musculus)

Body temperature

37.4ยบC

Age at sexual maturity

35 days

Mating age

45-60 days

Estrus cycle

4-5 days

Gestation period

19-21 days

Litter size

6-11

Weaning age

21 days

Housing environment Temperature

21ยบC

Relative humidity

50%.

Light-dark cycle

12-12 h

Daily food intake

4-5 g

Daily water intake

7 ml

Surface area

20 g = 46 cm2

Routes of drug administration

Oral Subcutaneous Intramuscular Intraperitoneal

Volume of drug administration

5-10 ml/kg body weight

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The rat (Rattus norvegicus)

Body temperature

37.5ยบC

Age at sexual maturity:

40-45 days

Mating age

70-150 days

Estrus cycle

4-5 days

Gestation period

21-23 days

Litter size

6-11

Weaning age

21 days

Housing environment Temperature

22ยบC

Relative humidity

55%.

Light-dark cycle

12-12 h

Daily food intake

10-20 g

Daily water intake

20 ml

Surface area

200 g = 325 cm2

Routes of drug administration

Oral Subcutaneous Intramuscular Intraperitoneal

Volume of drug administration

1-2 ml/kg body weight

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Laboratory Guide in Toxicology

The rabbit (Oryctolagus cuniculus)

Body temperature

38.3-39.5ยบC

Age at sexual maturity:

4-6 months

Mating age

6 months

Estrus cycle

Continuous

Gestation period

30-32 days

Litter size

6-8

Weaning age

45 days

Housing environment Temperature

10-18ยบC

Relative humidity

40-45%

Light-dark cycle

12-12 h

Daily food intake

15-300 g

Daily water intake

150-200 ml

Surface area

1.5 kg = 1270 cm2

Routes of drug administration

Oral Subcutaneous Intramuscular Intraperitoneal

Volume of drug administration

0.25-2 ml/kg body weight

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The chicken (Gallus domesticus) Body temperature

40.5ยบC

Age at sexual maturity:

5-6 months

Incubation period

21 days

Hatching conditions Temperature

38.4ยบC

Relative humidity

60%

Housing environment Temperature

Chicks 32-35ยบC Adults 18-21ยบC

Relative humidity

50%

Light-dark cycle

23 h light -1 h dark

Daily food intake Daily water intake Routes of drug administration (One day old) Routes of drug administration (Adult) Volume of drug administration (One day old) Volume of drug administration (Adult)

One-day chicks 5 g Adults 150 g One-day chicks 34 ml Adults 160 ml Oral Subcutaneous Intramuscular Intraperitoneal Oral Subcutaneous Intramuscular 5 ml/kg body weight.

0.25-1 ml/kg body weight

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Study questions

Laboratory Guide in Toxicology

• 1. Can you house mice and rabbits in the same room? Explain your answer.

• 2. What is the volume of intraperitoneal injection in a mouse weighing 32 g?

• 2. What is the volume of oral administration in a rat weighing 240 g?

• 3. What are the preferred routes of antibiotic administration in chicks?

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Laboratory Guide in Toxicology

Essential Calculations in Toxicology Solubility of substances

Solubility

Solvent for 1 part of solute

Very soluble

< 1 part

Freely soluble

1-10 parts

Soluble

10-30 parts

Sparingly soluble

30-100 parts

Slightly soluble

100-1000 parts

Very slightly soluble

1000-10000 parts

Practically insoluble

> 10000 parts

Percentage (w/v) 10% solution (w/v) of sodium chloride = 10 g of sodium chloride/100 ml of solution (i.e. add 10 g of sodium chloride, then complete the volume to 100 ml with water).

Molarity Gram molecular weight of solute/1000 ml of solution.

0.1 M dichlorvos (MW 221) = 22.1 g/1000 ml To convert % to molarity M = (g/L) / MW = (percent X 10) / MW Percent = (M X MW) / 10 Milligram â&#x20AC;&#x201C; milliequivalent conversions mEq/L = (mg/L) X valence / MW mg/L = (mEq/L) X MW / valence

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Useful household measures 1 drop = 1/20 ml 1 teaspoonful = 5 ml 1 table spoonful = 15 ml 1 cup = 250 ml Dilution of a concentrate solution V1 X C1 = V2 X C2 V1 and C1 are the volume and concentration of the first (concentrated) solution; V2 and C2 are the volume and concentration of the second (diluted) solution.

Note: The units of C1 and C2 must be the same on both sides of the equation, such as mg/100 ml.

Parts per million (ppm) 1 ppm = 1 mg/kg, 1 µg/g, 1 mg/L 1 ppb = 1 µg/kg, 1 µg/L 1 ppt = 1 ng/kg, 1 ng/L Percentage equivalents 1 ppm = 1 mg/kg = 1/1000000 % = 1/1000000 X 100 = 1/10000= 0.0001% 10 ppm = 0.001% 100 ppm = 0.01% 10000 ppm = 1% Consumption of toxicant in feed of animals Level in feed (ppm) X kg feed intake / body weight in kg = mg/kg body weight Toxic level in feed (ppm) = mg/kg of toxicant / % of body weight consumed as food/day

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Study questions

Laboratory Guide in Toxicology

1. Prepare physiological saline solution from 5 g of sodium chloride.

2. Dilute 10 ml of 1:1000 potassium permanganate solution to 1:10000.

3. Convert 350 ppm to percent.

4. Convert 7.5% aqueous solution of acetylcholine iodide into molarity.

5. Calculate the amount of zinc chloride consumed by a goat, knowing that: Zinc chloride level in feed = 150 ppm; feed intake was 2.5 kg; body weight was 35 kg.

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Laboratory Guide in Toxicology

Drug Residues Treatment of animals with drugs may result in the deposition of drug residues or its metabolites in tissues. Drug residues may result from feed additives or treatment with drugs such as antibiotics and anthelmintics. Environmental contaminants may also appear in animal tissues or products. Residues are expressed as ppm, ppb or ppt.

Acceptable daily intake (ADI) The amount of a drug or its metabolite when consumed during the entire life-time of a person that appears to be without risk to health.

No-effect â&#x20AC;&#x201C; level (NOEL) It is the maximum level of the drug that does not induce adverse effect. It is fed to the most sensitive laboratory animal (e.g. rats) for 2 years.

A rat consumes 15 g diet/day. e.g. 100 ppm = 100 mg/kg feed.

Total intake is 1.5 mg/rat (200 g) or 7.5 mg/kg body weight.

ADI for humans is derived by taking 1% of the NOEL:

.

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Tolerance level Maximum allowable concentration of a chemical in feed.

Negligible tolerance Insignificant toxicologically chemical residue.

Finite tolerance Measurable concentration of a chemical residue that is permitted in feed.

Zero tolerance No drug residue is allowed in feed, because of extreme toxicity or carcinogenicity.

Calculation of tolerance Human body weight 60 kg Total daily intake 1.5 kg food Food factor values: Muscle 1 Liver of cows 2 (relative to muscles) Liver of sheep 5 (relative to muscles) Milk 0.1 X concentration in muscles Food factor X 1/3 of total daily diet

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e.g. T=16.5 X 10 X 60 / 0.33 X1.5 = 0.2 ppm / day Withdrawal period It is the time required for a chemical residue to reach safe concentration as defined by the tolerance. It indicates the time of animal removal from medication until the time of slaughter.

It varies according to dose of the drug, dosage form, route of administration and animal species.

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Study questions

Laboratory Guide in Toxicology

What is meant by each of the following?   

ADI Tolerance Withdrawal time

Support your answers with examples from literature.

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Laboratory Guide in Toxicology

Toxicity Testing The potential toxicity of a chemical can be detected and evaluated by several tests in laboratory animals. These toxicity tests include: A. General tests 1. Acute poisoning: A single or multiple exposures of the animal to the chemical under study within 24 h resulting in acute signs of poisoning which may end in death of the test animal.

2. Subacute poisoning: Repeated exposure of the animal to the chemical for less than 30 days. The test chemical is administered daily or other specified intervals. Survival time is usually longer than that of the acute poisoning.

3. Subchronic poisoning: Repeated exposure of the animal to the chemical for 30-90 days. Additive tissue damage should be expected with this type of toxicity testing.

4. Chronic poisoning: Repeated exposure of the animal to the chemical for more than 90 days for upto a year or two, or even the life span of the test animal.

Mice, rats, rabbits, chicks or dogs are usually used under controlled conditions in the above mentioned tests. The preferred route of administration of the test substance is orally or parenterally. In chronic studies, however, the oral route is usually selected. On the basis of data obtained from the acute toxicity testing (LD50), the toxic agents can be rated according to the severity of toxicity as follows: Class

Oral LD50 (mg/kg) in rats

Example

Supertoxic Extremely toxic Highly toxic Moderately toxic Slightly toxic Practically non-toxic Relatively harmless

<0.1 <1 1-50 50-500 0.5-5 g 5-15 g > 15 g

TCDD Parathion Arsenic DDT Nitrate Salt Corn

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Furthermore, the chronicity factor can be calculated as follows:

C. F. = Acute LD50 / 90-Day LD50 Example: Chemical

Acute LD50 (mg/kg)

90-Day LD50 (mg/kg)

C. F.

Warfarin

1.6

0.077

20.8

Caffeine

192

150

1.3

C.F. > 2 indicates relatively cumulative toxicity.

B. Special toxicity tests These include specified toxicity tests on certain tissues or systems in the body of the laboratory animal.

Examples: Reproduction, fertility, teratogenicity, carcinogenicity, mutagenicity, endocrine glands, skin, muscles, eyes, liver, ears, lungs, kidneys, heart, testes, ovaries, brain and spinal cord, blood, behavior, potentiation, drug interaction as well as environmental toxicity tests on birds and fishes.

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Study questions

Laboratory Guide in Toxicology

• 1. What are the differences between acute and subchronic toxicity tests?

• 2. From the literature find three chemicals with cumulative toxicity.

• 3. Name a specific acute toxicity test that involves the eyes.

• 4. Name a specific chronic toxicity test that involves the liver.

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Laboratory Guide in Toxicology

Neurobehavioral Toxicology Behavior It is the adjustment of the animal to its environment under specified conditions. It simply means what the animal is doing.

Neurobehavioral toxicology The science that studies changes in the animal behavior induced by toxicants taking into consideration the behavioral, biological and neurochemical mechanisms by which they occur. Neurobehavioral toxicology provides information relating to risk assessment based on neurotoxic endpoints.

Test animals Usually rats, mice, chicks or other birds are used in neurobehavioral toxicology tests.

Behavioral functions examined Three different behavioral functions are usually studied in laboratory animals: 1. Motor.

2. Sensory.

3. Complex behavior (learning and memory).

Altered behavior in animals could be an indication of a direct or indirect toxic effect upon the nervous systems of the test animal.

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Examples of primary level of neurobehavioral tests for rats or mice:

Function

Behavioral test

Motor Spontaneous activity

Activity in open-field or activity wheel

Ataxia

Hindlimb splay

Muscular weakness

Forelimb grip strength

Fatigability

Swim endurance

Tremor

Frequency of occurrence

Sensory Visual, olfactory

Localization

Pain

Tail flick, writhing (abdominal stretching)

Orientation in space

Negative geotaxis at 30ยบ or 45ยบ

Cognitive Learning and memory

One-way avoidance

Responsiveness

Novel environment

Consumatory behavior

Food intake, water intake

Thermoregulation

Rectal temperature

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Laboratory Guide in Toxicology

Behavioral measures included in a functional observational battery in rats:

Home-cage and openâ&#x20AC;&#x201C;field

Manipulative

Physiologic

Posture

Ease of removal

Body temperature

Convulsion

Handling reactivity

Body weight

Tremors

Palpebral closure

Food intake

Palpebral closure

Approach response

Water intake

Lacrimation

Touch response

Respiration

Piloerection

Tail pinch response

Heart rate

Salivation

Righting reflex

Urination

Vocalization

Landing foot splay

Defecation

Rearing

Forelimb grip strength

Urination

Hindlimb grip strength

Defecation

Pupil response

Gait

Click response

Mobility Arousal Stereotypy

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Measures of neurobehavioral test battery according to functional domains in rats Autonomic Lacrimation Salivation Pupil response Defecation Urination Neuromuscular Gait score Mobility score Landing foot splay Forelimb grip strength Hindlimb grip strength Righting reflex Sensorimotor Tail pinch response Click response Touch response Approach response CNS Excitability Ease of removal Handling reactivity Clonic movements Tonic movements Arousal Vocalization

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Laboratory Guide in Toxicology

CNS Activity Home cage posture Palpebral closure Rearing Motor activity Physiological Body weight Body temperature Piloerection Food intake Water intake Defecation Urination Respiration

Examples of neurobehavioral tests in 7-day old chicks Open-field activity Latency to move Lines crossed Jumping Pecking Defecation Stress calls Tonic immobility test Hold the chick in both hands and place it on a wooden table for 15 seconds. Withdraw the hands and time the chick to upright itself and stand unaided.

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Procedure Perform these neurobehavioral tests in mice: 1. Open-field activity: The test measures the general locomotor activity, exploration (squares crossed) and rearing including the frequency of defecation and urination.

Use a open box, the arena of which is divided into 25 equal squares (Figure 1). Place a mouse in the center of the arena, and count the number of squares crossed, rearings, fecal boluses and urine pools during 3-min period.

.

Alternative test for activity (Activity wheel)

Figure 1: Open-field activity

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Laboratory Guide in Toxicology

2. Negative geotaxis: The test reflects vestibular function, neuromotor performance and coordination. The angle of the slope is 45º or 25º (Figure 2). Place a mouse in a head down position on the inclined surface. Time the mouse for completing a 180º – turn. The maximum time allowed is 60 seconds.

Negative geotaxis at 45°

Negative geotaxis at 25°

Figure 2: Negative geotaxis

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Study questions

1. Name five toxicants that affect animal behavior.

2. What is the value of monitoring animal behavior?

3. Referring to functional observational battery in rats, determine the quantal, rank order, descriptive and interval variables.

4. What is tonic immobility test in 7-day old chicks?

5. What can be measured in the open-field test in mice and chicks?

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Laboratory Guide in Toxicology

Determination of the Median Lethal Dose (LD50) Median lethal dose (LD50) The dose of a chemical that causes death in 50% of the test animals.

LC50: It is the concentration of the chemical (e.g. in the air or water) that causes death, usually within 4 hours, in 50% of the test animals.

Usually with new compounds, the starting point in toxicity assessment is the determination of chemical-induced lethality (e. g.

LD50). The LD50 depends on a quantal doseâ&#x20AC;&#x201C;response relationship, since lethality is precisely quantal and unequivocal.

Rats or mice are commonly used as experimental animals. Other animal species such as chicks and rabbits can also be used. The route of chemical administration is usually oral, subcutaneous or intraperitoneal in mice and rats. Factors that affect LD50 value are sex, age, diet, species, strain, temperature, crowding, stressors, diseases, etcâ&#x20AC;Ś Information obtained from the LD50experiment 1. Estimation of the LD50.

2. Type of toxic effects.

3. Onset of signs of poisoning.

4. Duration of poisoning.

5. Bioavailability of the test chemical by comparing the LD50 values obtained by different routes of administration.

6. Used with the median effective dose (ED50) to calculate the therapeutic index. TI = LD50 / ED50 Disadvantages 1. Large numbers of laboratory animals are used.

2. Usually, the slope of the curve is not reported.

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Procedure 1. Administer thiopental Na, intraperitoneally to adult mice, using 10 mice for each dose group (see the table below for the doses). The volume of administration is 10 ml/kg body weight.

2. Observe each dose group separately.

3. Record the number of deaths at each dose level by checking the heart beat 30 min after thiopental injection.

4. Plot % of lethality against the dose of thiopental on a semilog graph paper.

5. From the curve determine the value of LD50.

Tabulate your results in the following table.

Use the graph papers (linear and semilog) provided in the attachment to determine the LD50 value of thiopental Na in mice.

Dose (mg/kg, i.p.)

75 100 125 150 175 200 225 250

No. of mice used 10 10 10 10 10 10 10 10

No. of mice died

% Dead

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Laboratory Guide in Toxicology

Study questions

1. Determine the LD1 from the curve.

2. What is the cause of death in mice treated with a high dose of thiopental?

3. What are the information obtained from the experiment?

4. What are the alternatives to this type of LD50 experiment?

5. Plot the data using a linear graph paper. What is the main difference from the semilog graph paper?

6. Define TI.

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Laboratory Guide in Toxicology

Determination of Plasma Cholinesterase Activity by an Electrometric Method Cholinesterases hydrolyze esters of choline such as acetylcholine.

Two main types of cholinesterases exist: 1. Acetylcholinesterase (true cholinesterase; EC, 3.1.1.7). It is mainly found in the central nervous system, erythrocytes of mammals (not the birds!) and at the neuromuscular junction.

2.

Butyrylcholinesterase (pseudocholinesterase, nonspecific cholinesterase; EC, 3.1.1.8). It is mainly found in the plasma, liver and at synapses with acetylcholinesterase.

Physiologically, acetylcholinesterase hydrolyzes acetylcholine at the nerve endings into choline and acetic acid. The enzyme is inhibited to various extents by organophosphate and carbamate insecticides.

Approximately 30% decrease in enzyme activity indicates exposure of the animal or man to cholinesterase inhibitors.

Cholinesterase assay techniques Several assay techniques are available for the determination of cholinesterase activity in the plasma, erythrocytes, liver or brain tissues: 1. Electrometric technique (e. g. Michel' s method): It measures the pH change in the reaction medium following hydrolysis of acetylcholine into acetic acid and choline.

2. Hestrin method: It utilizes the reaction of acetylcholine with hydroxylamine and ferric chloride producing a reddish â&#x20AC;&#x201C; purple complex which is measured at 515 nm by a spectrophotometer.

3. Ellman's procedure: Acetylthiocholine iodide is hydrolyzed by acetylcholinesterase producing thiol groups. Quantification of the sulfhydryl groups can be achieved by coupling the reaction to 5,5-dithiobis â&#x20AC;&#x201C;(2nitrobenzoic acid) (DTNB) and measuring the colored product spectrophotometrically at 412 nm.

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F. K. Mohammad

Modified Electrometric Procedure for Measuring Cholinesterase Activity A modified simple, yet accurate, electrometric method is outlined below for measuring blood (plasma, serum, erythrocytes or whole blood) cholinesterase activity in laboratory animals (e. g. rats, mice or plasma or serum in chickens), (Figure 3).

Tissue cholinesterase activity can be measured too as outlined in figure 4.

Substrates The substrates that can be used in the reaction mixture with this electrometric technique include: Acetylcholine iodide Acetylthiocholine iodide Acetylcholine chloride Acetylcholine bromide Species variations Species differences should be expected when measuring blood (plasma, serum, erythrocytes or whole blood) or tissue cholinesterase activities in different animal species. Individual variations also exist.

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Laboratory Guide in Toxicology

3 ml distilled water + 0.2 ml aliquot of plasma, erythrocytes or whole blood + 3 ml barbital-phosphate buffer (pH 8.1)

Measure pH (pH1)

Add 0.1 ml 7.1% acetylcholine iodide

Incubate at 37˚C (e.g. 20 min in man, 30 min in rats)

Measure pH (pH2)

Cholinesterase activity (∆ pH/incubation time) = pH1-pH2- (∆ pH of blank, without sample) Figure 3: Steps for the electrometric determination of blood cholinesterase activity

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F. K. Mohammad

Homogenize tissue (e.g. brain, liver) 100 mg wet weight/3 ml barbital-phosphate buffer (pH 8.1)

3 ml distilled water + 0.2 ml aliquot of tissue homogenate + 3 ml barbital-phosphate buffer (pH 8.1)

Measure pH (pH1)

Add 0.1 ml 7.1% acetylcholine iodide

Incubate at 37˚C for 30 min

Cholinesterase activity (∆ pH/30 min) = pH1-pH2- (∆ pH of blank, without tissue sample) Figure 4: Steps for the electrometric determination of tissue cholinesterase activity

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Laboratory Guide in Toxicology

Tabulate your results as follows:

Sample

pH1

pH2

â&#x2C6;&#x2020;pH

0.2 ml plasma

Study questions

Blank (no plasma)

1. What is the value of measuring blood cholinesterase activity?

2. Which method is preferred for measuring cholinesterase activity in case of carbamate poisoning?

3. What is the physiological role of acetylcholinesterase in the nervous tissue?

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Laboratory Guide in Toxicology

Organophosphate and Carbamate Insecticides Poisoning Organophosphates and carbamates are widely used insecticides in veterinary practice against ectoparasites, and they have public health importance.

Examples of organophosphate insecticides: Dichlorvos, malathion, coumaphos, diazinon, parathion.

Examples of carbamate insecticides: Carbaryl, methomyl, propoxur, aldicarb.

Mechanism of toxic action Organophosphate insecticides irreversibly inhibit acetylcholinesterase activity leading to accumulation of acetylcholine at the nerve endings and subsequently causing parasympathetic overstimulation. Carbamate insecticides cause reversible inhibition of acetylcholinesterase.

Clinical signs of poisoning 1. Muscarinic effects: salivation, lacrimation, urination, defecation, miosis and dyspnea.

2. Nicotinic effects: muscle twitching and fasciculation, and paralysis.

3. Central nervous system effects: excitation, convulsions followed by depression.

4. The cause of death is respiratory failure.

Diagnosis 1. Measure blood (whole blood, plasma, serum or erythrocyte) cholinesterase activity. Approximately 30% decrease in enzyme activity is an indicative of exposure to acetylcholinesterase inhibitors.

2. Response to treatment with atropine sulfate.

3. Analyze the blood and tissues for cholinesterase inhibitors.

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Antidotes 1. Atropine sulfate 0.2 mg/kg to effect (mydriasis and dry mouth).

2. Pralidoxime (2-PAM) 20 mg/kg used in the early stage of organophosphate (but not carbamate) poisoning.

3. Diphenhydramine may reduce nicotinic effects.

4. Diazepam to control convulsions.

Procedure 1. Prepare 5% solution of dichlorvos: 1 ml dichlorvos 50% EC + 9 ml distilled water.

2. Administer 0.25 or 0.5 ml of 5% dichlorvos orally to one rat.

Observe the animal for the appearance of signs of poisoning.

3. To a second rat, administer dichlorvos as before, followed immediately by atropine sulfate 1% at 0.1 ml intraperitoneally.

Repeat atropine treatment every 15 minutes as needed. Observe the animal for the appearance of signs of poisoning.

Rat No.

Treatment

1

Dichlorvos

2

Atropine + Dichlorvos

Latency to onset of tremors (min)

Signs of poisoning

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Laboratory Guide in Toxicology

Study questions

1. What is the mechanism of antidotal actions of 2-PAM and atropine in case of organophosphate poisoning?

2. What are the differences between organophosphate and carbamate poisoning?

3. What is meant by atropinization?

4. What are the mechanisms of toxic action of carbamate and organophosphate insecticides in animals?

5. How can you treat organophosphate and carbamate poisonings in animals?

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Laboratory Guide in Toxicology

Neurotoxicity of Lindane Lindane, the gamma isomer of hexachlorocyclohexane is used as an insecticide in man and animals. It is considered to be a neurotoxicant.

Mechanism of toxicity Lindane exerts anti-GABA-ergic action by inhibiting GABA â&#x20AC;&#x201C; activated chloride ions flux via binding to picrotoxinin â&#x20AC;&#x201C; receptor on the GABA ionophore.

Signs of poisoning Excitation, impairment of motor activity, tonic-clonic convulsions, hypothermia, hyperesthesia, anorexia and abnormal posture.

Stimulation of the patient by sound, light or touching may precipitate convulsions.

Treatment No specific antidote exists. Use diazepam to control convulsions.

Procedure 1. Prepare 1% aqueous solution of lindane from a commercial insecticidal preparation (e. g. 20%).

2. Treat mice with lindane at 50, 75 or 100 mg/kg, orally by a gavage needle. The volume of administration is at 10 ml/kg.

3. Observe the mice for the appearance of signs of poisoning and any abnormal posture in the cage.

Mouse No.

Lindane (mg/kg, orally)

1

50

2

75

3

100

Latency to onset of convulsions (min)

Signs of poisoning

47

Study questions

F. K. Mohammad

1. In which way does lindane poisoning differ from that of the organophosphate insecticides?

2. What are the neurotoxic effects of lindane?

3. Do you recommend using lindane in clinical practice anymore?

Why?

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Laboratory Guide in Toxicology

Cadmium and General Anesthetics Cadmium (Cd) constitutes about 0.000011% of the earth's crust. It is a non-essential toxic metal which is considered to be one of the environmental pollutants. Cd is widely used in industries, e. g. ceramics, enamelware, plastics, batteries, coloring pigments, etc.

In the body, the highest Cd concentrations are found in the kidneys and liver. Approximately less than 5% of an oral Cd dose is absorbed. Absorption of inhaled Cd is much higher (10-40%).

Acute Cd intoxication causes hepatitis, testicular degeneration and central nervous system (CNS) effects manifested as depression with reductions in general locomotor activity. Cd also modifies the response of the animal to CNS active drugs such as sedatives, hypnotics and anesthetics. The kidneys are affected after chronic exposure to Cd. Procedure 1. Use two male adult mice.

2. Treat the first mouse with physiological saline solution at 10 ml/kg body weight, intraperitoneally. This will be the control.

3. Treat the second mouse with cadmium chloride at 2.5 mg/kg, injected intraperitoneally in a volume of 10 ml/kg.

4. Fifteen minutes later, treat both mice with the general anesthetic mixture ketamine (100 mg/ kg) and detomidine (0.6 mg/kg), intraperitoneally.

5. Determine the latency to onset of anesthesia (loss of righting reflex) and its duration (sleep time) in both mice.

Notes 1. Observe the writhing response in the Cd â&#x20AC;&#x201C; treated mice.

2. Keep the survived mice for the next laboratory period, and then observe the testicular size.

3. Try pentobarbital Na (35 mg/kg, i.p.) instead of detomidine â&#x20AC;&#x201C; ketamine mixture.

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Mouse No.

Treatment

1

Saline + ketaminedetomidine (control)

2

Cd + ketaminedetomidine

Latency to onset of anesthesia (min)

Duration of anesthesia (min)

Study questions

1. What are the antidotes of Cd? Are they effective clinically?

2. What are the toxic effects of Cd on the liver, kidney and testes?

3. Were the Cd-treated mice depressed or excited?

4. Is there any alternative alpha-2 adrenergic drug instead of detomidine? Name two.

5. What is the writhing response?

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Laboratory Guide in Toxicology

Aspirin â&#x20AC;&#x201C; Induced Gastric Irritation Aspirin (acetylsalicylic acid) is an analgesic, antipyretic and antiinflammatory agent. Aspirinâ&#x20AC;&#x201C;induced poisoning is characterized by hyperpnea, metabolic derangement and acidosis. Aspirin also irritates the gastric mucosa causing erosion, congestion, edema and hemorrhage.

Procedure 1. Fast two adult rats overnight. Allow free access to water.

2. Suspend aspirin in 1% carboxymethylcellulose at 25 g/100 ml.

3. Six hours before the laboratory period, administer aspirin to rat No. 1 at 500 mg/kg, orally by a gavage needle. Treat the rat No.2 (control) with the vehicle at 2 ml/kg, orally, and it will be used for comparison.

4. Observe the rats for any adverse effects.

5. Kill the rats with ether overdose and excise the stomach and front part of the small intestine. Incise the intestine and stomach along the lesser curvature and expose the mucosal surface.

6. Note and record any gross congestions and hemorrhages.

7. Wash the mucosa with physiological saline solution and observe any visible lesions.

Rat No.

Treatment

1

Aspirin

2

Vehicle (control)

Lesions

51

Study questions

F. K. Mohammad

1. How can you treat aspirin overdose in dogs?

2. What are the routes of aspirin excretion from the body?

3. What is the beneficial effect of coated tablets of aspirin?

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Laboratory Guide in Toxicology

Side Effects of Ketamine Ketamine is a dissociative general anesthetic commonly used with other sedatives or anesthetics. It antagonizes aspartate receptors in the brain causing functional disruption (dissociation) together with activating the limbic system in the brain.

Ketamine increases the heart rate and may depress respiration.

During ketamine anesthesia, muscle relaxation is not good, as it is poor, and patient suffers from rigidity. Many reflexes such as swallowing, coughing, corneal and pedal are maintained in animals.

The 2-adrenoceptor agonists xylazine, detomidine and medetomidine are used with ketamine to improve the quality of general anesthesia and to induce good muscle relaxation in animals.

Procedure 1. Use two adult rabbits.

2. Observe the animals before induction of anesthesia, and measure the respiratory rate for each one of them.

3. Inject rabbit No. 1 with ketamine HCl intramuscularly at 40 mg/kg.

4. Inject rabbit No. 2 with ketamine HCl intramuscularly at 40 mg/kg, followed immediately by xylazine HCl at 5 mg/kg, intramuscularly.

5. Time the onset and duration of anesthesia (loss of righting reflex).

6. Measure the respiratory rate of each rabbit at 15, 30 and 45 minutes after the injection.

7. Observe each rabbit for the quality of muscle relaxation and whether the reflexes (eye, pedal, swallowing) are maintained or not.

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Rabbit No. 1 Rabbit No. 2 (ketamine) (ketamine+xylazine)

Variable Respiratory rate/min 0 (pretreatment) 15 min 30 min 45 min Muscle relaxation* Reflexes* Swallowing Eye Pedal

Study questions

*Indicate (+) for the presence and (-) for the absence of muscle relaxation and/or reflexes.

1. Can you perform abdominal surgery with ketamine alone in animals? Explain the reason for your answer.

2. What are the side effects of ketamine anesthesia in dogs?

3. Name an antidote for ketaminexylazine combination.

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Laboratory Guide in Toxicology

Xylazine–Induced Diuresis Xylazine is a sedative and analgesic agent with muscle relaxant properties. It is combined with general anesthetics such as ketamine to produce balanced anesthesia in animals. Xylazine stimulates central presynaptic alpha 2-adrenoceptors resulting in reduced catecholamine synthesis and release from the nerve endings.

Xylazine–induced side effects in animals are characterized by hypotension, bradycardia, reduced intestinal motility, emesis (cats), decreased respiratory rate and frequent urination.

Yohimbine and atipamezole could be used to antagonize the effects of xylazine in animals and to enhance recovery from central nervous system depression and sedation.

Procedure 1. Inject rat No.1 with physiological saline solution at 1 ml/kg, intraperitoneally (control).

2. Inject rats No.2 and 3 with xylazine at 3 and 6 mg/kg, intraperitoneally, respectively. The volume of injection is 1 ml/kg.

3. Immediately after saline or xylazine injection, place each rat in a metabolic cage to collect urine for 3 hours.

4. Measure the volume of urine collected for each rat.

5. Calculate: µ

(µ µ µ

µ

µ µ

µ

6. Na and K concentrations are measured by a flame photometer.

Osmolality is measured by freezing point depression osmometer.

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F. K. Mohammad

Rat No.

Xylazine (mg/kg, i.p.)

Urine volume (ml) 1h

1

0 (control)

2 3

3 6

Xylazine (mg/kg, i.p.)

1

0 (control)

2

3

3

6

Study questions

Rat No.

Urine flow (µl/min)

Na conc. (µmol/ml)

2h

Total urine volume (ml)

3h

K conc. (µmol/ml)

Osmolality (mOsmol/kg)

Osmolal excretion (µOsmol/ min )

1. What is the mechanism of diuretic action of xylazine?

2. Do other alpha 2 – agonists possess diuretic actions?

3. What are the precautionary measures needed in treating dehydrated animals with xylazine?

4. What is the effect of xylazine on blood glucose level in animals?

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Laboratory Guide in Toxicology

Erythrocyte Fragility Test for Irritant Drugs Many parenteral drug preparations when administered intramuscularly in animals may cause local tissue irritation and damage. Irritant effects of these preparations may be monitored as follows: 1. Measuring serum or plasma creatine phosphokinase activity 10-24 h after intramuscular injection of the preparation.

2. Histopathological evaluation of muscle damage at the injection site.

3. In vitro erythrocyte hemolysis test.

4. Cytotoxic effect on cultured cells.

Erythrocyte fragility (hemolysis) test is a rapid and easy screening test for the assessment of local tissue toxicity of injectable preparations of drugs. Hemolysis in hypotonic or irritant solutions is an index of erythrocyte stability.

Several factors may contribute to local tissue irritating effects of drug preparations. These include the volume of injection, drug concentration, pH, buffering capacity of the solution, solvents and the excipients used in the pharmaceutical preparations.

Procedure 1. Prepare sheep erythrocytes suspension in physiological saline solution.

2. Prepare test tubes containing saline and the test substance (e. g. oxytetracycline 20%) as shown in the table.

3. Add one drop of erythrocyte suspension to the prepared test tubes containing saline and test substance.

4. Allow the tubes to stand for 2 hours at room temperature.

5. Record the concentration for the beginning of hemolysis and complete hemolysis

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F. K. Mohammad

Test tube No.

Saline (ml)

Test substance (ml)

1 2 3 4 5 6

0 0.2 0.4 0.6 0.8

1.0 0.8 0.6 0.4 0.2

1.0

0

(-ve control)

7

Study questions

(distilled water 1 ml, +ve control)

1. Name 5 drug preparations that cause muscle irritation after intramuscular Injection in: a. sheep b. dogs c. man 2. What are the factors that influence local tissue irritating ability of injectable drug preparations?

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Laboratory Guide in Toxicology

Pharmacovigilance Pharmacovigilance Pharmacovigilance is concerned with monitoring of the safety, quality and efficacy of marketed medicines in man and animals. It is a system for reporting adverse drug events. Veterinary pharmacovigilance is a monitoring program for adverse effects associated with the use of drugs in animals.

The primary purpose of veterinary pharmacovigilance is to detect problems or side effects with the use of animal drugs and vaccines once they are available in the market for use in clinical practice.

Pharmacovigilance also monitors adverse events in humans as a direct result of using or administering an animal drug, or accidental human exposure. Environmental effects or effects on wildlife are also considered. Extra label use of animal drugs and human drugs used in veterinary practice should be considered too. Veterinary pharmacovigilance ensures safe use of medicines in animals.

Adverse Drug Event (ADE) It is either an undesired side effect, or lack of a desired therapeutic or prophylactic effect. ADE includes any side effect, injury, toxicosis or allergic reaction (or lack of effect as expected) associated with use of an animal drug.

Adverse Drug Reaction (ADR) A reaction which is harmful and unintended that occurs at therapeutic doses usually used in animals for treatment of disease conditions as well as for prophylaxis and diagnosis of diseases. Lack of effectiveness is also a side effect, e.g. anesthetics, tranquilizers, anthelmintics, antibiotics (depends on more than one report or a group of reports). Usually further investigations are needed.

Pharmacovigilance is needed in every country because of the differences in the diseases and prescribing practice, genetic variations, diets, tradition, drug manufacturing processes and ways of drug distribution and use.

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Why pharmacovigilance is needed?

Monitoring is necessary since not all drug's effects are known at the time of its introduction into the market. Diverse patients are expected for drug usage. Tests in animals are insufficient to predict clinical safety. Patients used in clinical trials are selected and limited in number, the conditions of use differ from those in clinical practice and duration of trials is limited. From statistics point of view 5000 subjects are needed for detecting common ADR, and 30000 subjects are needed to be treated with a drug to be sure that we do not miss one patient with one an ADR which has an incidence of 1 in 10000 exposed individuals. Furthermore, chronic toxicity, information in young, elderly, drug-drug interactions are often incomplete or not available.

What are the ADR that should be reported?

1. ADR causing death.

2. Unexpected signs of toxicosis.

3. ADR not mentioned in the label of the medicinal preparation or package insert.

4. Off label use of drugs causing ADR in man or animals.

5. Veterinary drugs causing human illness.

6. Lack of expected efficacy.

7. Unsafe tissue residues resulting from drug usage in animals.

8. Adverse environmental effects.

9. Increased frequency of ADR.

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Laboratory Guide in Toxicology

Suggested ADR reporting form ADR REPORT IN ANIMALS Name of the veterinarian Date: Address: Name of the animal owner: Address: Name of the drug (or preparation): Batch No.: Manuf. Date: Exp. Date: Name of the Company: Animal species affected: Sex: Age: No. treated: No. affected: Dose: Route of administration: Duration of therapy: Reason of treatment: Nature of the adverse effect: Time of appearance of the ADR: Duration of ADR: Clinical examination and diagnostic tests performed: Drugs used with the main therapy: Humans affected: Environmental effects: Signature:

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F. K. Mohammad

Study questions

1. Examine prescription practice in the nearest clinic or hospital to you, and report any ADR. 2. Name the medicines that you have found to produce ADR not reported previously.

3. What is toxicovigilance?

4. What is the task of veterinary pharmacovigilance?

5. Name the pharmaceutical preparations that you have found to be ineffective in the clinical practice.

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Laboratory Guide in Toxicology

Polypharmacy Polypharmacy It is the use of multiple medications for the treatment of patients suffering from certain illnesses. That is, the use of medications more than the need of the patient. Elderly people are usually at higher risk of adverse drug effects resulting from polypharmacy. The number of prescribed drugs that constitutes polypharmacy is usually 4 or more.

The estimated risk of adverse drug events from 2, 5 and 7 drugs or more is 13, 58 and 82%, respectively (Fulton and Allen, 2005).

Factors that might lead to polypharmacy 1. Age of the patient. Multiple medications are used to treat multiple health-related conditions in the elderly.

2. Morbid medical conditions.

3. Urgent and recent hospitalization.

4. Difficulty in diagnosing the disease condition.

5. Multiple prescriptions by many physicians.

6. Changing prescription without stopping the previous one.

7. Lack of patient education about drug compliance.

Adverse effects of polypharmacy 8. Drug interactions and adverse drug reactions.

9. Drug waste.

10. Non-compliance.

How to avoid polypharmacy 1. Accurate diagnosis and medication.

2. Accurate medical history.

3. Link each medication to the required therapeutic response.

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F. K. Mohammad

4. Identify medications used to treat side effects of the main therapeutic initially prescribed.

5. Used drug-combination products.

6. Educate the patient about the medications when transferring him/her from the hospital to home.

7. Avoid unnecessary drugs.

8. Reduce the use of high risk drugs especially in the elderly.

9. Outweigh the potential benefits of drug combinations against the risk of harm.

10. Monitor any therapeutic failure to stop the medication.

Laboratory work (to be conducted at the university teaching hospital) 1. Collect the prescriptions that have more than two drugs from medical records of the hospital.

2. Categorize the drugs according to the therapeutic use.

3. Tabulate the mean medications used each day vs. the number of the patients treated.

4. From a pharmacology textbook obtain and tabulate the main side effects of the drugs used.

5. List any side effect seen in the patients.

6. Identify any unnecessary drug therapy.

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Laboratory Guide in Toxicology

Study questions

• 1. What are the main classes of drugs that can contribute to polypharmacy?

• 2. What are the benefits of polypharmacy?

• 3. What are the risks of harm associated with polypharmacy?

• 4. Is there polypharmacy in veterinary practice? Elaborate from your clinical experience.

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Laboratory Guide in Toxicology

Practical Aspects of Pharmacokinetics Pharmacokinetics Pharmacokinetics is quantitative time course of drug absorption, distribution, metabolism and excretion. Toxicokinetics may be used in case of a toxicant.

Volume of distribution (Vd) The volume of the body into which a drug or toxicant is apparently distributed. The apparent Vd is expressed as L/kg of body weight.

Large Vd implies distribution of the drug throughout the total body fluid or concentration in certain tissues.

Vd=Dose/Cp(0) Half-life (t1/2) It is the time needed for the drug concentration in the blood (plasma or serum) to decrease by 50%. The t1/2 guides the clinician in determining the interval of drug dosing. t1/2=0.693/kel Elimination rate constant (kel) It is the first order elimination rate constant which indicates the fraction of drug present at any time that would be eliminated per unit of time; e.g. kel of 0.1 h-1 means that 10% of the drug is eliminated in one hour.

Plasma clearance (Clp) It is the volume of plasma (or blood) cleared of drug by an organ in a unit of time.

Clp= Vd x kel

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F. K. Mohammad

Bioavailability It is the rate and extent of drug absorption into the systemic circulation. The response to a drug is usually related to the area under the plasma concentration vs. time curve (AUC). This area is a usual index of drug availability following extravascular administration.

Bioavailability of a drug after intravenous administration is considered to be 100%, as there is no absorption phase.

After a bolus intravenous injection of a drug, there would be usually a decline in plasma drug concentration with time.

Plasma Drug Concentration (mg/L)

1.92 1.76 1.6 1.44 1.28 1.12 0.96 0.8 0.64 0.48 0.32 0.16 0 0

1

2

3

4

5

6

7

8

9

10

11

12

Time (h)

Exponential decline of plasma drug concentration can be plotted on a semilogarithmic graph paper (log 10) to obtain a straight line:

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Laboratory Guide in Toxicology

Laboratory work Drug X was administered to a patient (40 kg, body weight) at 200 mg, i.v.

The following results were obtained: Time (h) Plasma concentration (mg/L) 1 20 2 15 4 6.9 8 3.3 12 1.3 Use a semilog paper to plot plasma concentration vs. time.

Calculate each of the following: Half-life.

Elimination rate constant.

Determine C0.

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F. K. Mohammad

Volume of distribution.

Plasma clearance of X. AUC0-∞ Do not forget the units!!

Study questions

• 1. Define elimination rate constant (Kel).

• 2. What is the value of measuring Vd? • 3. What is the equation that can be used to calculate t1/2 from the value of Kel and Clp from Vd and Kel ?

• 4. What are the benefits of measuring AUC for drugs?

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Laboratory Guide in Toxicology

Basic Analytic Toxicology Tests The veterinarian may be faced with cases of sudden death in animals suspected of being poisoned. Certain qualitative toxicologic screening tests can be performed for tentative initial diagnosis or to rule out certain poisoning as the probable cause of death. In all suspected cases of poisoning one part of the specimen should be sent to a well equipped certified veterinary diagnostic laboratory to perform complete toxicologic examination on a quantitative basis.

Basic equipment and supplies for analytical toxicology laboratory Analytical and top-pan balances.

Bench-top centrifuge.

Chemical supplies (buffers, NaCl, standards, etcâ&#x20AC;Ś) Cleaning supplies.

Distilled water.

Freezer.

Gas burner.

Glassware.

Incubator.

Microscope.

Nitrogen.

Osmometer.

Oven. pH meter pipettes.

Porcelain tiles.

Refrigerator.

Shaker.

Spectrophotometer (U/V, VIS) Thin-layer chromatography and its supplies.

Vortex-mixer.

Water-bath.

Water still.

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F. K. Mohammad

The steps needed in any systematic toxicological analyses are: 1. Sample preparation which tends to retain the poisonous substance through the sampling process. The preparation of the sample for further analysis includes homogenization, digestion, hydrolysis, extraction or derivatization.

2. Differential detection of the poison: This step identifies the poisonous substance by qualitative and quantitative analytical tests. These tests include color tests, spectroscopic methods, immunological assays, thin layer chromatography, gas chromatography and high performance liquid chromatography.

Usually more than one analytical method is needed for definite identification of the substance under examination. You should always compare the results obtained with authenticated standards.

Types of samples usually obtained for analytic toxicological tests 1.

2.

3.

4.

5.

Blood.

Body fluids (other than blood).

Excretory fluids and residues.

Tissues and other specimens.

Toxic plants.

The following toxicologic tests are preliminary screening procedures.

The results gained from these tests may be used for the purpose of selecting therapeutic measures only. However, the results should be confirmed by a certified toxicology laboratory.

Salicylate test a. Reaction reagent: 4 g ferric nitrate + 4 g mercuric chloride + 12 ml HCl (1M). Dilute to 100 ml with distilled water (Fe[NO3]3; 100 mmol/L; HgCl2 14.7 mmol / L; HCl 120 mmol/L). b. 1 drop of reaction reagent + 3 drops of serum. A violet color indicates the presence of salicylate(s) in the serum sample.

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Laboratory Guide in Toxicology

c. When the sample is urine: Boil the urine to remove diacetic acid which interferes with the reaction. Then use: 1 ml of urine + 5 drops of reaction reagent.

Alternative test for aspirin a. 2 ml of urine +1 ml of 10% ferric chloride solution. A purple color indicates the presence of salicylate. b. 5 ml of whole blood + 0.2 ml of concentrated HCl. Extract with ethylene dichloride. Discard the upper layer. Add 4 drops of 10% ferric chloride + 2 ml of distilled water and shake well. A purple color indicates the presence of salicylate. c. 1 drop of 10% ferric chloride solution + 2 drops of serum. A purple color indicates the presence of salicylate.

Acetaminophen test a. o-Cresol reagent: (0.096 mol/L). 10 ml of o-cresol diluted to 1 L with distilled water. b. 1 ml of urine + 1 ml of concentrated HCl. Heat in a boiling water bath for 10 min. Take 0.1 ml of this mixture and add 0.9 ml of o-cresol reagent. Add 2 ml of 4 M ammonium hydroxide.

A blue color indicates the presence of acetaminophen.

Phenothiazines a. Reaction reagent: 5 ml of ferric chloride solution (5%) + 45 ml of 20% w/w solution of perchloric acid + 50 ml of 50% v/v solution of nitric acid.

b. 1 ml of urine +1 ml of reaction reagent. Pink, red, orange, violet and blue colors indicate the presence phenothiazine derivatives. Beware! The colors are transient.

Alternative test for phenothiazine derivatives 2 ml of urine + 6 drops of concentrated sulfuric acid + 2 drops of 10% ferric chloride solution.

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Development of a light pink to purple color is an indication of the presence of phenothiazine tranquilizers in the sample.

Diphenylamine test Prepare 0.5% solution of diphenylamine in sulfuric acid (60% v/v). Apply few drops of this reagent to the biologic sample.

Development of a blue color indicates the presence of oxidizing compounds such as nitrate, nitrite, permanganate, manganese, chromate, bromate and chlorate.

Dithionite test for paraquat and diquat 1 ml of urine + 1 ml of 0.1% sodium dithionite in M sodium hydroxide (freshly prepared).

Development of a blue color indicates the presence of paraquat, whereas a green color indicates the presence of diquat in the sample.

Sulfonamides Dissolve a suitable amount of the sample in 0.1 M sodium hydroxide. Add 1% solution of copper sulfate drop by drop until the color is developed.

Development of green, blue or brown colors indicates the presence of sulfonamides.

Alternative test for sulfonamides Place a drop of urine or the unknown drug on a piece of filter paper. Place one drop of concentrated HCl on the sample.

Development of an intense orange color indicates the presence of sulfonamides.

Phenol a. 10 ml of urine + 1 ml of 20% ferric chloride. Development of purple color indicates the presence of phenol. b. Reaction reagent: 10 g mercury in 20 ml nitric acid. Dilute with equal quantity of distilled water. Stand for 2 hours and decant

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the excess of water. Boil 10 ml of urine with 1-2 ml of the reaction reagent. Development of a red color indicates the presence of phenol.

Strychnine a. 3 ml of filtered stomach content + 5 ml of concentrated sulfuric acid. b. Control: 3 ml of distilled water + 5 ml of sulfuric acid. c. Add a few crystals of potassium dichromate, and shake gently. d. Development of blue, violet, red and orange colors from the precipitating crystals indicates the presence of strychnine in the sample.

Cyanide a. 50 g of finely ground tissue or stomach content in 100-ml flask. b. Acidify with tartaric acid. c. In the neck of the flask, suspend a strip of filter paper moistened with 10% alcoholic solution of guaiac and 10% aqueous solution of copper sulfate. d. Stopper the flask and warm the contents for a 2-3 min; let stand for 30 minutes. e. Development of a blue color indicates the presence of cyanide.

Note: Blue color also develops by chloride, hydrogen, ammonia, oxides of nitrogen, bromine, chlorine and hydrogen peroxide.

Test for hydrocyanic acid in plant materials a. Preparation of picric acid test strips: Dip strips of filter paper in a solution containing 5 g sodium carbonate and 0.5 g picric acid in 100 ml of distilled water. Allow the strips to air-dry. b. Add 2-3 g of moistened shredded plant material to a test tube and add 4 drops of chloroform. c. Place one end of the filter paper strip in a spilt cork stopper and insert carefully into a test tube without touching the side of the tube. d. Incubate the test tube for 24 hours at 37ยบC. e. Development of a red to reddish brown color on the filter

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F. K. Mohammad

paper strip indicates the presence of hydrocyanic acid in the sample.

Heavy metals a. Place 25 g of the sample to be tested in a flask. b. Add 10 ml of concentrated HCl diluted with 90 ml of distilled water. c. Introduce into the flask a strip of bright copper foil or wire. d. Place a flask filled with cold-water over the flask, and boil gently for 30-45 minutes. e. Remove the copper strip or wire, wash it with distilled water and dry on a filter paper. f. Results: ď&#x201A;ˇ Dark discoloration indicates Arsenic or Bismuth. ď&#x201A;ˇ Silver deposit indicates Mercury. ď&#x201A;ˇ Dark color with a purple to blue violet sheen indicates Antimony. g. The black arsenic deposit is confirmed by placing the copper strip in 1 to 2 ml of 10% potassium cyanide solution.

Nitrate a. Diphenylamine reagent: Dissolve 0.5 g of diphenylamine in 20 ml distilled water. Complete the volume to 100 ml with sulfuric acid and cool the solution. b. Test reagent: 10 ml of diphenylamine reagent + 10 ml of 80% sulfuric acid. c. Add 1 drop to the sample under examination. Development of a green color indicates the presence of nitrates in the sample.

Flouride a. Add several drops of sodium hydroxide 1 M to the urine or stomach content samples. Dry at low heat in a glass dish. b. Add a small amount of powdered glass to the dry residue, and mix. c. Add several ml of concentrated sulfuric acid. Cover the dish with a small glass plate from the udersurface of which a drop of sodium chloride solution (5%) is suspended. When needed place ice on top of the plate to prevent evaporation of the

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Laboratory Guide in Toxicology

drop. d. Gently heat for 3-5 minutes. Remove the glass plate and examine the drop. e. If the specimen contains flouride, silicon flouride will be formed and appears as small, faintly pink and hexagonal crystals along the rim of the drop. The crystals should be examined under low power of the microscope to differentiate them from sodium chloride crystals.

Thallium a. Add 1 drop of 10% potassium iodide solution to 1 drop of weakly acidic urine. b. When a yellow precipitate appears, add 1 drop of 2% sodium thiosulfate. c. If thallium is present, the precipitate will not be dissolved.

Carbon monoxide a. Dilute 2 drops of whole blood with approximately 15 ml of distilled water so that the solution is faint pink in color. b. Add 5 drops of 20% sodium hydroxide. Shake quickly and observe the color. c. If the blood is normal or contains < 20% carboxyhemoglobin, the pink color will become straw yellow on shaking.

Persistence of the pink color for several seconds or more is an indication of the presence of > 20% carboxyhemoglobin.

Aflatoxins a. Blend 100 g of feed sample in a blender with 300 ml of methanol solvent (7:3, methanol:water) for 2-5 minutes. b. Stand until a layer of noncloudy liquid forms on the surface.

Place approximately 100 ml of this liquid in 500 ml separating funnel. Add 30 ml of benzene. Shake for 30 seconds, and then add 200 ml of distilled water.

Allow the mixture to separate and discard the lower layer.

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F. K. Mohammad

Place the upper layer in a beaker and evaporate to dryness. c. Resuspend the dry material with 0.5 ml of benzene. d. Spot on No.4 Whatman filter paper, allow to dry, and place under a long wave UV light. e. Blue fluorescence in the spot is an indication of the presence of aflatoxin.

Methanolic potassium hydroxide test Reagent: 20% solution of potassium hydroxide in methanol.

Add a few drops of the reagent to the solution of the sample in methanol. Heat until the color development is as follows: Color

Example of drugs

Red

Metronidazole

Orange-red

Fenitrothion

Yellow-orange

Nitrofurantoin

Yellow

Niclosamide, Nitroxynil

Marquis test Reagent: Prepare the reagent by adding 1 ml formaldehyde solution to 9 ml of concentrated sulfuric acid. Mix.

Add a drop of the reagent to the sample, and then observe the color development as follows:

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Laboratory Guide in Toxicology

Color

Red

Example of drugs

Nadolol, mephenesin, carbamate

Orange

Epinephrine (violet), amphetamine (brown), harmine, oxytetracycline, pethidine, spiramycin, trimethoprim

Yellow

Acriflavin (red), chlortetracycline (green), diphenhydramine, doxycycline, furaltadone

Green

Carbaryl

Blue

Clofibrate

Violet

Chlorpromazine, dihydrocodeine, promazine, promethazine

Brown

Erythromycin, ergotamine

Yellow-brown

Tylosin

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F. K. Mohammad

Sulfuric acid test Add few drops of concentrated sulfuric acid to the sample, and observe the color development as follows: Color

Example of drugs

Red

Fenitrothion

Violet-red

Oxytetracycline

Orange

Indomethacin, diphenhydramine, rotenone, stilbestrol

Yellow

Furosemide, quinidine, cortisone, estradiol, progesterone

Green

Phenothiazine

Brown-blue

Chlortetracycline

Yellow-brown

Tylosin

Orange-pink

Dexamethasone

No color

Testosterone

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Laboratory Guide in Toxicology

Mandelin's test Reagent: Dissolve 0.5 g of ammonium vanadate in 1.5 ml of distilled water. Dilute to 100 ml with concentrated sulfuric acid. Filter the solution through glass wool.

Add a drop of the reagent to the sample and observe the color development as follows: Color

Example of drugs

Red

Sodium cromoglycate

Blue

Xylazine, yohimbine (green), Doxapram

Orange

Levamisole (grey-green)

Yellow

Diphenhydramine, tylosin (yellow-brown)

Green

Niclosamide, obidoxime (blue), pentazocine, promethazine (violet)

Violet

Oxyclozanide (orange), oxytetracycline (redorange), strychnine

Brown

Tubocurarine

Orange-brown

Rifampicin, spiramycin

Yellow-brown

Trimethoprim, physostigmine

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F. K. Mohammad

Potential causes of changes in the color of urine Brown or black: nitrobenzene, phenols, rhubarb.

Yellow or orange: fluorescein, phenolphthalein,nitrofurantoin, senna, cascara.

Red or brown: aloin, phenothiazine derivatives, phenytoin, phenolphthalein, quinine, warfarin.

Blue or green: amitriptyline, indomethacin, phenols.

Smells mostly related to poisonous substances Bitter almond: cyanide.

Fruity alcohols: alcohols, esters.

Garlic: arsenic, phosphorus.

Mothballs: camphor.

Pears: chloral hydrate.

Petroleum: petroleum distillates, pesticide formulations.

Phenolic: disinfectants, phenols.

Stale tobacco: nicotine.

Shoe polish: nitrobenzene.

Sweet: chloroform, halogenated hydrocarbons.

Phytochemical screening of poisonous plants Plant preparation: Grind dried plant parts. Add distilled water at 25 ml/5 g plant material, stir and then incubate at 60 ÂşC for one hour.

Filter through Whatman No.1 filter paper. Centrifuge the filtrate for 15 minutes at 3000 rpm, and then use the supernatant for phytochemical analyses which are considered preliminary screening tests.

Alkaloids: Add 3 ml of Hagerâ&#x20AC;&#x2122;s reagent (saturated aqueous solution of picric acid) to 1 ml of the extract. The presence of alkaloids is indicated by the appearance of yellow color precipitate.

Steriods: Add 10 ml of chloroform to 1 ml of the extract. Add an equal volume of concentrated sulfuric acid on the side of the

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container. The presence of steroids is indicated when the upper layer turns red and the acid layer becomes yellow with green fluorescence.

Terpenoids: Add 2 ml of acetic anhydride and concentrated sulfuric acid to 2 ml of the extract. The presence of terpenoids is indicated by the development of blue greenish rings.

Fatty Acids: Add 5 ml of ether to 0.5 ml of extract, and mix . Allow to evaporate on a filter paper. Dry the filter paper. The presence of fatty acids is indicated by the appearance of transparency on the filter paper.

Tannins: Added 5 drops of 1% lead acetate to 2 ml of the extract.

The presence of tannins is indicated by the appearance of a yellowish precipitate.

Saponins: Add 20 ml of distilled water to 5 ml of the extract in a cylinder and shake vigorously for 15 minutes. The presence of saponins is indicated by the appearance of foam.

Anthocyanins: Add 2 ml of 2N HCl and ammonia to 2 ml of the extract. The presence of anthocyanins is indicated by the appearance of pink-red color which turns to blue-violet.

Leucoanthocyanins: Add 5 ml of isoamyl alcohol to 5 ml of the extract. The presence of leucoanthocyanins is indicated by the appearance of red color in the upper layer.

Coumarins: Add 3 ml of 10% NaOH to 2 ml of the extract. The presence of coumarins is indicated by the appearance of yellow color.

Carbohydrates: Add 5 ml of Benedictâ&#x20AC;&#x2122;s reagent to 1 ml of the extract Boil for 2 minutes and cool. The presence of carbohydrates is indicated by the appearance of red color percipitate.

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F. K. Mohammad

Glycosides: Add about 5 ml diluted sulfuric acid to 1 ml of the extract. Boil the mixture, filter and extract the filtrate with chloroform.

Treat the the chloroform layer is with 1 ml of ammonia. The presence of anthroquinone glycosides is indicated by the appearance of red color in the ammonia layer.

Flavonoids: Add few ml of ammonia to 5 ml of the extract. The presence of flavonoids is indicated by the appearance of fluorescence under UV or visible lights.

Proteins: Biuret test- Add 1 ml of 40% sodium hydroxide solution and 2 drops of 1% copper sulfate solution till a blue color is produced. Thereafter add to the 1 ml of the extract, and mix. The presence of proteins is indicated by the development of pinkish or purple violet color.

Gums and Mucilage: Add 10 ml of the extract slowly to 25 ml of absolute ethanol with constant stirring. Filter the solution through Whatman No.1 filter paper. Dry the filter paper in air. The presence of gums and mucilage is indicated by the appearance of swollen precipitate.

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Laboratory Guide in Toxicology

1. Name the qualitative tests used to

Study questions

identify anthelmintics.

2. Name the qualitative tests used to identify xylazine, chlorpromazine and morphine.

3. What are the main causes of foul smells in the urine? Give examples.

4. How can you identify saponins, proteins, alkaloids and tannins in plant extracts?

5. What are the requirements for developing an analytical toxicology laboratory?

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F. K. Mohammad

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Laboratory Guide in Toxicology

Common Antidotes Poison or drug overdose

Antidote

Acetaminophen

N-Acetylcysteine, Vit. C

Aflatoxin

Activated charcoal, Vit. E

Amitraz

Yohimbine

Amphetamine

Acetylpromazine maleate

Anticholinergic

Physostigmine

Arsenic

BAL, Succimer

Aspirin

Sodium bicarbonate

Beta Blockers

Atropine, Glucagon

Botulism

Guanidine

Caffeine

Diazepam

Carbamate insecticides

Atropine

Carbon monoxide

Oxygen

Copper

Penicillamine

Curare

Neostigmine

Cyanide

Na thiosulfate, Amyl nitrite

Detomidine

Atipamezole, Yohimbine

Diazepam

Flumazenil

2,4-Dichlorophenoxyacetate

Sodium bicarbonate

Digitalis

Digoxin immune FAB

Ethylene glycol

20% Ethanol, 4-Methyl pyrazole (5%), Thiamine

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F. K. Mohammad

Poison or drug overdose

Antidote

Fluoride

Calcium lactate

5-Fluorouracil

Thymidine

Folic acid

Leucovorin

Heparin

Protamine sulfate

Iron

Lindane

Deferoxamine CaEDTA, Penicillamine, Succimer Diazepam

Medetomidine

Atipamezole, Yohimbine

Mercury

Dimercaprol, Penicillamine

Morphine

Naloxone

Nitrate

Vit. C

Organophosphates

Atropine, 2-PAM

Paraquat

Bentonite, Fuller's earth

Pyrethroid

Diazepam, Methocarbamol

Strychnine

Diazepam

Tetanus toxin

Pentobarbital, Diazepam, Tetanus antitoxin

Thallium

Diphenylthiocarbazone

Vacor

Nicotinamide

Vit. D

Calcitonin

Warfarin

Vit. K

Xylazine

Yohimbine, Atipamezole

Zinc

CaEDTA, Penicillamine

Lead

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Laboratory Guide in Toxicology

Oral Median Lethal Doses (LD50) of Selected Chemicals in Rats Chemical

LD50 (mg/kg, orally)

Acetaldehyde

1900

Aldrin

39

Allethrin

680-860

Alpha chloralose

400

Aminopyrine

1700

Amitraz

600

Amobarbital

400

Amphetamine

60.5

Anilazine

>5000

Aspirin

1500

Atrazine

3000

Atropine

750

Banrot

5000

Blasticidin

56

Brodifacoum

0.27

Bromobenzene

2591

Caffeine

483

Carbachol

40

Carbaryl

850

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F. K. Mohammad

Chemical

LD50 (mg/kg, orally)

Carbofuran

8

Carbon tetrachloride

10054

Carboxin

3820

Chloral hydrate

500

Chloranil

4000

Chlordane

283

Chlordimeform

170

Chlorfenvinphos

10

Chloroneb

>5000

Chlorpromazine

493

Codeine

542

Crimidine

1.25

Crotoxyphos

74

Cyfluthrin

590-1189

Cypermethrin

1700

DDT

400

Deltamethrin

100

Diazinon

250

Dichlone

1300

2,4-Dichlorophenoxyacetic acid

300-1000

Dichlorvos

56

90

Laboratory Guide in Toxicology

Chemical

LD50 (mg/kg, orally)

Dicoumarin

542

Dieldrin

46

Digitoxin

24

2,4-Dinitrophenol

30

Dinocap

980

Diphenhydramine

500

Dithianon

638

Edifenphos

212

Ephedrine

160

Ethanol

13660

Ethylene dichloride

670-890

Fenamiphos

8

Fenvalerate

415

Flouroacetic acid

2.5

Hexachlorophene

165

Isopropyl alcohol

5-8 g/kg

Levamisole

480

Lindane

76

Malathion

1000

Meprobamate

918

Methanol

6-13 g/kg

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F. K. Mohammad

Chemical

LD50 (mg/kg, orally)

Methomyl

17

Methoxychlor

6000

Mirex

235

Monensin

35

Nicotine

55

Nitrofurazone

380-590

Papaverine

745

Paraquat

100

Parathion

13

Pentobarbital

118

Permethrin

4000

Phenoxybenzamine

2500

Phenylbutazone

1150

Phenylephrine

350

Propoxur

95

Pyriminil

12

Quinidine

1000

Red Squill

490

Rotenone

60

Sodium flouride

200

Strychnine

16

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Laboratory Guide in Toxicology

Chemical

LD50 (mg/kg, orally)

Tetraethyl lead

20

Thiram

780

Toluene

6.4-7.4 ml/kg

Toxaphene

80

Tripelennamine

570

Warfarin

186

Xylene

4 ml/kg

Zinc phosphide

46

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F. K. Mohammad

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Laboratory Guide in Toxicology

Appendix A. Specimens for toxicological analysis Chemical

Samples needed

Acetaminophen

Serum

Aflatoxin

Liver

Arsenic

Hair, liver, kidneys, urine

Cadmium

liver, kidneys

Carbamates

Brain, blood, liver

Carbon monoxide

Blood

Carbon tetrachloride

Serum, Liver

Chlorinated hydrocarbons

Fat, liver

Copper

Serum, Liver

Cyanide

Stomach content, blood

Lead

Hair, liver, kidneys, urine, blood

Mercury

Liver, kidneys

Nitrate

Stomach content, urine, blood

Organophosphates

Brain, blood, liver

Strychnine

Stomach content, urine, brain, liver

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F. K. Mohammad

B. Common anticoagulants

Chemical

Heparin

10 mg/100 ml blood

Oxalates (ammonium oxalate 6 mg, potassium oxalate 4 mg)

10 mg/ 5 ml blood

Sodium citrate

10 mg/90 ml blood

Sodium EDTA

10-20 mg/10 ml blood

Sodium fluoride

10 mg/1 ml blood

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Laboratory Guide in Toxicology

C. Concentration of common acids and bases Concentrated reagent

% (w/w)

ml required/L for 1 M solution

Hydrochloric acid (HCl)

36

85.5

Nitric acid (HNO3)

69.5

64

Sulfuric acid (H2SO4)

96

56

Acetic acid (CH3COOH)

99.5

56.9

Phosphoric acid (H3PO4)

85

68

Ammonium hydroxide (NH4OH)

58.6

66.5

Perchloric acid (HClO4)

70

85.7

Sodium hydroxide (NaOH) (saturated)

50

57-67

Potassium hydroxide (KOH) (saturated)

50

70

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F. K. Mohammad

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Laboratory Guide in Toxicology

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Non-Teratogenic Levels of 2,4-D and 2,4,5-T. PhD Dissertation, University of Missouri-Columbia, MO, USA. Mohammad, F.K., 1994. Effect of cadmium on detomidine – ketamine anesthesia in mice. Iraqi J. Vet. Sci., 7: 137-141.

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Application of an electrometric method for measurement of blood cholinesterase activities in sheep, goats and cattle treated with organophosphate insecticides. Journal of Veterinary Medicine (A) 54: 140143.

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Assessment of diphenhydramine effects against acute poisoning induced by the organophosphate insecticide dichlorvos in chicks. Hum. Vet. Med. Bioflux 4: 6-13.

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Plumb, D.C., 2002. Veterinary Drug Handbook. Iowa State Press, Ames, IA, USA. Plumlee, K., Editor, 2004. Clinical Veterinary Toxicology. Mosby, Inc., St. Louis, MO, USA. Prasad, B.,1985. Veterinary Pharmaceuticals. 2nd ed. CBS publishers and Distributors, Delhi, India.

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Woolley , D.E., Griffith, J.A.,1989. Kinetics and thresholds of several indices of lindane â&#x20AC;&#x201C; induced toxicity. Pharmacol. Biochem. Behav., 33:787-792.

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Index 2 2-PAM · 44, 88 A Acceptable daily intake · 17 Acetaminophen · 73, 87, 95 Acetylcholine · 3, 37, 43 Acetylcholine bromide · 38 Acetylcholine chloride · 38 Acetylcholine iodide · 38 Acetylcholinesterase · 37, 43 Acetylcholinesterase · 37 Acetylsalicylic acid · 51 Acetylthiocholine iodide · 37, 38 Acute poisoning · 21 ADE · 59 ADI · 17 ADR · 59, 60, 61 Adverse Drug Event · 59 Adverse Drug Reaction · 59 Aflatoxin · 78, 87, 95 Alkaloids · 82 Analytic Toxicology · I, 71 Anesthesia · 49, 50, 53, 55, 101 Anthocyanins · 83 Anthroquinone · 84 Antidote · 3, 87 Aspirin · 51, 87, 89

Atipamezole · 55 Atropine · 3, 43, 44, 87, 88, 89 AUC · 68 Autonomic · 28 B Behavior · 25 Bioactivation · 3 Bioavailability · 33, 68 Biuret test · 84 Butyrylcholinesterase · 37 C Cadmium · 49, 95, 104 Calculation · 18 Carbamate · I, 43, 87 Carbaryl · 43, 79, 89 Carbohydrates · 83 Carbon monoxide · 77, 87, 95 Cd · 49, 50 CF · 3 Cholinesterase · 37, 38 Chronic poisoning · 21 Chronicity factor · 3 Clinical toxicology · 3 Clp · 67 Coumarins · 83 Creatine phosphokinase · 57 Cyanide · 75, 87, 95

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D

H

Detomidine · 49, 50, 53, 87, 101 Diazepam · 44, 87, 88 Dichlorvos · 43, 44, 90 Dilution · 14 Diphenhydramine · 44, 81, 91 Diphenylamine · 74 Diquat · 74 Diuresis · 55

Half-life · 67, 69 Hazard · 4 Heavy metals · 76 Hemolysis · 57 Hestrin · 37 Household · 14 Hydrocyanic · 75, 76

E

Iatrogenic disease · 4 Interactions · 60, 63

Electrometric · 37, 38 Elimination rate constant · 67, 69 Ellman · 37 Emesis · 55 Environmental toxicology · 3 Exponential · 68

I

K K · 55, 56, 88, 100, 101, 103 kel · 67 Ketamine · 49, 50, 53, 54, 55, 101, 103

F

L

Fatty acids · 83 Favonoids · 84 Flouride · 76 Forensic · 4 Fragility · 57

LC50 · 33 LD50 · 3, 4, 21, 22, 33, 34, 89, 99 Lindane · I, 47, 88, 91 Locomotor · 30, 49

G

M

GABA · 47 Gums · 84

Mandelin's test · 81 Marquis test · 78

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Maximum tolerable dose · 4 Median lethal dose · 4, 33 Minimum toxic dose · 4 Molarity · 13 Motor activity · 47 Mouse · 47, 50 Mucilage · 84 Muscarinic · 43 N Na · 3, 34, 49, 55, 56, 87 Negative geotaxis · 26, 31 Neurobehavioral toxicology · 25 Neuromuscular · 28 Neurotoxicant. · 47 Nicotinic · 43 Nitrate · 21, 76, 88, 95 NOAEL · 4 NOEL · 4, 17

Phenothiazines · 73 Plasma clearance · 67, 70 Poison · 4, 87 Pollutant · 5 Polypharmacy · 63 Potassium hydroxide · 97 Potentiation · 22 ppb · 14, 17 ppm · 14, 17, 18 ppt · 14, 17 Pralidoxime · 44 Prescriptions · 63, 64 Proteins · 84 R Rabbit · 54 Rat · 44, 51, 56 Residues · 3, 17, 60, 72, 100 Risk · 5, 100 S

O Open-field activity · III, 29, 30 Organophosphate · 43 Osmolality · 55, 56 P Paraquat · 74, 88, 92 Pharmacokinetics · I, 67, 99 Pharmacovigilance · 59, 103 Phenol · 74

Salicylate · 72 Saponins · 83 Sedation · 55 Semilog · 34, 69 Sensorimotor · 28 Sheep · 18, 57, 101 Side effect · 3, 59, 64 Solubility · 13 Specimens · 95 Steroids · 83 Strychnine · 75, 88, 92, 95

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Subacute poisoning · 21 Subchronic poisoning · 21 Sulfonamides · 74 Sulfuric acid · 80, 97

V

T

W

t1/2 · 67 Tannins · 83 Terpenoids · 83 Test animals · 25 Thallium · 77, 88 Thiopental · 34 Tolerance level · 18 Tonic immobility test · 29 Toxicity · 5, 21, 99, 102 Toxicology · VII, 1, 3, 5, 7, 13, 25, 99, 100, 102, 103 Toxicosis · 5 Toxicovigilance · 5 Toxin · 5

Withdrawal period · 18 Writhing · 26, 49

Vd · 67 Volume of distribution · 67, 70

X Xylazine · 53, 54, 55, 56, 81, 88, 100, 101 Y Yohimbine · 55, 87, 88

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Attachments 1. Linear graph 2. Semilog graph

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