Forensic toxicology uses analytical chemistry, pharmacology, and clinical chemistry to aid in investigations of death, poisoning, and drug use. A toxicological analysis can be performed on various sample types to determine what toxic substances are present, in what concentrations, and their probable effects. Forensic toxicology can be separated into postmortem toxicology, human performance toxicology, and forensic drug testing. Different analytical techniques like immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry are used to detect drugs and metals in samples.

1. Forensic
Toxicology
Deepak chaudhary
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2. Forensic
Toxicology
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• Forensic toxicology is the use of toxicology and
disciplines such as analytical
chemistry, pharmacology and clinical chemistry to
aid medical or legal investigation of death,
poisoning, and drug use. The primary concern for
forensic toxicology is not the legal outcome of the
toxicological investigation or the technology
utilized, but rather the obtainment and
interpretation of results. A toxicological analysis
can be done to various kinds of samples. A
forensic toxicologist must consider the context of
an investigation, in particular any physical
symptoms recorded, and any evidence collected at
a crime scene that may narrow the search, such
as pill bottles, powders, trace residue, and any
available chemicals. Provided with this information
and samples with which to work, the forensic
toxicologist must determine which toxic
substances are present, in what concentrations,
and the probable effect of those chemicals on the
person.
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3. • forensic toxicology can be separated into 3 disciplines: Postmortem
toxicology, human performance toxicology, and forensic drug testing
(FDT).[3] Postmortem toxicology includes the analysis of biological specimens
taken from an autopsy to identify the effect of drugs, alcohol, and poisons. A
wide range of biological specimens may be analyzed including blood, urine,
gastric contents, oral fluids, hair, tissues, and more. The forensic toxicologist
works with pathologists, medical examiners, and coroners to help determine
the cause and manner of death. In human performance toxicology, a dose-
response relationship between a drug(s) present in the body and the effects
on the body are examined. This field of forensic toxicology is responsible for
building and implementing laws such as driving under the influence of
alcohol or drugs. Lastly, forensic drug testing (FDT) is the detection of drug
use among individuals in the workplace, sport doping, drug-related probation,
and new job applicant screenings.
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4. How certain substances affect your body
• Alcohol
• Alcohol enters your central nervous system through the blood stream through the lining within your
stomach and your small intestine. Once it is in your blood stream, is passes through your blood
brain barrier via blood circulation. The alcohol absorbed will reduce your reflexes, interfere with
nerve impulses, prolong muscle responses, and affect other parts of your body as well.[5]
• Marijuana
• Marijuana, like alcohol, is also absorbed into the blood stream and passed through the blood brain
barrier. However, the THC that is released from marijuana attaches to the CB-1 cannabinoid
receptors which causes all of the affects that you experience. This include, but not limited to, mood
changes, altered perception of time, and increased sensitivity.[6]
• Cocaine
• Cocaine is a stimulant unlike Marijuana or Alcohol. As soon as cocaine enters the bloodstream it
reaches the brain in minutes. Dopamine levels are increased intensely and the effects can last up to
about 30 minutes. The most common way to use cocaine is by snorting it through the nose but
other methods could be by smoking it in a crystal rock form. But because dopamine levels are
increased at such a rate this leads to an even worse come down leading to needing a higher dose
to get the same effect as the time before if taken again. This is how some addictions begin. Some

5. Sample
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6. Examples
Urine
A urine sample is urine that has come from the bladder and can be provided
or taken post-mortem. Urine is less likely to be infected with viruses such as
HIV or Hepatitis B than blood samples.[8] Many drugs have a higher
concentration and can remain for much longer in urine than blood. Collection
of urine samples can be taken in a noninvasive way which does not require
professionals for collection. Urine is used for qualitative analysis as it cannot
give any indication of impairment due to the fact that drug presence in urine
only indicates prior exposure.[9] Different drugs can also stay in your urine for
different amounts of time. For example, alcohol will stay within your urine for
7–12 hours, cocaine metabolites will stay for 2–4 days, and morphine will stay
for 48–74 hours. One drug that will stay in your urine for a varying amount of
time (dependent on the usage and frequency) is marijuana. For a single use,
it will stay for 3 days, moderate use (4 times per week) will stay for 5–7 days,
daily use of the drug will cause it to stay for 10–15 days, and a long-term

7. Blood
A blood sample of approximately 10 ml (0.35 imp fl oz; 0.34 US fl oz) is usually sufficient to screen and
confirm most common toxic substances. A blood sample provides the toxicologist with a profile of the
substance that the subject was influenced by at the time of collection; for this reason, it is the sample of
choice for measuring blood alcohol content in drunk driving cases.
Hair
Hair is capable of recording medium to long-term or high dosage substance abuse. Chemicals in the
bloodstream may be transferred to the growing hair and stored in the follicle, providing a
rough timeline of drug intake events. Head hair grows at rate of approximately 1 to 1.5 cm a month, and
so cross sections from different sections of the follicle can give estimates as to when a substance was
ingested. Testing for drugs in hair is not standard throughout the population. The darker and coarser the
hair the more drug that will be found in the hair. If two people consumed the same amount of drugs, the
person with the darker and coarser hair will have more drug in their hair than the lighter haired person
when tested. This raises issues of possible racial bias in substance tests with hair samples.[12] Hair
samples are analyzed using enzyme-linked immunosorbent assay (ELISA). In ELISA, an antigen must
be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.
Bone Marrow
Bone marrow can be used for testing but that depends on the quality and availability of the bones. So
far there is no proof that says that certain bones are better than others when it comes to testing.
Extracting bone marrow from larger bones is easier than smaller bones. Forensic toxicologists use bone
marrow to find what type poisons used. These poisons can include cocaine or ethanol.

8. Other
Other bodily fluids and organs may provide samples,
particularly samples collected during an autopsy. A common
autopsy sample is the gastric contents of the deceased, which
can be useful for detecting undigested pills or liquids that were
ingested prior to death. In highly decomposed bodies, traditional
samples may no longer be available. The vitreous humour from
the eye may be used, as the fibrous layer of the eyeball and the
eye socket of the skull protects the sample from trauma and
adulteration. Other common organs used for toxicology are the
brain, liver, and spleen.

9. Detection and
classification
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• Detection of drugs and pharmaceuticals in
biological samples is usually done by an initial
screening and then a confirmation of the
compound(s), which may include a
quantitation of the compound(s). The
screening and confirmation are usually, but
not necessarily, done with different analytical
methods. Every analytical method used in
forensic toxicology should be carefully tested
by performing a validation of the method to
ensure correct and indisputable results at all
times. The choice of method for testing is
highly dependent on what kind of substance
one expects to find and the material on which
the testing is performed. Customarily, a
classification scheme is utilized that places
poisons in categories such as: corrosive
agents, gases and volatile agents, metallic
poisons, nonvolatile organic agents, and
miscellaneous.
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10. • Immunoassays
• Immunoassays requires you to draw blood and use the antibodies to find a reaction with substances such
as drugs. The substances must be specific. It is the most common drug screening technique. Using the
targeted drug the test will tell you if it is positive or negative to that drug. There can be 4 results when
taking the test. Those results can be a true-positive, a false-negative, a false-positive, and a true-
negative.
• Gas chromatography-mass spectrometry
• Gas chromatography-mass spectrometry (GC-MS) is a widely used analytical technique for the detection
of volatile compounds. Ionization techniques most frequently used in forensic toxicology include electron
ionization (EI) or chemical ionization (CI), with EI being preferred in forensic analysis due to its detailed
mass spectra and its large library of spectra. However, chemical ionization can provide greater sensitivity
for certain compounds that have high electron affinity functional groups.
• Liquid chromatography-mass spectrometry
• Liquid chromatography-mass spectrometry (LC-MS) has the capability to analyze compounds that are
polar and less volatile. Derivatization is not required for these analytes as it would be in GC-MS, which
simplifies sample preparation. As an alternative to immunoassay screening which generally requires
confirmation with another technique, LC-MS offers greater selectivity and sensitivity. This subsequently
reduces the possibility of a false negative result that has been recorded in immunoassay drug screening
with synthetic cathinones and cannabinoids. A disadvantage of LC-MS on comparison to other analytical
techniques such as GC-MS, is the high instrumentation cost. However, recent advances in LC-MS have
led to higher resolution and sensitivity which assists in the evaluation of spectra to identify forensic

11. • Detection of metals
• The compounds suspected of containing a metal are traditionally
analyzed by the destruction of the organic matrix by chemical or
thermal oxidation. This leaves the metal to be identified and
quantified in the inorganic residue, and it can be detected using
such methods as the Reinsch test, emission spectroscopy or X-
ray diffraction. Unfortunately, while this identifies the metals
present it removes the original compound, and so hinders efforts
to determine what may have been ingested. The toxic effects of
various metallic compounds can vary considerably.

12. Principles of
dose-setting in
toxicology
studies
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Principles and concepts
• To achieve the regulatory goal of ensuring that
chemical uses are limited to the conditions
under which exposures are safe, dose-setting
for regulatory toxicology studies should be
aimed at identifying and characterizing the
dose range at which adverse effects are
unobservable by validated test methods. To
achieve this efficiently, we would propose that
the administered doses should cover the range
from very low (e.g., the low end of the
estimated human exposure level) up to, but
not exceeding, the dose that produces either:
i. Adverse effects and irreversible changes
that must be assumed to be adverse.
ii. A dose-disproportionate alteration in the
relationship between the administered dose
and the blood level of the chemical.
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13. Why identify and characterize the no-effect dosage
range?
• Practicality
• It is often assumed that the purpose of guideline toxicology studies is to identify all possible
adverse effects and to characterize their dose–response relationships, but we would contend that
in fact, current toxicology study designs are a compromise that attempt to identify the safe dose
range as well as to characterize adverse effects that are within, typically, 100–1000-fold greater
than expected human exposures, a dual focus that limits the ability of toxicology studies to serve
either purpose well. In practice, MTD doses may exceed human doses by even greater
magnitudes, further eroding plausible relationships to foreseeable human exposures. If
comprehensive testing for adverse effects were to be done thoroughly, each type of toxicology
study would need to incorporate many different treatment arms tailored to examine all organ
systems and processes within the dose ranges that the chemical affects each system. For
example, a reproductive toxicology study that attempts to test for effects on both anogenital
distance and fertility in the offspring would need to employ much larger animal numbers and more
treatment groups than currently required because statistical optimization would be different for
detecting biologically relevant changes in these different endpoints. Adequate dose–response
characterization would then require distinct administration protocols and separate control groups
for each adverse effect tested in that type of study, as well as many more dose levels than
currently required by OECD, U.S. EPA, and other international regulatory test guidelines. This
would expand the use of animals unnecessarily, raise the complexity of many types of toxicology
studies, and hence, increase costs and the potential for human error.

14. Why test doses up to the point of either toxicity or altered toxicokinetics, but not beyond?
Defining the safe dose range may be biologically incongruent with characterizing the
various adverse effects that occur across the entirety of the toxic dose range. This derives
from the fact that toxicity is a dynamic process with many causal factors, one of the most
critical being toxicokinetics. Neither toxicity nor its regulatory antecedent “hazard” arise
purely from the constituent atoms or the molecular structure of a chemical, and each
molecule of the chemical does not exhibit toxicity or hazard irrespective of the number of
molecules present. Thus, toxicity and hazard are not inherent or intrinsic to the chemical. In
the vernacular of chemistry, toxicity is not an “intensive” property of a chemical. To the
contrary, toxicity and hazard are “extensive” properties, meaning that in addition to a
chemical’s structure and physical nature, toxicity and hazard depend upon the quantity of
the chemical encountered, the route of exposure and the conditions under which a chemical
is encountered, and other factors such as the species, age, sex, behavior, and other
characteristics of the organism exposed to the chemical . Thus, the dose range and
conditions under which a chemical produces toxicity may provide little useful information
about the dose range and conditions necessary to assure a lack of relevant hazard or
adverse effects.

15. • Focusing toxicology studies exclusively on the safe dose range
rather than on the dose range that produces toxicity would be a
superior approach for several reasons. Above all, it is practical.
Human exposures to chemicals are not intended to pose hazards
or produce adverse effects; to the contrary, when exposure to
chemicals occurs, it is intended to be non-hazardous and without
adverse effects. Therefore, it is logical that the highest priority of
toxicity testing should be to identify and characterize the doses and
conditions that meet this intent. Focusing on the safe dose range is
also necessary from a logistical standpoint because ensuring
safety requires that the various biological targets that could be
adversely affected by a chemical are, in fact, not affected under
foreseeable conditions of exposure.

16. Assuring that the dose range and conditions have been identified under which
a chemical does not affect even one of its many possible biological targets is a
fundamentally different objective, and arguably a more difficult challenge,
than merely identifying that an adverse effect can be observed at some dose,
irrespective of its relevance to actual conditions of use and foreseeable
exposures. In fact, it is axiomatic and assured that all chemicals will produce
an adverse effect at some dose because all chemicals are toxic (i.e., hazardous)
under some conditions. Since the assurance of no adverse effects is the most
critical goal of toxicology testing, it is prudent to expend sufficient resources
to ensure that those conditions are thoroughly defined rather than attempting
to also address questions less relevant to safety, such as characterizing the
various effects that might occur at doses beyond the safe dose range.

17. Example #1: aspirin
Salicylates are not unique in this respect. The CNS-depressant effects of
ethanol are also high-dose effects that occur secondary to saturation of
metabolic capacity and the resultant change from first-order to zero-order
kinetics S toxicity of ethanol, for which it is intentionally consumed as a
social inebriant, depends upon sufficient concentrations in brain to perturb
nerve cell membrane viscosity, slow neurotransmission, and inhibit the
activity of GABAergic neurons and other receptor signaling pathways in the
CNS . At low consumption rates, ethanol does not reach CNS-depressant
levels in brain due to first pass liver metabolism, which prevents its
concentrations from accumulating in blood.

18. The rate-limiting step in ethyl alcohol metabolism is its conversion to acetaldehyde via the enzyme alcohol
dehydrogenase (ADH), a liver enzyme with high affinity (a very low Km) but low capacity that becomes
saturated with consumption of one or two standard alcoholic beverages per hour, or about 14–28 g ethanol
per hour in an adult male. At consumption levels below this, the rate of ethanol metabolism is proportional
to the blood level (i.e., elimination behaves as a first-order process) because sufficient ADH is present to
quantitatively convert ethanol to acetaldehyde. Thus, at low consumptions levels, blood ethanol
concentrations remain consistently very low. If ethanol consumption exceeds the available ADH, the capacity
of this rate-limiting enzyme is saturated and ethanol metabolism becomes increasingly dependent upon
CYP2E1, an inducible enzyme with higher capacity but lower affinity for alcohol (high Km). Under these
conditions, ethanol metabolism as well as its disappearance from the blood becomes independent of the
blood ethanol concentration. Elimination then behaves as a zero-order process equal to the maximum
capacity of the enzymes that metabolize ethanol. Consequently, blood ethanol concentrations increase
disproportionately, causing CNS concentrations to reach depressant levels . Without saturation of alcohol
metabolism by ADH, rates of alcohol consumption typical in social settings would have little acute effect on
people other than to increase urination frequency.

19. Most relevant to the point of this paper, if the hazard identification and risk problem
formulation questions are intended to understand human health effects associated with
chronic, high-dose human ethanol consumption, MTD animal toxicity testing would indeed
be appropriate (although unjustified given the very large human cohort available for study
of diseases associated with high-dose ethanol consumption). In contrast, if hazard
identification and risk problem formulation is intended to address the very much lower
ethanol exposures from occupational and other environmental scenarios, then chronic
toxicity testing based on an MTD is clearly not relevant. In fact, MTD-based testing would
provide misinformation because the hazards and risks associated with a sub-KMD-based
dosing strategy consistent with realistic occupational and general environmental exposures
are well-separated from intentional high-dose chronic drinking scenarios and their
consequent kinetic differences. Importantly, series of papers would incorrectly imply that
toxicity and hazard associated with very high-dose ethanol consumption informs hazard,
toxicity and risk from much lower consumption levels; it certainly does not, even though
MTD studies will inform toxicity and hazards of chronic ethanol abuse scenarios.

20. Changes in the relationship between
administered-dose and blood concentration
are critical
• For purposes of toxicological interpretation and regulatory toxicology study designs, it is critical to
understand how the region of the administered dose / blood concentration curve at which
toxicological effects are measured relates to the kinetics of the chemical, and whether this region is
below, above, or within the area of maximum curvature, i.e., the KMD region. It is not to be inferred,
however, that adverse effects cannot occur below a KMD, or that adverse effects will always
manifest just above a KMD; the point is that toxicity and its underlying mechanisms can be
expected to differ with a change in kinetics. Effects produced at doses either above or below the
KMD region are most clearly interpretable and relevant for the purpose of defining the non-
hazardous dose range. Results produced at doses within the KMD range or across doses that span
it are likely ambiguous for the purpose of establishing safety as well as for making inferences about
the potential modes of action underlying toxic effects. On the other hand, toxicity observed at doses
clearly above the KMD region can be unambiguously interpreted if the KMD region is well separated
from the range of foreseeable human exposures. Such toxicity lacks quantitative and mechanistic
relevance to humans and requires no further experimental attention as it represents an adverse
effect confounded by overloading of the animal’s physiological and metabolic capacity. In this

21. • A final point that should not be lost or mischaracterized: not every chemical
exhibits a point of saturation, a change in slope, or a KMD in its administered-
dose/blood-concentration relationship. For new regulatory toxicity testing, it is
critical to know which chemicals do, and which do not exhibit those kinetic
characteristics and to incorporate this understanding into toxicological study
designs. For existing toxicology data that were generated without utilizing
kinetic understanding in the study design, interpretations about relevant
hazards and adverse effects would be informed, and potentially corrected, by
kinetic data. Failing to perform kinetic studies and the understanding that can
be gleaned from them will ensure that regulatory toxicology studies continue
to maximize uncertainty, inefficiency, waste of animal lives, and animal
suffering.

22. • Withdrawal symptoms and frequent association with other
psychoactive substances take on considerable importance, particularly with
ethyl alcohol. For some authors Methadone Maintenance Treatment is
thought to impede capacity to drive, until psychic stabilisation (> 1 year) and
secure absence of the co-use of other psychoactive substances.
Buprenorphine in healthy subjects increases reaction time in a laboratory test
but not in a simulation driving test.
• Studies that have evaluated all medications employed in heroine dependence
therapy have shown that the parameters of competence (deviation from the
lateral standard position, speed and capacity to steer round a bend, reaction
to stimuli), do not present a difference in treated patients vs controls, except
in the case of combined intake of ethylic alcohol

23. Alcohol,
drugs and
driving
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• The effects of acute intake of ethyl alcohol vary
depending on the levels of ethanolemia (in mg%mL
o g/L) and the characteristics of the subject.
Alcohol can induce sedation and reduction of
anxiety, dyslalia, ataxia, impaired judgement and
disinhibition. Alcohol has psycho-behavioural
effects linearly correlated to its blood
concentration. The 50 mg%mL limit, fixed by most
driving codes as the limit for drunk driving, is not
predictive of the disabling effects of lower
concentrations, more evident in the adolescent and
elderly population. In any case, the multiplication of
risk by 3, 10 and 40 times applies when haematic
concentrations exceed 80, 100 and 150 mg%mL,
respectively. Driving with levels greater than 150
mg%mL substantiate the identification of alcohol
abuse or dependency problems, in need of social-
rehabilitative intervention.
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24. Conclusion
• Activity in the field of Forensic Toxicology is identified with the detection,
identification and quantification of xenobiotics in biological and non
biological matter. A synopsis of such analytical phases leads to the
interpretation of results through a rigorous evaluative criteriology in relation
to different regulatory areas.
• The two main areas where the analysis of biological material applies are
«forensic Toxicology of the dead» and «forensic Toxicology of the living
person».

25. • Forensic Toxicology of the dead is devoted to determine the presence of
xenobiotics in liquids and tissues and evaluate the possible causal or
concausal role in the determination and dynamics of the death.
• Forensic Toxicology of the living person is committed to determine the
presence of xenobiotics in the biological specimen (blood, urine, air inhaled,
hair, etc.) and in evaluating the possible causal or concausal role of
incapacity and/or deviations in behaviour (see suitability to drive, WDT,
doping, etc.), or rather harm to the person.
• Obligation in the above-mentioned areas is complex because of «pre-
analytical» and «analytical» variables. Among the pre-analytical variables are:
quantity of dose ingested, frequency and means of ingestion, interval
between intake and sample taking, the sample collection procedure, the
interval between sample taking and analysis.

26. • Among the analytical variables are: elevated number of analytes,
large variety of chemical structures, of volatility, functional groups,
hydrophilic/lipophilic ratios, values of pKa or pKb; wide ranges of
concentration in liquids and biological tissues, dependent on dose
intake; the way the specimens are stored; the possible lack of
pharmacokinetic and pharmacodynamic studies; the diversity of
biological matrices and potential analytical interferences produced
by exogenous, endogenic and putrefactive substances.
• The complexity of those variables ensures that every analysis may
be given as an individual case for which there are no rules
applicable to all xenobiotics and all situations.

27. • With the diffusion of environmental toxins and the clandestine drug market, the
forensic toxicology laboratory is also committed to the analysis of non-
biological material. In this context, Forensic Toxicology can provide to
institutions and society information and awareness on the appearance of new
drugs; identification of the major channels of drug distribution in the local and
national black market; identification of the means adopted by traffickers to
bypass systems of control; information on substances used in the cutting or
treatment of the drug; suggestions for timely legislative adaptations.
• With the main objective of providing scientifically based evidence, the
complexity of all the above outlined roles of forensic toxicology entails the
need for the adoption of quality assurance systems, ascertainement
methodologies and evaluation criteriologies.

28. Thank you
DEEPAK CHAUDHARY
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