Toxicology testing, also known as safety assessment, or toxicity testing, is conducted to determine the degree to which a substance can damage a living or non-living organism. It is often conducted by researchers using standard test procedures to comply with governing regulations, for example for medicines and pesticides. Much toxicology is considered to be part of the field of preclinical development. Stages of in vitro and in vivo research are conducted to determine safe doses of exposure in humans before a first-in-man study. Toxicology testing may be conducted by the pharmaceutical industry, biotechnology companies or contract research organizations. Drug development is a high-risk enterprise. The typical new drug takes 10-12 years to get to market and costs up to $500 million. Pharmaceutical companies face continually increasing challenges in drug development— shorter product life cycles, global competition, as well as daunting technical and regulatory hurdles. Meanwhile, as a result of the Human Genome Project and high throughput drug development methods, there are many more drug candidates to test. Thus, there is growing pressure on pharmaceutical and biopharmaceutical companies.

1. A Literature Review on Toxicity Studies:
Toxicity studies is Bound to Make an Impact in
Pharmaceutical Sciences

2. Contents Overview
 Introduction
 Literature Reviews
 Definitions
 Testing Strategies
 The Importance of Toxicity studies
 Regulatory Studies
 Stages of possible toxicity of an ingested
substance
 Toxicity Tests
 Single Dose Acute Toxicity Testing for
Pharmaceuticals
 Principle of Toxicity Studies
 Instruments Used
 Testing Procedures
 Observation
 Chronic Toxicity Studies
 Proposals For Clinical Study
 External Factors That May Influence
Susceptibility To Toxicity
 Conclusion
 References
2

3. Introduction
Toxicology
'Toxicology' traditionally known as the 'science of poisons' basically is defined as the study of
the effects of chemical agents on biological material with special emphasis on the harmful
effects. After gaining relevant information on the harmful effects of a compound the levels
for its safe usage or the degree of its safeness is established, which is also known as its
(compound) Biosafety level. Toxicology testing, also known as safety assessment, or toxicity
testing, is conducted to determine the degree to which a substance can damage a living or
non-living organism.
It is often conducted by researchers using standard test procedures to comply with
governing regulations, for example for medicines and pesticides. Much toxicology is
considered to be part of the field of preclinical development. Stages of in vitro and in vivo
research are conducted to determine safe doses of exposure in humans before a first-in-
man study. Toxicology testing may be conducted by the pharmaceutical industry,
biotechnology companies or contract research organizations.
3

4. Literature Reviews
1. Literature review on duckweed toxicity testing.
Wuncheng Wang
Water Quality Section, Illinois State Water Survey, Box 697, Peoria, Illinois 61652 USA
(2005) Science Direct, USA.
Duckweed commonly refers to a group of floating, flowering plants of the family Lemnaceae. Duckweed plants
are fast growing and widely distributed. They are easy to culture and to test. Some reports suggest that
duckweed plants are tolerant to environmental toxicity. Other studies, however, indicate that duckweed plants are
as sensitive to toxicity as other aquatic species. Duckweed plants are especially suitable for use in complex
effluent bioassays, and for testing herbicide pollution in the aquatic environment, lake and river pollution,
sediment toxicity, and the like. Duckweed and algae represent different levels of complexity in the plant kingdom.
They complement each other as phytotoxicity test organisms, instead of mutually excluding each other. Many
duckweed species have been studied, primarily of the Lemna and Spirodela genera. Lemna minor and L. gibba
have been recommended as standard test species. Differences in duckweed test methodology occur with regard
to test types, test vessels, control tests, nutrient media, end points, and applications [1].
4

5. 2. Literature review on higher plants for toxicity testing.
Wuncheng Wang
Water Quality Section, Illinois State Water Survey, Box 697, Peoria, Illinois 61652 USA
(1990) Science Direct, USA.
Phytotoxicity tests using higher plants in general are infrequently used as a part of ecotoxicology. Many reports
assess herbicide toxicity merely on the basis of faunal species tests. This is inadequate because the herbicide
is much greater on flora than on fauna. Environmental pollution by herbicides was likely to have been quite wide-
spread during the past years (1964–1984) when the use of herbicides grew five-fold. When herbicides reach non-
target areas, they can cause unacceptable harm to non-target species, plants in particular. The toxicity of
to algal species is not likely to be identical to that of higher plants, so that algal species may not serve as a
surrogate species for the toxicity evaluation. Currently there are two promising phytotoxicity tests. Common
duckweed is an aquatic species and sensitive to toxicity. Duckweek test can be used with static, renewal, or flow-
through methods. The latter two are especially useful for unstable compounds or samples. Seed germination and
root elongation tests are versatile and can be tested in water, wastewater, sediment, and slurry. Many recent
activities in these areas suggest that phytotoxicity tests are a valuable part of ecotoxicology [2].
5

6. 3. Studies on cadmium toxicity in plants: A review
P Das, S Samantaray, GR Rout
Regional Plant Resource Centre, Bhubaneswar-751015, India
(1998) Science Direct, USA.
Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate
the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens
for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the
mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk
treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA
69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA
ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the species Mycoplasma arginini and M. orale.
Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations
of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these
antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%)
chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or
infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of
three-quarters is a reasonable expectation [3].
6

7. 4. Quantifying Synergy: A Systematic Review of Mixture Toxicity Studies within Environmental Toxicology.
Nina Cedergreen
Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark
(2014) Plos One.
Cocktail effects and synergistic interactions of chemicals in mixtures are an area of great concern to both the public and regulatory authorities. The
main concern is whether some chemicals can enhance the effect of other chemicals, so that they jointly exert a larger effect than predicted. This
phenomenon is called synergy. Here we present a review of the scientific literature on three main groups of environmentally relevant chemical
toxicants: pesticides, metal ions and antifouling compounds. The aim of the review is to determine 1) the frequency of synergy, 2) the extent of
3) whether any particular groups or classes of chemicals tend to induce synergy, and 4) which physiological mechanisms might be responsible for
synergy. Synergy is here defined as mixtures with minimum two-fold difference between observed and predicted effect concentrations using
Concentration Addition (CA) as a reference model and including both lethal and sub-lethal endpoints. The results showed that synergy occurred in
3% and 26% of the 194, 21 and 136 binary pesticide, metal and antifoulants mixtures included in the data compilation on frequency. The difference
between observed and predicted effect concentrations was rarely more than 10-fold. For pesticides, synergistic mixtures included cholinesterase
inhibitors or azole fungicides in 95% of 69 described cases. Both groups of pesticides are known to interfere with metabolic degradation of other
xenobiotics. For the four synergistic metal and 47 synergistic antifoulant mixtures the pattern in terms of chemical groups inducing synergy was less
clear. Hypotheses in terms of mechanisms governing these interactions are discussed. It was concluded that true synergistic interactions between
chemicals are rare and often occur at high concentrations. Addressing the cumulative rather than synergistic effect of co-occurring chemicals, using
standard models as CA, is therefore regarded as the most important step in the risk assessment of chemical cocktails [4].
7

8. 5. Sucrose Acetate Isobutyrate (SAIB): Historical Aspects of its use in Beverages and a Review of Toxicity Studies Prior to
1988.
R.C.Reynolds1, cIChappel2
1Corporate Health, Safety, and Environment, Eastman Kodak Company, Rochester, NY 14652, USA
2Botany Hill, Oakville, Ontario, Canada L6J 6J5
(1998) Science Direct, USA.
Sucrose acetate isobutyrate (SAIB), a mixture of esters of sucrose with a composition approximating the name sucrose diacetate hexaisobutyrate, has been used
over 30 yr in many countries as a `weighting' or `density-adjusting' agent in non-alcoholic carbonated and non-carbonated beverages. As part of the
of safety of SAIB as a direct food additive in human diets, a program of toxicity testing was started in the late 1950s that culminated in extensive studies of SAIB in
rodents, monkeys and humans over the last decade. This review summarizes the toxicity data, accrued up until 1988, that precede the safety studies published
elsewhere in this issue. SAIB has been shown to have very low acute and chronic toxicities in rats, monkeys, and, except for effects on the liver, in dogs at feeding
levels of up to 10% in the diet. Slight effects seen in rats and monkeys at levels of 10% in the diet are unlikely to be directly caused by exposure to SAIB. In dogs,
however, SAIB causes decreases in bromosulfophthalein (BSP) and indocyanine green (ICG) elimination from the serum immediately following a single dose,
indicative of interference with biliary excretion. On repeated feeding in dogs, SAIB caused increases in serum alkaline phosphatase levels, but enzymes indicative
toxic effects on the liver were unaffected. On prolonged feeding to dogs, SAIB caused changes in liver morphology revealed by electron microscopy. All of these
effects were reversed when SAIB was withdrawn from the diet. The no-effect level for these effects in dogs was near 5 mg/kg body weight, but these effects were
seen in rats fed up to 4 g/kg body weight/day, monkeys fed up to 10 g/kg body weight/day, or humans fed up to 20 mg/kg body weight/day. The toxicity and
pharmacological studies in dogs, rats and monkeys suggest that the effect of SAIB on biliary excretion and liver morphology in dogs is essentially pharmacological
rather than toxicological in nature and that the difference between the effects in dogs at levels as low as 5 mg/kg body weight/day, and the lack of effects in rats
monkeys at levels up to 10 g/kg/day is not merely a quantitative difference between species, but an absolute qualitative difference [5].
8

9. 6. Biodistribution and toxicity of engineered gold nanoparticles: a review of in vitro and in vivo studies.
Nikolai Khlebtsov1,2 and Lev Dykmana1
1Saratov State University, 83 Ul. Astrakhanskaya, Saratov 410012, Russian Federation
2Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, 13 Pr. Entuziastov, Saratov 410049, Russian
Federation
(2011) Chemical Society Reviews.
Recent advances in wet chemical synthesis and biomolecular functionalization of gold nanoparticles have led to a dramatic expansion of their potential biomedical
including biosensorics, bioimaging, photothermal therapy, and targeted drug delivery. As the range of gold nanoparticle types and their applications continues to increase,
safety concerns are gaining attention, which makes it necessary to better understand the potential toxicity hazards of these novel materials. Whereas about 80 reports on the in
vivo biodistribution and in vitrocell toxicity of gold nanoparticles are available in the literature, there is lack of correlation between both fields and there is no clear
of intrinsic nanoparticle effects. At present, the major obstacle is the significant discrepancy in experimental conditions under which biodistribution and toxicity effects have
evaluated. This critical review presents a detailed analysis of data on the in vitro and in vivo biodistribution and toxicity of most popular gold nanoparticles, including atomic
clusters and colloidal particles of diameters from 1 to 200 nm, gold nanoshells, nanorods, and nanowires. Emphasis is placed on the systematization of data over particle types
parameters, particle surface functionalization, animal and cell models, organs examined, doses applied, the type of particle administration and the time of examination, assays
evaluating gold particle toxicity, and methods for determining the gold concentration in organs and distribution of particles over cells. On the basis of a critical analysis of data,
arrive at some general conclusions on key nanoparticle parameters, methods of particle surface modification, and doses administered that determine the type and kinetics of
biodistribution and toxicity at cellular and organismal levels [6].
9

10. Definitions
Toxicity : Any toxic (adverse) effect that a chemical or physical agent might produce within a living organism.
Toxicology
Types of Toxic Studies
Acute toxicity : It refers to those adverse effects occurring following
oral or dermal administration of a single dose of a substance, or
multiple doses given within 24 hours, or an inhalation exposure of 4
hours.
Sub acute Toxicity: It resembles acute toxicity except that the
exposure duration is greater, from several days to one month.
Sub chronic toxicity: It is the toxic exposures repeated or spread
over an intermediate time range (1 – 3 months)
Chronic Toxicity: It is the exposures (either repeated or continuous)
over a long (greater than 3 months) period of time.

11. Testing Strategies
A number of different types of data are used in order to establish the safety of chemical
substances for use in foods. These include:
 Consideration of the chemical structutre and any intended biological activity (e.g. anti-
oxidant).
 In vitro models, such as cell cultures or tissue slices.
 Laboratory animals.
 Human volunteers.

12. The Importance of Toxicity studies
12
• Before a study reaches Phase I clinical trials, scientists spend years conducting preclinical research. One vital part of this
includes performing toxicology research on a particular medication or pharmaceutical product. The U.S. Food and
Drug Administration (FDA) put great emphasis on the importance of preclinical safety evaluations.
• According to the FDA’s guidance for industry on preclinical safety evaluation, pharmacokinetic research needs to
include experimental medicines or biopharmaceutical products that represent drugs meant for toxicity testing. This
administration route should be similar to the one which will be used in clinical trials.
• During toxicity testing, systemic exposure should be monitored, along with patterns of absorption. Before beginning a
clinical trial, it will be necessary to provide information on absorption, clearance, and disposition of each compound in
animal models in order to anticipate safety of exposure in human subjects.
• Additionally, the methods for evaluating absorption, clearance, and disposition should be equivalent in animal and
human models. It is important to determine the metabolism of tested drugs and how they will react within the human
body.

13. Regulatory Studies
13
 Acute Toxicity
 Subacute Toxicity (Repeated Dose)
 Sub chronic And Chronic Exposure
 Chronic Exposure
 Drug Disposition/Pharmacokinetics (ADME)
 In Vitro Permeation Studies
 In Vivo Absorption Studies
 Irritation and Sensitization
 Immunotoxicity
 Reproductive Toxicity
 Genotoxicity/Mutagenicity
 Adjuvant Safety and Immunogenicity
 Safety Pharmacology

14. Stages of possible toxicity of an ingested substance
Not bioavailable
Chemical in food
Modified by gastrointestinal secretion or
microflora
Ingestion
Excretion
Absorbed intact
LIVER

15. Stages of possible toxicity of an ingested substance
Interaction with cell
constituents/cells
Excretion
Repair
Reactive metabolites
Circulatory system
Unchanged
Toxicity
Stable metabolites
Extrahepatic organs/systems (further
metabolic activation and/or detoxication
possible)
LIVER
Excretion

16. Toxicity Tests
Acute oral toxicity - Single dose study to define extent of toxicity in absence
of other data.
Short-term toxicity - Repeated daily doses for 14-28 days to provide
indications of toxic potential.
Subchronic toxicity - Repeated daily doses for 90 days to provide information
on major site(s) of toxicity and effects, and to indicate suitable dose levels for
chronic studies, usually in two species, rodent and non-rodent.
Chronic toxicity and carcinogenicity - Repeated daily doses for 2 years in
rodents, providing the data most frequently used in deriving the ADI.

17. Genetic toxicity - Short-termed tests for capacity to interact with DNA and to cause
mutations or chromosome changes, using a variety of endpoints in bacterial and
mammalian systems, in vitro and in vivo.
Reproductive and developmental toxicity - Repeated daily doses before, during and
after gestation to determine effects on male and female fertility and on the developing
fetus and neonate and possible inheritable effects. Usually involves a multi-generation
study in a rodent and developmental toxicity in two species.
Immunotoxicity - Investigations on the structure and function of the tissues and
cells involved in the immune response (included in short-term and subchronic
studies).
Neurotoxicity - Investigations on the structure and function of the nervous system,
and on behaviour (included in short-term and subchronic studies).

18. Single Dose Acute Toxicity Testing for Pharmaceuticals
 Acute toxicity studies in animals are usually necessary for any pharmaceutical intended for human
use.
 It is useful in choosing doses for repeat-dose studies, providing preliminary identification of target
organs of toxicity, and, occasionally, revealing delayed toxicity.
 Acute toxicity studies may also aid in the selection of starting doses for Phase 1 human studies, and
provide information relevant to acute overdosing in humans.
 Acute toxicity is the toxicity produced by a pharmaceutical when it is administered in one or more doses
during a period not exceeding 24 hours.

19. Principle of Toxicity Studies
PRINCIPLE OF TOXICITY STUDIES
 Standard operating procedures (SOP’s) and NIH guidelines should
be thoroughly followed for these studies.
 It should be performed by well trained and qualified staff.
 These should comply with norms of good laboratory practices.
 The test substances and systems should be properly characterized and
standardized.
ANIMAL PROTECTION Studies should be designed so that the maximum amount of information is
obtained from the smallest number of animals.To avoid causing excessive pain or tissue damage in the
animals, pharmaceuticals with irritant or corrosive characteristics should not be administered in
concentrations that produce severe toxicity solely from local effects.

20. Instruments Used
20
For various Biochemical, Histopathological and Hematological parameters the toxicology
division houses a variety of instruments like :
- Haematology Analyzer - Biochemistry Analyzer
- Various microscopes including inverted microscope
- Urine Analyzer
- Inhalation instrument (nose only)
- CO2 Incubator
- Automatic tissue processor
- Deep freezer
- Microtome

21. Testing Procedures
 The test compound should be administered to animals to identify doses causing no adverse effect
and doses causing major (life- threatening) toxicity.
 The use of vehicle control groups should be considered
 Acute toxicity studies in animals should ordinarily be conducted using two routes of drug
administration: (1) The route intended for human administration, and (2) intravenous administration, if
feasible.
 Studies should be conducted in at least two mammalian species
(Rodents and non rodents)

22. Observation
 Animals should be observed for 14 days after pharmaceutical administration. All mortalities, clinical
signs, time of onset, duration, should be recorded.
 Also reversibility of toxicity should be recorded.
 Gross necropsies should be performed on all animals, including those sacrificed, moribund, found dead,
or terminated at 14 days.
 Clinical pathology and histopathology should be monitored at an early time and at termination (i.e.,
ideally, for maximum effect and recovery).
 The toxicity studies should be designed to assess dose-response relationships and pharmacokinetics to
develop the lead compound.


23. Chronic Toxicity Studies
 It is the ability of the substance or mixture of substances to cause harmful effects over an extended
period, usually upon repeated and continuous exposure.
 The result of chronic toxicity study in animals should suggest signs and symptoms of adverse
reactions to look for in man.

24. Proposals For Clinical Study
Certain basic studies should be considered mandatory when examining drug
action in vivo.
1. Make an assessment of drug exposure in the animal, preferably by measurement of
plasma drug concentrations at several time points, and after several different
doses.
2. Perform a measurement of the degree of plasma protein binding of the drug, ex vivo
if possible, and if not, then in vitro. This is vital if comparisons are to be made of a
drug action in more than one species.
3. Obtain knowledge on the metabolism of the drug and determine whether active
metabolites could be influencing the results being obtained.

25. 4. Ensure that the dose schedules to be used are relevant to the way that the drug will
be used in humans to allow translational value to the clinic.
5. Consider whether there is a temporal mismatch between the exposure information
being gained and the outcome measure being investigated because this might give
insight into the mechanism of action of the compound under investigation.
6. Consider the determinants of target engagement whenever the information
above has been gathered.

26. External Factors That May Influence Susceptibility To Toxicity
DIETARY FACTORS
Alcohol
Carbohydrates
Essential elements
Fat
Protein
Pyrolysis products (formed during cooking)
Trace elements
Vitamins
ENVIRONMENTAL FACTORS
Drugs of abuse
Heavy metals
Industrial pollutants
Pesticides
Petroleum products
Pharmaceuticals
Pyrolysis products (as pollutants)
Tobacco smoke

27. Conclusion
27
The integrated overview and conclusions should clearly define the characteristics of the human
pharmaceutical, as demonstrated by the nonclinical studies, and arrive at logical, well-argued conclusions
supporting the safety of the product for the intended clinical use. Taking the pharmacology,
pharmacokinetics, and toxicology results into account, the implications of the nonclinical findings for the
safe human use of the pharmaceutical should be discussed, especially as it applies to labeling.
Roughly 80% of candidate drugs will fail during the preclinical phase of their development. A well thought out
nonclinical pharmacology/ toxicology program is critical for the long-term effectiveness of a drug development
effort. Because they help drug developers avoid clinical trials that are destined to fail, nonclinical studies can
be a major tool for reducing drug development costs.
However, agencies have struggled to incorporate recent scientific and technologic advances in toxicology,
basic human biology, molecular biology, pharmacokinetics, dose-response modeling, imaging, computation,
and other relevant fields, so many of the current requirements are still based on approaches that originated
more than 40 years ago. In addition, more sophisticated exposure assessments have identified different
durations and routes of exposure for various populations, such as residential exposures of toddlers, that
require more toxicology data for risk assessment.

28. 28
Proposed strategies for improving toxicity testing can be difficult to compare directly. Some
strategies aim at meeting regulatory mandates and therefore focus on specific needs. The
different purposes of those testing strategies contribute to major differences between them.
However, there can also be important differences between testing strategies and approaches of
initiatives and proposals that try to fulfill the same risk-management needs. The committee
elected to focus primarily on the major aspects of the reports reviewed, rather than critiquing the
details. Most of the reviewed reports describe initiatives or proposals that are still under
development, some of which are sometimes presented with few details; some reports were
available to the committee only as drafts. Studies should be designed so that the maximum
amount of information is obtained from the smallest number of animals.
To avoid causing excessive pain or tissue damage in the animals, pharmaceuticals with irritant or
corrosive characteristics should not be administered in concentrations that produce severe
toxicity solely from local effects.

29. References
1. Wang W (1990) Literature review on duckweed toxicity testing. Environ Res. 52(1): 07-22.
2. Wang W (1991) Literature review on higher plants for toxicity testing. INT J ENVIRON POLLUT 59(4): 381–
400.
3. P Das, S Samantaray, GR Rout (1998) Studies on cadmium toxicity in plants: A review. INT J ENVIRON
POLLUT 98(1): 29-36.
4. Nina Cedergreen (2014) Quantifying Synergy: A Systematic Review of Mixture Toxicity Studies within
Environmental Toxicology. PLoS One 9(5): 10-12.
5. RC Reynolds, Chappel (1998) Sucrose Acetate Isobutyrate (SAIB): Historical Aspects of its use in
Beverages and a Review of Toxicity Studies Prior to 1988. Food Chem Toxicol 36(2): 81-93.
6. Khlebtsov N, Dykman L (2011) Biodistribution and toxicity of engineered gold nanoparticles: a review of
in vitro and in vivo studies. Chem Soc Rev. 40(3): 1647-1671.
29

30. 30