Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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A harmonized classiﬁcation was issued recently with ISO 14644-1, with

reference to the classiﬁcation terminology and also with units (now in m

Rooms classiﬁed as ISO class 5, 7, or 8, correspond respectively to US

categories 100, 10.000, or 100.000 (SBCC, 2005). The key element of the

ISO class 5 is laminar air ﬂow, a mass of air that ﬂows at a controlled and

low rate and so drags particles without turbulence. These are generally

represented by air ﬂow cabinets (islands of ISO class 5).

Another point to consider when designing a clean room is the material

used in construction. Internal surfaces (walls, ceilings, and ﬂoors) should

be formed by unbroken surfaces made of smooth materials with no

shedding particles, therefore allowing easy cleaning and disinfection.

Corners should be round. The installations should have a minimum

amount of shelving and equipment designed to reduce dust and facilitate

cleaning. Recommended materials are polished stainless steel or solid

resins.

The ﬂow of materials and people must be carefully managed. Following

GMP principles, materials should pass through an airlock that does not

allow both doors to be open simultaneously. Used material and products

should be removed by an independent exit. Personnel movement occurs

through separate rooms with separate doors for entry and exit.

The garments used by the workers should be made of synthetic fabrics

that minimize contamination risks. Operators should be trained for their

work and also for the maintenance of clean conditions and hygiene.

Cleaning is a key factor in the maintenance of good conditions and reduces

or eliminates potential contaminants that can affect the product. Contami-

nants can have various sources: live particles, inert particles (dusts, glass,

ﬁle dust, lubricants, etc.) or cross-contamination when different processes

or products share the same clean area. To avoid problems, the cleaning

procedures should be written and approved, validated, and executed by

trained operators.

14.5.2 Biosafety

Cell cultivation is a tool for the production of viral vaccines, tumor

antigens, mAbs, and recombinant proteins. These organisms, their pro-

ducts, or adventitious agents that may be present in the production

processes can potentially be transmitted to the operators, to the environ-

ment, and to the outside. Protection measures must be taken to reduce this

risk, so that containment must be a central issue regarding the design of

facilities, bioreactors, and downstream processing equipment. To work

with GMOs (genetically modiﬁed organisms) requires adherence to spe-

ciﬁc rules adopted by the regulatory agency of each country, for example,

NIH (2002) in the USA and CTNBio (1997) in Brazil. When working

with non-pathogenic recombinant DNA in mammalian cells (risk class I),

biosafety level is classiﬁed as BSL-1 or BSL-1/’’large volume’’, when the

working volume exceeds 10 L and is in a closed system like a bioreactor.

Some procedures, such as sampling, reagent addition, or liquid transfer,

are critical and should be performed with particular care to avoid contam-

ination. Exhaust gases from bioreactors should be ﬁltered through HEPA

ﬁlters or incinerated. GMO-containing bioreactors should only be opened

354 Animal Cell Technology

after adequate sterilization. Waste liquid that could contain live recombi-

nant or pathogenic organisms should be decontaminated before discarding.

Simultaneous processing can be accepted for some products like mAbs

and recombinant proteins once closed systems are used.

14.6 Cell banks

The production of heterologous proteins for therapeutic use requires

selection of the producer cell line, based on yield, monoclonality (for

proteins), product quality, stability, and absence of contaminants like

bacteria, molds, mycoplasmas, and viruses. Progress in the production of

biopharmaceuticals by cell culture is due mainly to the use of diploid cells

and continuous cell lines, together with the maintenance of cells by cryo-

preservation. It is important to guarantee that the expression system

chosen is able to generate the product in a consistent and economically

feasible way (Levine and Castillo, 1999).

The cells used for a process must be held in a cell bank, guaranteeing a

continuous supply during the lifespan of the product. According to the

FDA (1993a), the master cell bank (MCB) is deﬁned as a large number of

aliquots containing cells with uniform composition and derived from a

single tissue or cell and conserved in liquid nitrogen (–1968C). The MCB

is used for the preparation of the working cell bank (WCB) that is used for

the manufacture of production batches. For the preparation of the banks,

cells are cultured to an amount sufﬁcient to dispense into hundreds of

individual vials.

The documentation related to the cell banks should include the inven-

tory of all vials, their identity, and their location. There must be a

monitoring program for the stability of the cryopreserved cells in the

MCB, including viability and growth characteristics. The cell banks must

be generated in a controlled environment, observing GMP requirements.

While preparing the banks no other cell line or virus should be cultivated

simultaneously in the same laboratory or by the same operators. Access to

the stored vials should be controlled. The storage temperature should be

monitored and the liquid nitrogen level veriﬁed in all the storage units,

with records of deviations and any correcting actions.

The manufacturer must establish the maximum cultivation span for a

particular cell in vitro (passage number). The characterization must in-

clude the growth rate, morphology, speciﬁc yield, and quality of the

molecule of interest (Wiebe and May, 1990). The post-production cells

(PPCs) must be removed from the bioreactor and tested at least once to

evaluate whether new contaminants were introduced or induced by the

cultivation conditions. Changes in the culture medium or production scale

require new evaluation of the PPCs to determine any effects on the yield

and product consistency (Levine and Castillo, 1999).

14.6.1 Cell bank qualiﬁcation

The cell bank qualiﬁcation must consider many factors including the

presence and identiﬁcation of contaminants (FDA, 1993a; ICH, 1998a;

Regulatory aspects 355

WHO, 1998). Usually an extensive characterization is required for the

MCB, including tests for the establishment of cell properties and stability

of those properties during the production process. The WCB must be

subjected to tests such as microbial sterility, including mycoplasma detec-

tion tests and routine tests for virus detection (in vitro and in vivo), in

order to identify contaminants that might be introduced by the culture

media or manipulation. This is important because the composition of

culture media is changing, with the trend to substitute animal sera for

chemically deﬁned or animal component-free formulations. For cell line

selection for the production of biologicals, FDA (1997) and WHO (1998)

do not allow the use of cells obtained from carriers of transmissible

diseases or diseases of unknown etiology. Cells from neural origin can

contain or are able to amplify the causative agent for spongiform encepha-

lopathy and therefore must not be used for the production of biologicals.

Cell line characterization

The cell line should have a detailed history, including origin (organ, tissue,

age, gender, and donor species) and a description of the methods used for

its screening, history of passages, and culture media used. The cell charac-

teristics that are identiﬁed include: karyotype, isozyme proﬁle, growth

rate (generation time), morphology, tumorigenicity, or genetic markers

(DNA ﬁngerprinting). When dealing with recombinant products, the

documentation should include information about the DNA insert that

codes for the product, preparation method, and the complete DNA

sequence (vector), including the regulatory sequences and antibiotic resis-

tance markers. The history must contain the cloning details of the

recombinant cell and the methodology used, as well as insert copy number

and the physical status of the vector inside the host cell, whether integrated

or extrachromosomal. The nucleotide sequence of the insert that codiﬁes

the expressed product, as well as the restriction map of the expression

vector, must be analyzed at the characterization phase of the MCB and at

least once in the PPCs (FDA, 1985; ICH, 1995, 1998a; Onions, 1997).

Cells such as human epithelial cells used for attenuated vaccine production

need to be tested for tumorigenicity (FDA, 1993a). Whenever cells are

characterized as non-tumorigenic, in vivo and in vitro testing are per-

formed either with MCB or WCB cells propagated up to or over the

cultivation time allowed for production, according to the methodology

described by WHO (1998).

Adventitious agent testing

Cells in culture are susceptible to microbial contamination from various

sources. The cell line itself can contain viruses derived from the animal it

originates from. Other sources of animal contamination are sera, additives

to the culture media, or porcine trypsin used for detachment of adherent

cells. Operators represent another contamination source. Bacterial and

fungal contamination should be tested in 1% of the total number of

ampoules, but not less than in two ampoules of MCB and WCB, according

to the protocols described in the European, Japanese, or American

356 Animal Cell Technology

Pharmacopeia (ICH, 1998a). The absence of mycoplasmas must be veriﬁed

in the MCB and WCB ampoules by at least two different methods (FDA,

1993a). Mycoplasma contaminations are rather common and persistent

while not causing perceptible morphological changes. They can cause

chromosomal alterations, as well as changes in cellular metabolism and

viral replication. According to the literature, the most common sources of

contamination are oral human microbiota due to operator contamination,

and bovine microbiota originating from sera (Timenetsky et al., 1992).

Virus testing

Routine testing for adventitious viruses

Cells derived from MCB or WCB must be tested for adventitious viruses

by in vitro and in vivo methods. For the in vitro test, indicator cells can be

used to verify cytopathic or morphological changes when challenged with

the product, the cells used for production or other related materials, like

culture supernatant or lysed cells. The in vivo testing for viral agents that

induce pathologies can be carried out by the intramuscular inoculation of

cells or cell lysates into adult and newborn mice and guinea pigs. An

alternative is the intracerebral inoculation of mice with the test material.

Any abnormality observed in the clinical state or mortality of the animals

indicates the possibility of infection that needs to be investigated. For egg

inoculation, viable cells should be injected into the allantoid cavity and

yolk sac of embryonated chicken eggs. Cells from MCB and WCB are

accepted when neither animals nor eggs show evidence of any viral agent

(Onions, 1997; WHO, 1998).

Testing for speciﬁc viruses

Species-speciﬁc viruses potentially present in mouse, hamster, and rat cell

lines should be tested through assays for antibody production in the

respective animals (MAP, HAP, or RAP). In this assay the cells are

inoculated into an animal of the corresponding species, which is bled after

28 days to evaluate the presence of antibodies against the speciﬁc viruses

(WHO, 1992, 1998; ICH, 1997). For the lymphocytic choriomeningitis

virus, murine cytomegalovirus, murine rotavirus, thymic virus, and lactic

dehydrogenase virus, speciﬁc tests are recommended (Onions, 1997). The

MCB should not be used for production if any potentially human disease-

causing virus or polyomavirus is found. Other viruses present do not

prevent cells from being used, since removal or inactivation procedures

during the downstream processing can be validated. For human cell lines

the tests should include Epstein–Barr virus, cytomegalovirus, hepatitis B

and C, herpes 6 virus, and any other viruses derived from the donor’s

anamnesis. Hybrid cells derived from two species should be tested for

viruses speciﬁc to both species (FDA, 1997; WHO, 1998). Animal-derived

reagents like sera or trypsin should be certiﬁed through appropriate testing

(WHO, 1990). Speciﬁc detection tests are required for bovine diarrhea

virus, bovine polyomavirus, and prions. Prions are agents of bovine

spongiform encephalopathy (BSE) and present a particular risk. It is

recommended to use sera exclusively from non-endemic countries of BSE

or, even better, not to use animal-derived reagents at all (WHO, 2003).

Regulatory aspects 357

Today, many media suppliers offer serum-free and/or animal component-

free media, thus decreasing contamination risks.

Testing for retroviruses

Retroviruses are associated with oncogenic and degenerative diseases and

may be present in some cell lines. They represent a problem since some

retroviruses are able to replicate in human cells. Cells from the MCB and

cells cultivated over the regular in vitro cultivation period (passage num-

ber) should be tested for rodent retroviruses by electronic microscopy

and infectivity on susceptible cells (transformation focus detection in

S+L- cells, as well as syncitium and plaque formation in XC cells). If

infectivity is not detected and electronic microscopy does not reveal

retroviruses or retrovirus-like particles, a reverse transcriptase assay can be

used to detect non-infectious retroviruses (ICH, 1997). Myelomas used

for production of mAbs have the inherent capacity of producing infectious

retroviruses, and therefore a suitable test should be performed in several

supernatant harvests and lot-to-lot consistency should be demonstrated.

At the end of a typical cultivation process the burden of endogenous viral

particles should be determined. These data are important for planning the

puriﬁcation process, including steps for removal or inactivation of retro-

viruses (FDA, 1997; WHO, 1998). The CHO cell line expresses defective

retroviral particles; although CHO retroviruses are not infectious, recom-

bination is frequent and DNA repair can occur. Thus, for the puriﬁcation

of CHO-derived recombinant proteins, it is recommended to include one

or more robust steps for retrovirus removal or inactivation. For cells other

than murine, speciﬁc assays for retroviruses are necessary. Cells from

human origin should be tested for human T-lymphotropic retrovirus

(HTLV-1 and -2) and for the lentiviruses HIV-1 and HIV-2 (FDA, 1997;

Onions, 1997).

14.7 Validation

The concept of validation came up in the 1970s in association with

sterilization procedures and was extended to all steps of pharmaceutical

manufacturing procedures. Validation means proving that any and all

procedures, processes, equipment, material, operations, and systems com-

ply with the expected performance. Well-planned and well-conducted

validation studies constitute GMP principles once they guarantee a con-

sistently safe and efﬁcacious ﬁnal product. Validation is important for

companies, ﬁrst for QA, and also for cost reduction, decreasing failures,

rejection, reworks, recalls, and complaints. The positive aspect of valida-

tion is an increase in productivity, as a consequence of a well-controlled

process. Validation is required by the regulatory agencies of many coun-

tries.

14.7.1 General aspects

A validation plan (Validation Master Plan) is a GMP requirement, attest-

ing control of all critical operation aspects. Consistent results must be

358 Animal Cell Technology

demonstrated in at least three studies with representative production runs

showing acceptable and comparable results. Unlike other GMP require-

ments, validation itself does not improve the process. It conﬁrms that the

process is adequately developed and under control. Validation processes

require the mutual collaboration of all related sectors, such as develop-

ment, production, engineering, maintenance, QA, and QC (ANVISA,

2003).

Design Qualiﬁcation (DQ) is the ﬁrst validation element of a new

facility system or equipment, where adherence to the user’s speciﬁcations

and to GMP rules is demonstrated. Installation Qualiﬁcation (IQ) follows

with the veriﬁcation of adequacy of the area, installation of equipment

pipelines, utilities, instrumentation, and conformity of the material used to

the project speciﬁcations. At the Operational Qualiﬁcation (OQ) phase,

carried out after installation of all equipment, it is veriﬁed whether the

system, when in operation, complies with the acceptance criteria deﬁned

in the validation plan. Once the OQ phase is successfully ﬁnalized it is

possible to proceed with the calibration procedures, operation and clean-

ing, operator training, and preventive maintenance program. After IQ and

OQ are concluded, it is time for the Performance Qualiﬁcation (PQ), with

the aim of verifying that what was designed, built, and operated results in

a product that meets the expected speciﬁcations. Production and QC

personnel are specially trained for these assessments. The tests can be done

with the product of interest or a placebo, and are related to all operations,

from raw material reception to product release (EC, 2001).

Process validation

Process validation is usually performed before commercialization (Pro-

spective Validation). Exceptionally, it may be necessary to validate pro-

cesses during routine production (Concurrent or Simultaneous

Validation). Processes already in use can also be validated (Retrospective

Validation). To validate a process, a previous validation of facilities,

systems, equipment, and analytical methods (ICH, 1996a), as well as

previous training of operators (WHO, 1996; EC, 2001), are required.

Cleaning validation

The efﬁciency of cleaning procedures should be veriﬁed through validated

analytical testing with enough sensitivity for residues or detection of

contaminants at acceptable levels. It is important that all surfaces poten-

tially in contact with the product be cleaned by previously validated

methods. The FDA proposes that manufacturers set limits for active

principle residues after cleaning and in the product to be obtained at a

subsequent production run (FDA, 1993b). A multipurpose plant should be

specially monitored to avoid cross-contamination (Doblhoff-Dier and

Bliem, 1999).

Regulatory aspects 359

Changes in validation

Any change in material, product components, equipment, environment,

methodology, or assays that could affect product quality or process

reproducibility should be documented. It should be guaranteed that, after

the alterations, the process results in a product that meets the speciﬁca-

tions previously approved. All changes that might affect product quality

or process reproducibility require revalidation and therefore should be

subject to permission by the regulatory agencies (WHO, 1996; EC, 2001).

14.7.2 Biological products

The norms for medicinal production are particularly stringent. Biological

products are composed of complex molecules, produced by cell lines with

a relatively recent history, and difﬁcult to characterize. Tests performed

only on the ﬁnal product do not guarantee consistency of production. The

puriﬁcation procedures should be planned and validated for the removal

of potential contaminants from diverse sources: cells, culture media,

equipment, and reagents used in the puriﬁcation or even degradation

products derived from the protein itself. There are examples of products

with unexpected risks that have caused serious problems such as blood

contamination by HIV-1 virus between 1980 and 1985 (Bloom, 1984) or

the presence of residual infectious viruses in the poliomyelitis vaccine due

to inefﬁcient inactivation (Lubiniecki et al., 1990).

Potential risks associated with animal cell-derived products

Among the products derived from animal cells, the potential contaminants

are associated with: (i) the cell itself (whole cells, viruses, and other

transmissible agents, cellular DNA and growth-promoting proteins); (ii)

raw materials (adventitious agents such as bacteria, molds, mycoplasma,

viruses, foreign proteins, and endotoxins); (iii) reagents used in the process

(antibiotics, resin ligands, solvents, sanitizers, inducers, and nutrients).

The production planning must include steps for the removal or inactiva-

tion of potential risk factors. It is important to identify the introduction,

reduction, or concentration of a given risk factor and its content during

the process and in the ﬁnal product.

The regulatory authorities evaluate safety aspects when the application

for registration is submitted. During product development it is possible to

discuss further with the authorities with the aim of establishing safety

limits (Lubiniecki et al., 1990; WHO, 1998).

Viruses and other transmissible agents

Virus identiﬁcation can start during cell bank characterization and con-

tinues until the ﬁnal product is obtained. It is essential to demonstrate that

the viruses are not co-puriﬁed with the product (FDA, 1993a, 1997; ICH,

1997). The efﬁciency of removal or inactivation procedures should be

demonstrated on a small scale through spiking experiments, by the use of

the viruses themselves, when identiﬁed and possible, or with model viruses

which mimic any possible variants: large or small, DNA or RNA genome,

360 Animal Cell Technology

enveloped or not, and with variable resistance to physicochemical agents.

At least one or preferably more steps for virus removal or inactivation

should be included in the puriﬁcation process. The validation is carried

out by deliberate contamination of the supernatant with high titers of the

model viruses followed by performing the complete puriﬁcation protocol.

The viral burden is quantiﬁed at each step of the process, and the

cumulative virus reduction capacity is usually expressed on a logarithmic

scale (FDA, 1997).

Cellular DNA

Cells derived from primary cultures or diploid cell lines have been used

for a long time for viral vaccine production and the residual cellular DNA

is not considered of risk to the users. On the other hand, continuous cell

lines, which can be cultivated indeﬁnitely due to deregulation of growth

control genes, are thought to pose risks to the users due to the possible

transmission of transformation characteristics (Barone et al., 1992; Bert-

hold and Walter, 1994). However, Wierenga et al. (1995) injected a human

tumor DNA into monkeys over an 8-year period without tumor induc-

tion. The quantity of DNA administered to a patient undergoing blood

transfusion can reach 450 g (Duxbury et al., 1995), which is 10

times

higher than the limit allowed in products derived from continuous cell

lines. These facts, taken together with other evidence (Kurt, 1995; Petric-

ciani and Horaud, 1995; Temin, 1990), led to the argument that the risk

associated with residual cellular DNA is low when the amount is 100 pg

per dose or less. An important aspect is that residual contaminant DNA

usually occurs in small fragments of 200–500 bp (Rodrigues et al., 1997),

thus not codifying a functional gene.

According to WHO (1998), DNA from a continuous cell line can be

considered a cellular contaminant, instead of a signiﬁcant risk factor that

requires reduction to extremely low levels. Therefore, upper limits of

10 ng per dose are acceptable for products generated from continuous cell

lines. Only in speciﬁc situations that might be considered harmful, for

example, when infectious retroviral pro-virion sequences are present, the

acceptable limit per dose should be assigned by the regulatory authorities.

Levels of cellular DNA should be measured in the supernatant harvest, in

the intermediate puriﬁcation steps and in the ﬁnal product to determine its

initial concentration and whether it was removed or concentrated. The

removal efﬁciency must be determined based on several runs to ensure

conﬁdence in the data. Validation of the process excludes the need for

residual cellular DNA testing in the bulk product (FDA, 1997; WHO,

1998).

Other contaminants

The presence of transforming proteins that induce the proliferation of

different cell lines and that are coded by oncogenes represent a limited

risk, as they are secreted in small amounts and are quickly inactivated

when administered in vivo (Petricciani, 1988; Lupker, 1998). Analytical

tests to assess purity as well as the puriﬁcation process should be validated

to demonstrate the capacity to remove host cell proteins to acceptable

levels. Once the lot-to-lot consistency has been demonstrated, the test can

Regulatory aspects 361

be dispensed with for release of the bulk product (WHO, 1998). Other

potential contaminants that should be monitored are endotoxins, animal

sera-derived proteins or protein fragments, and degradation products or

other contaminants derived from the puriﬁcation process, such as resin

ligands, detergents, or salts. The puriﬁcation steps should be designed to

effectively remove those contaminants and the assays for their quantiﬁca-

tion should be validated (ICH, 1996a).

14.8 Stability

Stability is deﬁned as the period of time during which the product

maintains, within the speciﬁed limits, the same characteristics and proper-

ties displayed when it was manufactured. Environmental factors like tem-

perature, air, light, ionic content, and humidity can affect the drug’s

quality (Ugwu and Apte, 2004). Proteins are susceptible to proteolysis,

denaturation, aggregation, fragmentation, and chemical modiﬁcations such

as oxidation and changes in the disulﬁde bonds. The degradation process

directly affects the biological activity of the product and might cause

immunogenicity, among other adverse effects. The shelf-life is determined

by stability tests that guarantee that the product is active, pure, and safe

during a speciﬁed period, if stored under the prescribed conditions. The

stability tests should establish identity, purity, potency, and modiﬁcations

of the product. Tests to demonstrate stability include: (i) potency of

biological activity in vivo or in vitro, compared to reference material; (ii)

purity and molecular characterization of the active component and degra-

dation products, by SDS-PAGE, capillary electrophoresis, immunoelec-

trophoresis, western blotting, isoelectric focusing, chromatography

(reverse phase, ion exchange, hydrophobic interaction, or afﬁnity), peptide

mapping, circular dichroism, or mass spectrometry; (iii) physical charac-

teristics and appearance (color, opacity, pH, visible particulates in the

solution or after reconstitution of the lyophilized material); (iv) sterility

testing or alternatively package integrity testing; (v) assays to quantify

additives, stabilizers, preservatives, or excipients.

For registration, the stability tests supporting the proposed shelf-life

must be performed in three consecutive product batches in their ﬁnal

package. The data obtained on a pilot scale are accepted for registration

with a commitment by the manufacturer, after approval, to submit the ﬁrst

three batches of the commercial-scale production for stability testing. The

shelf-life of biological products determines the frequency at which the

tests should be repeated. For a product with a shelf-life longer than 1 year,

the tests should be repeated every 3 months for the ﬁrst year, every 6

months for the second year, and annually after that. The stability should

be monitored after the beginning of commercialization with enough

batches and a frequency to allow a tendency analysis (ICH, 1996b; EC,

2004). Accelerated tests (Tydeman and Kirkwood, 1984), with samples

stored at temperatures above the speciﬁcations, can help in the identiﬁca-

tion of degradation products and evaluate the capacity of analytical tools

used in the stability testing.

362 Animal Cell Technology

14.9 Clinical trials

The same process that will be used for commercial-scale production

should be adopted to produce the material used in the preclinical and

clinical trials. Signiﬁcant differences in the production process used to

obtain this material can invalidate the clinical trial results for the commer-

cial product, unless the comparability is demonstrated by identity, purity,

stability, and potency tests (ICH, 1998b).

14.9.1 Preclinical studies

Preclinical studies of a new drug give a good indication of its safety before

it is used in volunteers. The main objectives of a preclinical trial are to

determine the initial safe dose for humans, to deﬁne the dose regime, to

identify potential toxicity, the organs that may be affected, the severity of

adverse effects, and to identify safety parameters for the clinical monitor-

ing. The preclinical studies use two approaches: in vitro and in vivo. The

species speciﬁcity, immunogenicity, and pleiotropic activity should be

taken into consideration for biopharmaceuticals (ICH, 1998b).

Biological activity/pharmacodynamics

The biological activity can be assessed through in vitro assays with cell

lines with the objective of determining which effects can be related to the

clinical efﬁcacy. The animal species selected for toxicity assays should

consider the species speciﬁcity of the biopharmaceutical. Mammalian cell

lines can be used to anticipate speciﬁc in vivo activity on other species

including the human. For mAbs, immunological properties must be stud-

ied, including the antigen speciﬁcity, as well as complement binding and

cross-reactivity with tissues other than the target. The FDA recommenda-

tions (1997) for mAbs list a series of human tissues that must be included

in the cross-reactivity testing. A positive result with a non-target human

tissue requires an extensive animal study. Preclinical studies for the

evaluation of pharmacological and toxicological properties may involve a

large number of animals. Consequently, economical and ethical considera-

tions have encouraged the development of alternative in vitro systems

using cell culture. The guidelines for these studies are being constantly

revised by the regulatory authorities.

Toxicity studies

Toxicity studies include the evaluation of acute and chronic effects and

reproductive toxicity, as well as mutagenic and carcinogenic activity

(CNS, 1997; ICH, 1998b; Walsh, 2004a).

Acute and chronic toxicity

Acute toxicity is determined by the administration of one high dose of the

product to rodents, usually rats and mice of both genders. Two delivery

routes are chosen, one being that proposed for normal product use. The

animals are monitored and any deaths are analyzed in detail.

Regulatory aspects 363