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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5855_Библиотеки_им_академика_М_И_Перельмана.pdf
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
Chromosomal DNA
Ribosomes
Fimbriae
Plasmid DNA
Flagella
Capsule
Cell wall
Plasma membrance
Cytoplasm
membrane glycoprotien
(peplomer)
genetic material
(c) Viruses
capsid
Cell wall
matrix
lipid envelope
Vacuole
Septum
Nucleus
(b) Fungi(a) Bacteria
Figure 1: Structures of (a) bacteria, (b) fungi, and (c) viruses adapted from references 21–23
by a plasma membrane, see Figure 1a, [21, 24]. Most bacteria have
a cell wall made up of a continuous peptidoglycan. Gram-positive
bacteria have a thicker peptidoglycan than gram-negative bacteria.
Some bacteria also possess flagella which enable bacterial motility,
and fimbriae and capsule which enable attachment to surfaces. The
capsule also provides additional protection to the cell. Some bacteria
may produce spores which are highly resistant to chemicals.
Fungi consist of yeast and mold. Unlike bacteria, fungi are eukaryotic. Their cells consist of nucleus, mitochondrion, Golgi apparatus and vacuole, which are membrane-bound, in a cytoplasm
surrounded by a plasma membrane and cell wall, see Figure 1b [22].
The cell wall of fungi is typically composed of mannoproteins, chitins and glucans [25]. Fungi produce spores that are less resistant
than bacterial spores.
Hand Sanitizers
Viruses are obligate parasites consisting of genetic material, either
DNA or RNA, enclosed within a capsid composed of protein, see
Figure 1c [23, 26]. Structurally, viruses are described as enveloped
when their capsid is enclosed by an outer lipo- protein envelope, or
non-enveloped. The envelope consists of peplomers for attachment
to the host organism. Some pathogenic viruses include, for example,
those belonging to the families Adenoviridae, Coronaviridae and Her-
pesviridae [26]. The coronavirus is part of the Coronaviridae family.
It can be clearly seen that bacteria, fungi and viruses possess characteristic structures that may dier in chemical composition. Therefore, depending on their mechanisms of action, some compounds
are eective only against certain microorganisms.
403
Antimicrobial compounds and their applications in hand sanitizers
The antimicrobial compounds are classified based on their chemical
composition, see Table 1. They may show one or more mechanisms
of action against one or more types of microorganisms.
Ethanol and isopropyl alcohols are ABHS recommended by WHO.
Some studies have shown that alcohols are sporicidal [9, 28] or
sporostatic [29]. Other studies have shown that alcohols lack activity
against fungi [30, 31] including two common fungal species found
in indoor air, namely Aspergillus fumigatus and Penicillium chrysoge-
num [30]. With conflicting findings, the eectiveness of alcohols on
fungi cannot be ascertained, but their activities on bacteria and
viruses are conclusive.
Table 1: Summary of antimicrobial compounds and their spectra of activity
Classes of antimicrobial
compounds
Examples Spectrum of activity Mechanism(s) of action
Alcohols Ethanol,
isopropyl
alcohol
Broad spectrum activity against
non- sporulating gram-negative
bacteria Weaker activity against
non-sporulating gram-positive
bacteria due to thicker peptidoglycan layer
Some activity against enveloped
viruses Ethanol has stronger and
broader activity than isopropanol
May have some activity against
fungi. Mechanism of action is
not clear
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
Precipitate proteins and dissolve
lipids in plasma membrane,
thereby disrupting its function.
Bactericidal eect [27]
Inhibit ribosomes, hence inter-
fering with protein synthesis.
Bactericidal eect [27]
1. Target viral envelope [27]
2. Target capsid and genetic
material which determine the
production of viral envelope
thus, inactivating the virus [27]
1. Sporicidal eect [9, 28]
2. Inhibit fungal spore germina-
tion, suggestive of sporostatic
eect [29]
3. No fungicidal or fungistatic
activity [30, 31]
Quaternary Ammonium
Compounds (QAC)
Benzalkonium
chloride (BKC),
benzethonium
chloride
Broad spectrum activity against
non- sporulating gram-positive
bacteria
Some activity against some non-
sporulating gram-negative bacteria and some enveloped viruses
Weak activity against fungi
Biguanides Chlorhexidine
gluconate (CG)
Broad spectrum activity against
non-sporulating gram-positive
bacteria and enveloped viruses
Weak activity against non-spor-
ulating gram-negative bacteria
and fungi
Halogens Chlorine Broad spectrum activity against
gram-positive and gram-negative
bacteria
Broad spectrum activity against
viruses
Disrupt plasma membrane of
microorganisms [32, 33]
Mechanisms of action against
bacteria and viruses similar to
those of alcohols [34]
Highly bactericidal [34]
Highly oxidizing on cell wall
and plasma membrane, hence
destroying protein and interfer-
ing with protein activity
[35, 36 ]
Inactivate RNA genetic material.
Viricidal activity [35, 36]
(Continued )
Hand Sanitizers
405
Table 1: (Continued )
Classes of antimicrobial
compounds Examples Spectrum of activity Mechanism(s) of action
Iodine Broad spectrum activity against
bacteria and enveloped viruses
Activity against spores not ascer-
tained
Phenols Chloroxylenol Broad spectrum activity against
bacteria and enveloped viruses,
with the exception of Pseu-
domonas aeruginosa
Penetrate into microorganisms
swiftly to destroy genetic
material, resulting in cell death.
Microbicidal activity [9, 36]
Induce leakage of intracellular
component by deactivating
enzymes, damaging plasma
membrane or inhibiting cell
wall synthesis [9, 36, 37]
Some activity against fungi Damage plasma membrane,
thereby inducing leakage
of intracellular components [36,
38]
Bisphenols Triclosan Broad spectrum activity against
bacteria (except Pseudomonas
aeruginosa)
Peroxygens/Peroxides Hydrogen per-
oxide
Broad spectrum activity against
gram-positive bacteria, viruses,
Interfere with plasma membrane
[36 ] Bactericidal eect [36]
Sporostatic [36]
Act as oxidant on cell lipids, pro-
teins and DNA [9, 36]
yeast and spore-forming bacteria
DNA: Deoxyribonucleic acid; RNA: ribonucleic acid.
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
Hand Sanitizers
Quaternary ammonium compounds (QAC) are usually dissolved
in an aqueous medium to produce NABHS, which are non-volatile.
A commonly used QAC, benzalkonium chloride, is regarded as one
of the safest and most ecacious synthetic biocides [28]. It is thought
to interfere with the plasma membrane of microorganisms, thereby
inducing leakage of its cellular contents [28]. Another example of a
QAC that was widely employed is benzethonium chloride. It was,
however, banned from use in hand sanitizers by FDA in 2019 due to
insucient ecacy data. Although it has not been found to cause
harm, its use in hand sanitizers may mislead consumers on its eectiveness and result in false protection [39].
Biguanides are also employed in NABHS. The most notable example is chlorhexidine gluconate (CG) which is commonly used in
hospitals. CG is highly bactericidal against gram-positive bacteria,
microbiocidal against enveloped viruses and has some weak activity against gram-negative bacteria and fungi [9, 34]. Several studies demonstrated its rapid kill of two commonly found bacteria,
namely Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)
[36, 40–42]. However, CG lacks sporicidal activity and is ineective
against spore-forming bacteria. CG was reported to cause damage
to the plasma membrane of yeast cells, leading to the leakage of
intracellular components and cell death [36, 43–46].
407
Chlorine and iodine are examples of halogens used for antisepsis.
Chlorine is more commonly used to disinfect water. Iodine, commonly available as povidone-iodine, is more often used for skin disinfection before and after surgery. Both halogens have broad activity
against bacteria and viruses. Some studies on chlorine have postulated activity against spores [36, 47–49]. Povidone-iodine, which is
used at 5%–10% in formulations for skin application, inactivates but
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
does not kill spore-forming bacteria [9]. At 2%, povidone-iodine is
eective against E. coli but a higher concentration of 7.5%–10% is
needed against non-enveloped viruses [50].
Chloroxylenol is used in antimicrobial hand soaps and surgical
hand scrubs. Chloroxylenol exerts strong activity against bacteria
and enveloped viruses, with the exception of Pseudomonas aerugi-
nosa (P. aeruginosa) [9, 51–53]. However, chloroxylenol is not used in
hand sanitizers despite its strong ecacy and safety for cosmetic use
because studies have concluded that chloroxylenol has less immediate ecacy and less residual activity compared to CG and povidone-iodine [8, 51, 53–56].
Triclosan is an example of a bisphenol with strong bactericidal activity against gram-positive bacteria and mycobacteria. In the past, triclosan was used in hand sanitizer formulations. However, in 2019,
it was banned under the FDA Consumer Antiseptic Rub Proposed
Rule [57], after studies highlighted toxicities of triclosan, such as
decreased thyroid hormone levels [58] and breast cancer with longterm use [59, 60]. In fact, a study also found that the eectiveness of
triclosan products was similar to plain soap [61]. Therefore, the risks
from triclosan use outweigh its benefits, limiting its application in
hand sanitizer formulations.
Hydrogen peroxide is a peroxygen which damages cell lipids, proteins and DNA of microorganisms [9, 36]. Although it damages
these structures, it exhibits little antimicrobial eectiveness when
used alone; hence, it is often combined with other active ingredients such as alcohol [8]. Today, possible damage to fibroblasts
and risk of bleeding limits its use [62]. FDA recommends the use
of ABHS or BKC-containing NABHS in the COVID-19 pandemic,
Hand Sanitizers
while Centers of Disease Control and Prevention (CDC) recommends the use of ABHS [63]. However, studies have shown that CG
and povidone-iodine are also useful against the coronavirus [9]. At
a concentration of 0.12%, CG is postulated to demonstrate antiviral activity against the coronavirus [9, 64]. Nasal povidone-iodine
has also been shown to pre- vent perioperative spread of COVID-19,
though the ecacy of povidone-iodine in hand sanitizers has yet
to be proven [9, 65].
Test methods used to evaluate ecacy/eectiveness
of hand sanitizers
There are no compendial methods (pharmacopeial standards) for
evaluating the ecacy of hand sanitizers and the choice of method
is left to the discretion of manufacturers. Standards such as the
European Standards (EN) and the American Society for Testing
and Materials (ASTM) standards are more commonly employed.
Within these standards, there are dierent methods for hand sanitizers including EN1500, EN1040, ASTM-E1174 and ASTM-E2755, see
Table 2.
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EN1500, ASTM-E1174 and ASTM-E2755 are in vivo tests while
EN1040 is an in vitro test. In vivo tests are preferred as they are more
realistic. The microbial load, which will have an impact on the outcome, is not always clearly stated. The exposure time to the test
hand sanitizer is currently not standardized. It is 30 seconds for the
in vivo tests but 5 minutes for the in vitro test. EN1500 has a control
to exclude confounding factors but ASTM-E1174 and ASTM- E2755
do not include any control. The acceptance criteria are dierent,
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
Table 2: Comparison of test methods for evaluating ecacy of hand sanitizers
Test method Principle of test method
EN1500 • Evaluates the in vivo ecacy by measuring the
number of viable bacteria remaining on contaminated fingertips after exposure to hand sanitizer
• Test organism: E. coli
• Test subjects assigned to use control (with no anti-
microbial activity) or the test hand sanitizer
• Known number of test organism placed on the fingertips of test subjects. Hands (including contaminated fingertips) rubbed twice with 3 mL of test
hand sanitizer (or control) each time. The viable
count of test organism remaining on fingertips is
determined
• Acceptance criteria: pass a non-inferiority statistical test of log-reduction with reference to the
control
EN1040 • Quantitative suspension test to assess in vitro bac-
tericidal activity
• Test organism: P. aeruginosa or S. aureus
• Test product challenged with test organism (1.5–5
× 107 cfu/mL)
• Te st hand sanitizer and test organism (1.5–5 ×
107 cfu/mL) are mixed together at 20 ± 1°C for
5 mins. 1 mL of the suspension is added to 9 mL of
a neutralizer solution to quench the bactericidal
activity, and the resultant mixture incubated for
2 days to allow any surviving bacteria to grow
• Acceptance criteria: 5-log-reduction factor
ASTM-E1174 • Liquid suspension test to assess in vivo bactericidal
activity
• Test organism: Serratia marcescens (S. marcescens) or
E. coli
References
[66, 67]
[68–70]
[67, 71]
(Continued)
Hand Sanitizers
Table 2: (Continued )
Test method Principle of test method References
• Liquid suspension containing 5 × 108 to 1 × 109
cfu/mL of bacteria is spread on hands. Within 10
sec, 3 mL of test hand sanitizer is added to hands
and continuously rubbed for 30 sec. Place hand in
a glove filled with 75 mL of neutralizer. Uniformly
massage hand for 1 min and withdraw a known
volume for plating. The procedure is repeated
ten times and the viable count is determined,
respectively
• Acceptance criteria: 2-log-reduction factor after
first application and 3-log-reduction factor after
tenth application of test hand sanitizer
ASTM-E2755 • Similar procedure to ASTM-E1174, except: Test
organism: S. marcescens or S. aureus Volume of test
hand sanitizer used is 1.5 mL instead of 3 mL
[7 2]
411
with EN1040 being the most stringent. The test organisms used are
also not similar, with some organisms known to be more susceptible than others. ASTM-E1174 is recommended in the FDA Tentative
Final Mono- graph 2016 revised version [8] and is more widely used
in the US and Canada [67]. Nevertheless, the choice of test method
lies with the discretion of the manufacturer of the hand sanitizer.
It can be clearly seen that the test methods apply only to bacteria.
Additional tests should be performed to encompass a wider range
of ‘microorganisms’ to support the claim of being eective against
99.9% of microorganisms. The ASTM-E1838 is a finger pad method
for viruses [73]. In contrast, the ASTM-E2613 is a finger pad method
for fungi [74] while the ASTM-E2011 is a whole hand method
for viruses [75]. In principle, manufacturers should consider these