Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

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Table 10.2 Antibacterial activity of commonly used disinfectants and antiseptics

Class of compound

Alcohols

Ethanol/isopropyl

Aldehydes

Glutaraldehyde

Formaldehyde

Biguanides

Chlorhexidine

Halogens

Hypochlorite/

chloramines

lodine/iodophor

Peroxygens

Peracetic acid

Hydrogen peroxide

Phenolics

Clear soluble fluids

Chloroxylenol

Bisphenols

Quaternary ammonium

compounds

Benzalkonium

Cetrimide

Activity against:

Bacterial

Mycobacteria spores

+ -

+ +

+ -

— —

General level*

of antibacterial activity

Intermediate

High

Intermediate

High

Intermediate, problems

with Ps. aeruginosa

High

Intermediate

High

Low

Low, poor against

Ps. aeruginosa

Intermediate

* Activity will depend on concentration, time of contact, temperature, etc. (see Chapter 11) but these

are activities expected if in-use concentrations were being employed.

resistant but this resistance is of much lesser magnitude than for bacterial spores.

The ability to rapidly destroy pathogenic fungi such as the opportunistic yeast,

Candida albicans, and filamentous fungi such as Trichophyton mentagrophytes, and

spores of common spoilage moulds such as Aspergillus niger, is put to advantage in

many applications of use. Many disinfectants have good activity against these fungi

(Table 10.3).

2.2.5

Viruses

Susceptibility of viruses to antimicrobial agents can depend on whether the viruses

possess a lipid envelope. Non-lipid viruses are frequently more resistant to disinfectants

and it is also likely that such viruses cannot be readily categorized with respect to their

sensitivities to antimicrobial agents. These viruses are responsible for many nosocomial

infections, e.g. rotaviruses, picornaviruses and adenoviruses (see Chapter 3), and it

may be necessary to select an antiseptic or disinfectant to suit specific circumstances.

Certain viruses, such as Ebola and Marburg which cause haemorrhagic fevers, are

highly infectious and their safe destruction by disinfectants is of paramount importance.

Chemical disinfectants, antiseptics and preservatives 205

Table 10.3 Antifungal activity of disinfectants and antiseptics (adapted from Scott et al. 1986)

Antimicrobial agent

Phenolic (0.36%)

Chlorhexidine gluconate (0.02%,

alcoholic)

Iodine (1%, alcoholic)

Povidone-iodine (10%, alcoholic and

aqueous)

Hypochlorite (0.2%)

Cetrimide (1%)

Chlorhexidine gluconate (0.05%) +

cetrimide (0.5%)

Chlorhexidine gluconate (0.5%, aqueous)

Time (

imin) to

Aspergillus

niger

give >99.99% kill*

Trichophyton

mentagrophytes

>20

Candida

albicans

* Initial viable counts were ca. 1 x 10

There is much concern for the safety of personnel handling articles contaminated

with pathogenic viruses such as hepatitis B virus (HBV) and human immunodeficiency

virus (HIV) which causes acquired immune deficiency syndrome (AIDS). Some

agents have been recommended for disinfection of HBV and HIV depending on the

circumstances and level of contamination; these are listed in Table 10.4. Disinfectants

must be able to treat rapidly and reliably accidental spills of blood, body fluids or

secretions from HIV infected patients. Such spills may contain levels of HIV as high as

infectious units/ml. Recent evidence from the Medical Devices Agency evaluation

of disinfectants against HIV indicated that few chemicals could destroy the virus in a

Table 10.4 Chemical disinfection of human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Adapted from

ACDP (1990) and Anon (1991)

206 Chapter 10

Disinfecting agent

Chlorine-releasing preparations,

e.g. hypochlorite lOOOOppm av. Cl

at least 30min at room temperature

Hypochlorite 1000ppm av. Cl

Aldehydes, e.g. glutaraldehyde

2% (w/v), 30min at room

temperature

Alcohol 70% ethanol, at least 2min

for HIV but evaporation a

problem

Application

Spillage of HIV contaminated

blood and body fluid

Minor contamination of

inanimate surfaces

Reserved for non-corrosive

treatment of delicate items

Limited application

Comment

Use fresh solution

Deteriorates on storage and

may be adversely affected

by organic matter

Corrosive to metals

Bleaches fabrics

Must be freshly activated

Not recommended for surface

decontamination due to

vapour toxicity (see Table 10.5)

Use alternative if possible as

activity in presence of protein

questionable

short time in the presence of high serum levels; only two of 13 products (glutaraldehyde

and dichloroisocyanurate) were effective under the most stringent test conditions.

The virucidal activity of chemicals is difficult to determine in the laboratory. Tissue

culture techniques are the most common methods for growing and estimating viruses;

however, antimicrobial agents may also adversely affect the tissue culture: see also

Chapter 11.

2.2.6 Protozoa

Acanthamoeba spp. can cause acanthamoeba keratitis with associated corneal scarring

and loss of vision in soft contact lens wearers. The cysts of this protozoan, in particular,

present a problem in respect of lens disinfection. The chlorine-generating systems in

use are generally inadequate. Although polyhexamethylene biguanide shows promise

as an acanthamoebacide, only hydrogen peroxide-based disinfection is considered

completely reliable and consistent in producing an acanthomoebacidal effect.

2.2.7 Prions

Prions (small proteinaceous infectious particles, also known as unconventional slow

viruses) are a unique class of infectious agent associated with causing spongiform

encephalopathies such as bovine spongiform encephalopathy (BSE) in cattle and

Creutzfeldt Jakob disease (CJD) in humans. There is considerable concern about the

transmission of these agents from infected animals or patients. Risk of infectivity is

highest in brain and spinal cord tissues. There are still many unknown factors regarding

destruction of prions. It appears that they are resistant to most disinfectant procedures

and that autoclaving or exposure to 1N sodium hydroxide is required for decontamination.

However, current advice is to destroy surgical instruments where procedures involve

brain, spinal cord or eye in patients with confirmed or suspected CJD.

2.3 Intended application

The intended application of an antimicrobial agent, whether for preservation, antisepsis

or disinfection, will influence its selection and also affect its performance. For example,

in medicinal preparations the ingredients in the formulation may antagonize preservative

activity. The risk to the patient will depend on whether the antimicrobial is in close

contact with a break in the skin or mucous membranes or is introduced into a sterile

area of the body.

In disinfection of instruments, the chemicals used must not adversely affect the

instruments, e.g. cause corrosion of metals, affect clarity or integrity of lenses, or change

texture of synthetic polymers. Many materials such as fabrics, rubber, plastics are capable

of adsorbing certain disinfectants, e.g. quaternary ammonium compounds (QACs), are

adsorbed by fabrics, while phenolics are adsorbed by rubber, the consequence of this

being a reduction in concentration of active compound. A disinfectant can only exert

its effect if it is in contact with the item being treated. Therefore access to all parts of an

instrument or piece of equipment is essential. For small items, total immersion in the

disinfectant must also be ensured.

Chemical disinfectants, antiseptics and preservatives 207

2.4 Environmental factors

Organic matter can have a drastic effect on antimicrobial activity either by adsorption

or chemical inactivation, thus reducing the concentration of active agent in solution or

by acting as a barrier to the penetration of the disinfectant. Blood, body fluids, pus,

milk, food residues or colloidal proteins, even present in small amounts, all reduce the

effectiveness of antimicrobial agents to varying degrees and some are seriously affected.

In their normal habitats, microorganisms have a tendency to adhere to surfaces and

are thus less accessible to the chemical agent. Some organisms are specific to certain

environments and their destruction will be of paramount importance in the selection

of a suitable agent, e.g. Legionella in cooling towers and non-potable water supply

systems, Listeria in the dairy and food industry and hepatitis in blood-contaminated

articles.

Dried organic deposits may inhibit penetration of the chemical agent. Where

possible, objects to be disinfected should be thoroughly cleaned. The presence of ions

in water can also affect activity of antimicrobial agents, thus water for testing biocidal

activity can be made artificially 'hard' by addition of ions.

These factors can have very significant effects on activity and are summarized in

Table 10.5.

2.5 Toxicity of the agent

In choosing an antimicrobial agent for a particular application some consideration must

be given to its toxicity. Increasing concern for health and safety is reflected in the

Control of Substances Hazardous to Health (COSHH) Regulations which specify the

precautions required in handling toxic or potentially toxic agents. In respect of

disinfectant use these regulations affect, particularly, the use of phenolics, formaldehyde

and glutaraldehyde. Toxic volatile substances, in general, should be kept in covered

containers to reduce the level of exposure to irritant vapour and they should be used

with an extractor facility. Limits governing the exposure of individuals to such substances

are now listed, e.g. 0.7mg/m

(0.2 ppm) glutaraldehyde for both short- and long-term

exposure. The aldehydes, glutaraldehyde less so than formaldehyde, may affect the

eyes and skin (causing contact dermatitis), and may induce respiratory distress. Face

protection and impermeable nitrile rubber gloves should be worn when using these

agents. Table 10.5 lists the toxicity of many of the disinfectants in use and other concerns

of toxicity are described for individual agents below.

Where the atmosphere of a workplace is likely to be contaminated, sampling and

analysis of the atmosphere may need to be carried out on a periodic basis with a frequency

determined by conditions.

3 Types of compound

The following section presents in alphabetical order by chemical grouping the agents

most often employed for disinfection, antisepsis and preservation. This information is

summarized in Table 10.6.

208 Chapter 10

Table 10.5 Properties of commonly used disinfectants and antiseptics

Class of

compound

Alcohols

Ethanol

Aldehydes

Glutaraldehyde

Biguanides

Chlorhexidine

Chlorine

compounds

Hypochlorite

Iodine

preparations

lodophors

Phenolics

Clear soluble

fluids

Black/white

fluids

Chloroxylenol

QACs

Cetrimide and

benzalkonium

Chloride

Effect of

organic

matter

Slight

Severe

Slight

Moderate/

severe

Severe

pH optimum

pH8

pH7-8

Acid/neutral

Acid pH

Alkaline pH

Toxicity and

OES*

Avoid broken

skin, eyes

OES: 1000ppm/1900

mgrrr

, 8h only

Respiratory complaints and

contact dermatitis reported

Eyes, sensitivity

OES: 0.2 ppm/0.7 mg rrr

10min only

Avoid contact with eyes and

mucous membranes

Sensitivity may develop

Irritation of skin, eyes and

lungs

OES: 1ppm/3mgirr

10min; 0.5ppm/1.5mgm"

Eye irritation

OES:0.1ppm/1mg

, 10min only

Protect skin and eyes

Very irritant

Sensitivity. May

irritate skin

OES: 10ppm/38 mgrrr

10min; 5ppm/19mgnrr

, 8h

Avoid contact with eyes

Other factors

Poor penetration,

good cleansing

properties

Non-corrosive,

useful for heat

sensitive

instruments

Incompatible with

soap and anionic

detergents

Inactivated by hard

water, some

materials and plastic

Corrosive to metals

May corrode

metals

Adsorbed by

rubber/plastic

Greatly reduced by

dilution

Adsorbed by

rubber/plastic

Incompatible with

soap and anionic

detergents

Adsorbed by fabrics

* From the Control of Substances Hazardous to Health (COSHH) Regulations (1988).

OES, occupational exposure standard; QAC, quaternary ammonium compound.

Chemical disinfectants, antiseptics and preservatives 209

Table 10.6 Examples of the

Antimicrobial agent

Acids and esters.

e.g. benzoic acid,

parabens

Alcohols,

e.g. ethyl or isopropyl

Aldehydes,

e.g. glutaraldehyde

Biguanides,

e.g. chlorhexidinef

(gluconate, acetate etc)

Chlorine,

e.g. hypoclorite

Hydrogen peroxide

main antimicrobial

groups as antiseptics, disinfectants and preservatives

Antiseptic activity

Concentration

50-90%

in water

10%

0.02%

0.2%

0.5% (in 70%

alcohol)

1.0%

4.0%

<0.5%

avCI

1.5%

3-6%

Typical formulation/

application

Skin prep.

Gel for warts

Bladder irrigation

Mouthwash

Skin prep.

Dusting powder,

cream dental gel

Pre-op. scrub in

surfactant

Solution for skin and

wounds

Stabilized cream

Solution for wounds

and ulcers, mouthwash

Disinfectant Activity

Concentration

50-90%

in water

2.0%

0.05%

0.5% (in 70%

alcohol)

1-10%

3.0%

Typical formulation/

application

Clean surface prep.,

thermometers

Solution for instruments

Storage of instruments,

clean instrument

disinfection (30min)

Emergency instrument

disinfection (2min)

Solution for surfaces and

instruments

Disinfection of soft contact

lenses

Preservative activity

Concentration

0.05-0.1%

0.25%

0.0025%

0.01%

Typical formulation/

application

For oral and topical

formulations

Solution for hard

contact lenses

Eye-drops

* Also used in combination with other agents e.g. chlorhexidine, iodine.

t Several forms available having x% chlorhexidine and 10x% cetrimide.

QAC, quaternary ammonium compound.

Iodine compounds,

e.g. free iodine,

povidone-iodine

Phenolics,

e.g. tar acids (clear

soluble phenolics),

non-coal tar

(chloroxylenol),

bisphenol (triclosan)

QACs,

e.g. cetyltrimethyl

ammonium

bromide (cetrimide)

1.0%

2.5%

7.5%

10%

0.5%

1.3%

2.0%

0.1%

0.5%

1.0%

Aqueous or alcoholic

(70%) solution

Mouthwash

Dry powder spray

Scalp and skin

cleanser

Pre-op. scrub, fabric

dressing

Dusting powder

Solution

Skin cleanser

Solution for wounds

and burns

Cream

Skin solution

10.0%

1-2%

0.1%

1.0%

Aqueous or ale. solution

Solution

Storage or sterile

instruments

Instruments (1 h)

0.01%

Eye-drops

3.1

Acids and esters

Antimicrobial activity, within a pharmaceutical context, is generally found only in the

organic acids. These are weak acids and will therefore dissociate incompletely to give

the three entities HA, H

and A" in solution. As the undissociated form, HA, is the

active antimicrobial agent, the ionization constant, K

, is important and the pK

of the

acid must be considered especially in formulation of the agent.

3.1.1 Benzoic acid

This is an organic acid, C

COOH, which is included, alone or in combination with

other preservatives, in many pharmaceuticals. Although the compound is often used as

the sodium salt, the non-ionized acid is the active substance. A limitation on its use is

imposed by the pH of the final product as the pK

of benzoic acid is 4.2 at which pH

50% of the acid is ionized. It is advisable to limit use of the acid to preservation of

pharmaceuticals having a maximum final pH of 5.0 and if possible less than 4.0.

Concentrations of 0.05-0.1 % are suitable for oral preparations. A disadvantage of the

compound is the development of resistance by some organisms, involving in some

cases metabolism of the acid resulting in complete loss of activity. Benzoic acid also

has some use in combination with other agents, salicylic acid among others, in the

treatment of superficial fungal infections.

3.1.2 Sorbic acid

This compound is a widely used preservative as the acid or its potassium salt. The pK

is 4.8 and, as with benzoic acid, activity decreases with increasing pH and ionization.

It is most effective at pH 4 or below. Pharmaceutical products such as gums, mucilages

and syrups are usefully preserved with this agent.

3.1.3 Sulphur dioxide, sulphites and metabisulphites

Sulphur dioxide has extensive use as a preservative in the food and beverage industries.

In a pharmaceutical context, sodium sulphite and metabisulphite or bisulphite have a

dual role acting as preservatives and antioxidants.

3.1.4 Esters of p-hydroxybenzoic acid (parabens)

A series of alkyl esters (Fig. 10.1) of/?(4)-hydroxybenzoic acid was originally prepared

to overcome the marked pH-dependence on activity of the acids.

These parabens, the methyl, ethyl, propyl and butyl esters, are less readily ionized

having pK

values in the range 8-8.5 and exhibit good preservative activity even at pH

212 Chapter 10

Fig. 10.1 p-Hydroxybenzoates (R is methyl, ethyl, propyl, butyl

or benzyl).

levels of 7-8, although optimum activity is again displayed in acidic solutions. This

broader pH range allows extensive and successful use of the parabens as pharmaceutical

preservatives. The agents are active against a wide range of fungi but are less active

against bacteria, especially the pseudomonads which may utilize the parabens as a

carbon source. They are frequently used as preservatives of emulsions, creams and

lotions where two phases exist. Combinations of esters are most successful for this

type of product in that the more water-soluble methyl ester (0.25%) protects the aqueous

phase whereas the propyl or butyl esters (0.02%) give protection to the oil phase. Such

combinations are also considered to extend the range of activity. As inactivation of

parabens occurs with non-ionic surfactants, due care should be taken in formulation

of these.

3.2 Alcohols

3.2.1 Alcohols used for disinfection and antisepsis

The aliphatic alcohols, notably ethanol and isopropanol, which are used for disinfection

and antisepsis, are bactericidal against vegetative forms, including Mycobacterium spp.,

but are not sporicidal. Alcohols have poor penetration of organic matter and their use is

therefore restricted to clean conditions. They possess properties such as a cleansing

action and volatility, are able to achieve a rapid and large reduction in skin flora and

have been widely used for skin preparation prior to injection or other surgical procedures.

However, the contact time of an alcohol-soaked swab with the skin prior to venepuncture

is so brief that it is thought to be of doubtful value.

Ethanol (CH

OH) is widely used as a disinfectant and antiseptic. The presence

of water is essential for activity, hence 100% ethanol is ineffective. Concentrations

between 60 and 95% are bactericidal but a 70% solution is usually employed for the

disinfection of skin, clean instruments or surfaces. At higher concentrations, e.g. 90%,

ethanol is also active against most viruses, including HIV. Ethanol is also a popular

choice in pharmaceutical preparations and cosmetic products as a solvent and

preservative.

Isopropyl alcohol (isopropanol, CH

CHOH.CH

) has slightly greater bactericidal

activity than that of ethanol but is also about twice as toxic. It is less active against

viruses, particularly non-enveloped viruses, and should be considered a limited-spectrum

virucide. Used at concentrations of 60-70%, it is an acceptable alternative to ethanol

for preoperative skin treatment and is also employed as a preservative for cosmetics.

3.2.2 Alcohols as preservatives

The aralkyl alcohols and more highly substituted aliphatic alcohols (Fig. 10.2) are

used mostly as preservatives. These include:

1 Benzyl alcohol (C

OH). This has antibacterial and weak local anaesthetic

properties and is used as an antimicrobial preservative at a concentration of 2%, although

its use in cosmetics is restricted.

2 Chlorbutol (trichlorobutanol; trichloro-r-butanol; trichlorobutanol). Typical in-use

concentration: 0.5%. It has been used as a preservative in injections and eyedrops. It is

Chemical disinfectants, antiseptics and preservatives 213

214 Chapter 10

Fig. 10.2 Structural formulae of alcohols used in preserving and disinfection: A, 2-phenylethanol;

B, 2-phenoxyethanol; C, chlorbutol (trichlonw-butanol); D, Bronopol (2-bromo-2-nitropropan-l,3-

diol).

unstable, decomposition occurring at acid pH during autoclaving, while alkaline

solutions are unstable at room temperature.

3 Phenylethanol (phenylethy 1 alcohol; 2-phenylethanol). Typical in-use concentration:

0.25-0.5%. It is reported to have greater activity against Gram-negative organisms and

is usually employed in conjunction with another agent.

4 Phenoxyethanol (2-phenoxyethanol). Typical in-use concentration: 1%. It is more

active against Ps. aeruginosa than against other bacteria and is usually combined

with other preservatives such as the hydroxybenzoates to broaden the spectrum of

antimicrobial activity.

5 Bronopol (2-bromo-2-nitropropano-l,3-diol). Typical in-use concentration: 0.01-

0.1%. It has a broad spectrum of antibacterial activity, including activity against

Pseudomonas spp. The main limitation on the use of bronopol is that when exposed to

light at alkaline pH, especially if accompanied by an increase in temperature, solutions

decompose, turning yellow or brown. A number of decomposition products including

formaldehyde are produced. In addition, nitrite ions may be produced and react with

any secondary and tertiary amines present forming nitrosamines, which are potentially

carcinogenic.

Aldehydes

A number of aldehydes possess antimicrobial properties, including sporicidal activity;

however, only two, formaldehyde and glutaraldehyde, are used for disinfection. Both

these aldehydes are highly effective biocides and their use as 'chemosterilants' reflect

this.

Glutaraldehyde

Glutaraldehyde (CHO(CH

)

CHO) has a broad spectrum of antimicrobial activity and

rapid rate of kill, most vegetative bacteria being killed within a minute of exposure,

although bacterial spores may require 3 hours or more. The latter depends on the

intrinsic resistance of spores which may vary widely. It has the further advantage

of not being affected significantly by organic matter. The glutaraldehyde molecule