Pulizia e disinfezione nel nuovo Annex 1: intervista a Tim Sandle

For disinfection to be effective, prior cleaning to remove surface contamination should be performed. Cleaning programmes should effectively remove disinfectant residues. More than one type of disinfecting agent should be employed to ensure that where they have different modes of action, their combined usage is effective against bacteria and fungi. Disinfection should include the periodic use of a sporicidal agent.”

Once again, we spoke to Tim Sandle, microbiologist, author and science journalist, known as one of the leading experts in the field, to discuss in the most appropriate way what challenges the pharmaceutical industry is facing in adapting cleaning & disinfection to the Annex 1 requirements.

Rotation of disinfectants

The latest version of Annex 1 adds in paragraph 4.33: “more than one type of disinfecting agent should be employed to ensure that where they have different modes of action, their combined usage is effective against bacteria and fungi”. Thus, it seems that more products effective against bacteria and fungi should be used in addition to a sporicidal agent. Is the two-product rotation (one bactericide/fungicide and one sporicide) – already in use by many pharmaceutical companies – acceptable?

When cleanroom disinfectants are selected many users opt to select two or more disinfectants. If a sporicide is a regularly used agent, then two disinfectants should be sufficient. 

Rotation is important for several reasons:

• Most disinfectants do not have a complete spectrum of activity effective against all microorganisms (spectrum of activity is the ability of the disinfectant to kill different types of microorganisms and microorganisms which are in different physiological states). The disinfectants commonly used are often effective against vegetative cells but are not sporicidal. To maintain an effective contamination control, the elimination of bacterial endospores through a sporicidal disinfectant is recommended (these are sometimes referred to as high-level disinfectants). Here a sporicide would be used in rotation with a non-sporicidal disinfectant.
• The disinfectants with the formulations which are effective against the greatest range of microorganisms are often expensive. With this, many manufacturers use a general broad-spectrum disinfectant daily or weekly with a sporicidal disinfectant used weekly or monthly (a decision often based on the results of microbiological environmental monitoring and the characterization of the isolated microorganisms).
• Some disinfectants, such as sporicides, are corrosive. While the risk to surfaces can be reduced through rinsing, rotation is sometimes undertaken in order to reduce the risk of damage to cleanroom equipment and working benches.
• Rotating two disinfectants can also reduce the possibility of resistant strains of microorganisms developing. Whilst the phenomenon of microbial resistance is an issue of major concern for antibiotics there are few data to support development of resistance to disinfectant. Nonetheless, it remains a regulatory expectation.

Thus, the reasons for rotation are approached from either a desire to widen the mode of action or to address anticipated regulatory concerns.

Effective cleaning & disinfection 

Cleaning and disinfection affect floors, walls, ceilings, but also machinery and equipment, and often hard-to-reach surfaces. Which cleaning and disinfection methods are most effective in this respect?

These depends on the circumstances, however good application techniques are important, for example with floor cleaning and disinfection:

      • Either roll all of the floor surface with a tacky roller to remove any loose debris and fibers, moving any equipment to one half of the room nearest exit door, or wipe the floor using a neutral detergent solution and mop and bucket.
      • Use a use disinfectant impregnated mop wipes and mops (alternatively a mop with a ready prepared disinfectant solution could be used).
      • Using overlapping mop strokes, wipe down half the floor area, working methodically around the area starting at furthest reach point and working towards yourself in a straight line. Replace the mop wipe if the wipe becomes visibly contaminated or dry.
      • NOTE: Particular attention should be paid to corners and edges.
      • Leave the disinfectant solution on the surface for the manufacturer’s recommended contact time to allow the disinfectant to be effective.
      • Remove the mop head and discard to waste.
      • Fit a second mop head with ethanol impregnated mop wipe (alternatively a mop with a ready prepared alcohol solution could be used).
      • After the allotted time, using the fresh mop head wipe down using overlapping mop strokes to remove any residue, working methodically around the area starting at furthest reach point and working towards yourself in a straight line. Replace the mop wipe if the wipe becomes visibly contaminated or dry.

Use of fumigation

Where disinfection is difficult, is it advisable to use fumigation?

Yes, and this is always advisable following events that can lead to high rates of contamination, such as maintenance works or a facility shutdown. Fumigation-based approaches are advantageous for decontaminating the inside of buildings because they are easily dispersed, penetrate into difficult to access areas, decontaminate the interior volume of the space (not only surfaces) and are less labour intensive than many spray based approaches. Optimal agents are hydrogen peroxide and chlorine dioxide.

Attention needs to be paid to the concentration and dwell time, and with controlling temperature and humidity, as well as the mechanism of dispersing the fumigant. Each of these factors contributes to the overall efficacy.

“The movement of material or equipment from lower grade or unclassified area to

higher-grade clean areas should be subject to cleaning and disinfection commensurate with the risk and in line with the CCS.”

The Contamination Control Strategy refers to risk-commensurate cleaning and disinfection.


The validation of detergent and disinfectant products, as well as the validation of cleaning is a very important step: what are the mandatory activities?

Validation studies are broken down into three sections: suspension tests (phase 1 and 2) to evaluate the reduction of a known organism population inoculated directly into a sample of the liquid disinfectant, surface tests (phase 2) that assess a disinfectant’s ability to reduce the number of challenge organisms on an inoculated surface, and field trials (phase 3) a final assessment of the environmental monitoring data to validate the approach.

Suspension methods evaluate the reduction= of a known organism population inoculated directly into a sample of the liquid disinfectant. Following inoculation and the observation of a predetermined contact time, samples of the inoculated substance are removed, neutralized and evaluated for survivors as compared to an untreated control suspension. Since the simulation of organism films on the specific environmental surface types are not accounted for in this method, it is recommended that suspension-based tests be used only for initial disinfectant screening purposes.

Surface testing involves aliquoting onto a surface coupon a mix of the challenge organism and, where required, an interfering substance (such a protein, to simulate dirty conditions). The surface coupon will be fashioned from a representative surface in the cleanroom (here several different materials will require testing in order to show how the disinfectant performs). To this an amount of the test disinfectant is added. The solutions are left for the required contact time. Once the contact time has elapsed, the coupon is transferred to a neutralizer solution. Then, as with the suspension test once sufficient time has been given for neutralization, microbial survivors are assessed by plating out or filtering the disinfectant neutralizer solution using a microbial culture method.

A possible array of surfaces to consider (this will be facility dependent) are:

      • Stainless steel
      • Glass
      • Aluminum
      • Epoxy
      • Enamel
      • Acrylic
      • Mipolam
      • Vinyl
      • Hardwood
      • Plastic
      • Plexiglas
      • Chromium

Once the testing has been performed, a report should be generated that must conclude the disinfectant efficacy test outcome in relation to the acceptance criteria. If a disinfectant passes the test, deeming it suitable for use, cleanroom procedures must reflect the practices adopted during the qualification, such as disinfectant concentration, contact time and method of application to surfaces. The disinfectant’s final adoption must then be based on a follow-up assessment or field trial, which includes an evaluation of microbial counts and species recovered.

Use of sterile concentrates

“Disinfectants and detergents used in grade A and grade B areas should be sterile prior to use. Disinfectants used in grade C and D may also be required to be sterile where determined in the CCS. Where the disinfectants and detergents are diluted / prepared by the sterile product manufacturer, this should be done in a manner to prevent contamination and they should be monitored for microbial contamination. Dilutions should be kept in previously cleaned containers (and sterilized where applicable) and should only be stored for the defined period. If the disinfectants and detergents are supplied “ready-made” then results from certificates of analysis or conformance can be accepted subject to successful completion of the appropriate vendor qualification.”

In-house prep: in the latest Annex 1 version there is more focus on both in-house preparation of detergents and disinfectants and on the use of WFI water.

What do you think about the use of sterile concentrates in cleaning and disinfection  procedures?

The use of a concentrate that is made up or the use of ready-to-use solutions, is a choice of each user. Both methods are effective.

In particular, many companies, switching to under-isolator production, are considering the use of concentrated products for Grade C/D outdoor environments. Would you recommend their use? If so, with which procedure? 

Yes, there are no concerns, especially within lower grades of cleanrooms. The important point is to ensure the dilution is correct and that the correct grade of water is used.

Training of personnel

Personnel training: with the latest Annex 1, and the increasingly stringent rules on cleaning and disinfection, personnel training is a key point. Are there also changes in training methods with the new Annex 1?

The Annex could go further with training. Appropriate training for all personnel dealing with disinfectants is vital. This training should include all relevant staff including contract personnel working within the facility. Documentation of this training is essential.  All personnel should understand the importance of cGMP. For a new employee, this training could be part of their initial GMP induction. A pre-determined program of training should be in place and documented as well as a system to measure the effectiveness of the training. Personnel should all be trained in good cleaning techniques using the appropriate equipment. All staff should be provided with appropriate clothing in order to perform this operation, e.g., cleanroom clothing in manufacturing areas with adequate PPE. Procedures should also be in place if spillage occurs with any of these agents.

Training programs could include:

      • The importance of disinfection in relation to GMP, its necessity in preventing microbial proliferation, cross contamination and the importance and significance of good disinfection routines.
      • The importance of the correct handling of disinfectants, whether it is during the disinfectant’s preparation, testing or use.
      • Basic microbiology and how contamination is transferred in the workplace.
      • An understanding of the properties of disinfectants and their correct application.

A refresher program should also be in place, ensuring that once personnel are trained, their knowledge is kept up to date. Many companies now incorporate this into regular GMP refresher training, recommended to be undertaken on a periodic basis. Adequate documentation of these updates in an employee training record is essential.

Cleaning & Disinfection in Grade C & D areas

Finally, the CCS in the new Annex 1 extends to all areas at risk of contamination.

Also in paragraph 4.35 it is stated that “disinfectants used in grade C and D may also be required to be sterile where determined in the CCS”. This implies increased attention also for non-sterile areas and for producers of cosmetics, ointments and biological intermediates with low bacterial load etc. For the latter, the challenge will certainly be significant. What are the essential activities to adapt?

Some good practices for the adoption and use of disinfectants, in accordance with GMP, are:

      • Written procedures should be in place.
      • Responsibilities for cleaning should be assigned. Often this is interpreted as the need to have independent cleaning staff separate from those involved in product manufacture.
      • Staff must be trained in cleaning techniques and have a training record.
      • Details of cleaning frequencies, methods, equipment, and materials must be recorded in written procedures. This may relate to an approved supplier specification.
      • The cleaning of equipment and materials must take place at regular intervals.
      • Inspection of equipment for cleanliness before use should be part of routine operations.
      • A cleaning log should be kept. The purpose is to keep a record of the areas cleaned, agents used, and the identity of the operator.
      • The microorganisms isolated (the microbiota) from environmental monitoring programs should be examined for resistant strains. Some isolates from these reviews should be incorporated into disinfectant efficacy studies.
      • The monitoring for microbial contamination in disinfectant and detergent solutions should be periodically undertaken.
      • The storing of disinfectant and detergent solutions should be for defined (and short) periods.
      • Room use should be recorded after each operation.
      • There should be a technical agreement with the company who supplies the disinfectant. Ideally the disinfectants purchased should be lot tracked.
      • SOPs containing references to disinfectants, cleaning agents, materials and equipment used and calibration of equipment are accessible. Here the cleaning and disinfection methods are clearly defined.
      • The SOP should include cleaning method details e.g., wiping front to back or top to bottom with overlapping strokes.
      • Cleaning method, e.g., double bucket, rinsing wipe action, contact time and cleaning of cleaning materials must also be included in the
      • Documentation of rotation, rationale, and frequency, needs to be set out.
      • Cleaning logs also need to be in place and available.

In compliance with GMP, cleaning and disinfection procedures should be included in any audit program as it maintains assurance that contamination control procedures are adequate and in control.


The Contamination Control Strategy underlying the new Annex 1 finds its backbone in Quality Risk Management: assessing risks on a scientific basis and offering a commensurate response to them is the real key to adapting to the new requirements.

Dr. Tim Sandle is a pharmaceutical microbiologist, science writer and journalist. He is a chartered biologist and holds a first class honours degree in Applied Biology; a Masters degree in education; and has a doctorate from Keele University.

Interview by Cristina Masciola, AM Instruments – Marketing & Communication Manager