General Laboratory Safety

There are many categories of hazards that might be encountered in a laboratory setting, and
situations can change frequently. Even after you have identified and controlled all current risks,
it is vital that you remain open to the possibility that new unexpected dangers can arise. Ensure
your assessment covers chemicals (chemicals being used and chemicals that could be created
from the work you will be performing), flammable solvents, biological reagents, gases,
equipment being used, others working in the space, lifting and handling etc. Review the controls
measures regularly to ensure they remain adequate for risks encountered / being created.

Before entering the laboratory

 Familiarize yourself with the local rules. These will normally be in the form of a notice
board at the entrance to or at designated points within the lab which indicates the basic
minimum requirements. This can include details of PPE requirements, specific access
requirements e.g. no pace makers etc. Ensure you follow these rules at all times.
 The Safety Data Sheet (SDS) for any materials you are required to handle. Pay particular
attention to the sections on handling and storage; exposure controls and accidental release
(spillage). There is Guidance on how to interpret Safety Data Sheets on the H&S website.
 The location of safety and emergency equipment such as fire extinguishers, hand wash
basin, eye wash and shower, first aid and spill response kits, fire alarm activation points,
telephone and emergency exits.
 Emergency spill response procedures for the materials you will handle, emergency
reporting procedures and telephone numbers.
 The location of designated and alternative escape routes.
 Any first aid measures which are specific to the chemical you are working with e.g.
phenol.

During laboratory work

Good Laboratory Practice- Principles

 Follow Departmental and local Laboratory Rules at all times.

 Use protective equipment as directed and remove before leaving the laboratory
 Wear your fastened lab coat when your risk assessment requires it (required in all labs
handling chemicals & biological)
 Where directed by the Safety Data Sheet and/or directed by your risk assessment, wear
appropriate gloves when handling materials and replace if damaged. NB: Nitrile may not
always be appropriate and breakthrough times must be adhered to.
 Never pipette by mouth, use pipette fillers.
  Tie back long hair
 Remove gloves before leaving the lab and do not touch your face when wearing gloves
 Cover any cuts and grazes with waterproof plasters
 Use appropriate eye protection when directed (all chemical laboratories are classed as eye
protection zones, the lab noticeboard will provide this information)
 Wear ‗sensible‘ shoes in the laboratory – not open-toed sandals or flip flops
 Never eat, drink, smoke or apply cosmetics in the laboratory
 Never manipulate contact lenses in the laboratory, except in an emergency
 Wash hands before leaving the laboratory and apply a suitable hand moisturiser
 Keep work area clean and tidy – including fume cupboards and microbiological safety
cabinets
 Keep personal items in the storage area provided, not on benches, lab floors or in
gangways
 The risk assessment must determine whether chemical reactions are carried out in fume
cupboards or whether work with biological agents is carried out in a microbiological
safety cabinets

Use of Fume Cupboards

 All chemical reactions assessed as posing a risk to the users and others in the space must
be carried out in a fume cupboard
 Keep the interior tidy. Do Not block the rear air vents
 Keep reactions / work and chemicals at least 15 cm inside the front of the cupboard
 Large pieces of equipment restrict air flow, use lab jacks to maintain dynamic flows
 Wear appropriate PPE as determined by risk assessment
 Keep the sash down as far as comfortable while working
 Do not put your head into the cupboard while working
 Ensure lightweight items such as tissues, disposable gloves and filter papers are not
drawn into the ducts
 Only items which are required for the current experiment should be available. All other
items should be removed to their appropriate storage location.
 Keep sash closed when not actively working in the cupboard
 Always close the sash at night
 Perform Daily ‗User‘ checks.
These include checking:
o Sash is running smoothly and the auto-descend operates (where fitted)
 o Air flow is good – check indicator on cupboard
o Lights working
o Fire Trace pressure gauge in the ‗green‘ (where installed)

There are other routine checks required which need to be documented in the ‗Log Book‘.
Further details can be found on the Health and Safety website. All fume cupboards must be
tested by a competent person at least once in 14 months and date of last test readily visible on the
fume cupboard. Do not use a fume cupboard where more than 14 months has lapsed.

Use of Microbiological Safety Cabinets (MSC’s)

Any work with a biological agent that poses an airborne infection risk must be conducted
in an MSC.
Before you start work, turn the MSC on, remove the night door and allow the airflows to
stabilise for 5 minutes.
Correct MSC function is dependent on airflows within the cabinet not being disrupted
 Keep clutter to a minimum
 Keep centrifuges, etc. out
 Do not use Bunsen burners
 Keep grilles at the front, back and sides clear
 Do not use if the airflow alarm sounds
 Clean down the work surface frequently, and especially after you have spilt
anything on it
 Leave the MSC empty when finished
 Keep lab doors shut.

MSCs must always be tested by a competent engineer for correct function in any 14
month period (as a minimum). The date of last test must visible.

Before leaving the laboratory

 Perform a safety check at the end of each experiment and before leaving the lab.
 Isolate services / supplies are necessary.
 Return unused materials, equipment and apparatus to their correct storage locations
 Dispose of all waste appropriately using the correct waste streams. Where necessary
label materials and ensure the SDS is available. Departments will have local waste
management arrangements which must be followed.
 Remove defective or damaged equipment immediately, and arrange to have it repaired
or replaced via departmental procedures
  Decontaminate any equipment or work areas that may have been in contact with
hazardous materials
 Leave behind protective clothing (lab coats, gloves, etc.) and wash hands thoroughly
before leaving the laboratory
 Where required, display ‗Continuous Running‘ and/or ‗Overnight Reaction‘ cards for
on-going experiments
 Close the door and ensure the laboratory is secure if you are the last one to leave

Other laboratory hazards

As well as the hazards presented by chemicals and biological agents in use in a
laboratory, there are other hazards which also need to be considered:-

Glassware
The most common laboratory incidents, involve handling glassware. The resulting
injuries can be cuts, burns and even poisoning when cut by contaminated glassware. These can
be avoided by following a few simple rules when handling glassware and ensuring the correct
type of glass is used for the activity. Ensure you have received proper instructions before you use
glass equipment designed for specialized tasks.

Here are few safety rules:

 Wear appropriate gloves when handling glassware, ensure they offer grip and cut
resistance.
 Store glassware carefully so as not to damage it or yourself.
 When inserting tubing into the side arm of a flask, condenser etc., grease or wet the
tubing. (Acetone works well on vinyl tubing). Ensure the tubing is the correct
diameter.
 When removing the tubing from glassware, do not attempt to pull it off.
 Lay the glass item on the lab bench (if possible). Cut the tubing near the end of the
glass. Always cut away from your body
 Next, slice the tubing lengthwise and slide the material off the glass connection.
 Substitute plastic connections for glass whenever possible to decrease the risk of
injury.
 Use glassware for vacuum work that is designed for that purpose.
Heating devices

 Ensure all equipment is fit for purpose and maintained.

 Portable electrical equipment will display a sticker to indicate when it was last checked
for electrical safety.
 Carry out a pre-use check and test any safety devices before using and don‘t use any
equipment that is damaged.
 Heating devices can include: Hotplates, Tube & Box Furnaces, Heating Mantles, Hot-
Air Guns, Oil Baths etc.
 Improper use of any one of these could result in fire or burns to the user.
 Always turn off the equipment when no longer required.

Vacuum systems

 Familiarize yourself with the operations of the vacuum system.

 Make sure you have the correct types of vessel / glassware for the level of vacuum
being created.
 Ensure that glassware is plastic coated / meshed or that there is a physical barrier
between you and the glass. NB: the sash of the fume cupboard may provide this
protection.
 Always use a trap on the suction line to prevent liquids from being drawn into the
pump.
 If gases or vapours are being drawn through the pump, a cold trap should be used in the
suction line to prevent contamination of the pump oil.
 Place a tray under the pump to catch any oil drips.

Gases - Compressed gas cylinders

You must be trained to handle cylinders and how to attach the correct regulator before
handing any gas cylinder.

 The gases contained in the cylinder can present a range of hazards, toxic, corrosive,
asphyxiant, oxygen enrichment etc.
 The space in which they are used may need specialist monitoring systems.
 Ensure any risk assessment for the space is adhered to and your risk assessment of the
activity covers these materials where necessary.

Gases – Cryogenic Liquids

You must be trained to handle cryogenic liquid gases
  The typical cryogenic liquids in use at the University are Liquid Nitrogen and Liquid
Helium.
 The hazards associated with the handling and use of cryogenic liquids are:
o Asphyxiation due to oxygen depletion
o Cold Burns
o Cold effect on the lungs
o Ice-plug formation leading to high velocity ejection or container explosion
o Explosion of container due to rapid expansion
 The space in which they are used must have specialist monitoring systems. Always
check the oxygen level on the monitoring panel before entering the laboratory. DO
NOT enter any space where there is a visible or audible alarm.
 Ensure any risk assessment for the space is adhered to and your risk assessment of the
activity covers these materials where necessary.

Other laboratory equipment

 All laboratory equipment including instrumentation should be captured in relevant risk

assessments.
 The manufacturer‘s safety and technical information and the user manual should be
consulted to aid the development of these assessments as it will provide information on
the system requirements, details of any known hazards and how to operate the
equipment safely. However, the user manual will not be able to answer many specific
questions regarding the use of the equipment in its actual environment or the substances
and materials being used in the equipment. Hence, the risk assessment needs to cover
these and will also help to identify any specific training requirements for the users.
 Full details on the requirements for work equipment can be found on the Health and
Safety web pages
 There may be other laboratory hazards such as biological reagents, extremes of
temperature, volatile materials. All these hazards need to be covered by your
assessment for the activity you are planning.

What is GMP?

Good Manufacturing Practices or GMP is a system that consists of processes, procedures

and documentation that ensures manufacturing products, such as food, cosmetics, and
pharmaceutical goods, are consistently produced and controlled according to set quality
standards. Implementing GMP can help cut down on losses and waste, avoid recall, seizure, fines
and jail time. Overall, it protects both company and consumer from negative food safety events.
 GMPs examine and cover every aspect of the manufacturing process to guard against any
risks that can be catastrophic for products, such as cross-contamination, adulteration, and
mislabeling. Some areas that can influence the safety and quality of products that GMP guideline
and regulation address are the following:

 Quality management
 Sanitation and hygiene
 Building and facilities
 Equipment
 Raw materials
 Personnel
 Validation and qualification
 Complaints
 Documentation and recordkeeping
 Inspections & quality audits

What is the difference between GMP and cGMP?

Good Manufacturing Practices (GMP) and current Good Manufacturing Practices

(cGMP) are, in most cases, interchangeable. GMP is the basic regulation promulgated by the US
Food and Drug Administration (FDA) under the authority of the Federal Food, Drug, and
Cosmetic Act to ensure that manufacturers are taking proactive steps to guarantee their products
are safe and effective. cGMP, on the other hand, was implemented by the FDA to ensure
continuous improvement in the approach of manufacturers to product quality. It implies a
constant commitment to the highest available quality standards through the use of up-to-date
systems and technologies.

What is the difference between GMP and cGMP?

Good Manufacturing Practices (GMP) and current Good Manufacturing Practices

(cGMP) are, in most cases, interchangeable. GMP is the basic regulation promulgated by the US
Food and Drug Administration (FDA) under the authority of the Federal Food, Drug, and
Cosmetic Act to ensure that manufacturers are taking proactive steps to guarantee their products
are safe and effective. cGMP, on the other hand, was implemented by the FDA to ensure
continuous improvement in the approach of manufacturers to product quality. It implies a
constant commitment to the highest available quality standards through the use of up-to-date
systems and technologies.

Guidelines and Basic Concepts

GMP guidelines are a set of principles that help manufacturers implement an effective
manufacturing process and ensure that quality is built into the organization and the processes
involved. GMP guidelines are customarily flexible, with countries having their own legislation to
comply with local GMP guidelines and principles. But almost all regulations are derived from
the basic concept and guidelines which are:

Quality management
The principle of quality management is to ensure that manufactured products are fit for
their intended use, comply with requirements and does not place consumers at risk due to
inadequate safety, quality, or efficacy measures. To achieve this quality objective, quality
assurance, good manufacturing practices, quality control, and quality risk management should be
comprehensively and correctly implemented.

Quality assurance
The system of quality assurance aims to ensure that manufactured products are designed
and developed in a way that meets the requirements for Good Manufacturing Practice.

Good Manufacturing Practice for Products

As a part of quality assurance, good manufacturing practice is concerned with production
and quality control. It aims to mitigate the risks that are inherent in the production process. Its
basic requirements according to WHO‘s Good Manufacturing Practices for Pharmaceuticals state
the following:

 All manufacturing processes are clearly defined, systematically reviewed in the light of
experience, and shown to be capable of consistently manufacturing medicinal products of
the required quality and complying with their specifications and/or marketing
authorization;
 Critical steps of manufacturing processes and significant changes to the process are
validated;
 All necessary facilities for GMP are provided including i. appropriately qualified and
trained personnel; ii. adequate premises and space; iii. suitable equipment and services; iv.
correct materials, containers, and labels; v. approved procedures and instructions;
 Instructions and procedures are written in an instructional form in clear and unambiguous
language, specifically applicable to the facilities provided;
  Operators are trained to carry out procedures correctly;
 Records are made, manually and/or by recording instruments, during manufacture which
demonstrate that all the steps required by the defined procedures and instructions were in
fact taken and that the quantity and quality of the product were as expected. Any significant
deviations are fully recorded and investigated;
 Records of manufacture including distribution which enable the complete history of a batch
to be traced are retained in a comprehensible and accessible form;
 The distribution (wholesaling) of the products minimizes any risk to their quality;
 A system is available to recall any batch of product, from sale or supply;
 Complaints about marketed products are examined, the causes of quality defects
investigated and appropriate measures are taken in respect of the defective products and to
prevent re-occurrence
Quality control
Quality control is a part of Good Manufacturing Practice that focuses on sampling,
specification, and testing. It checks the organization, documentation, and release procedures to
ensure that products go through the required tests before being released for sale or supply.

Quality risk management

Quality risk management is a systematic process of assessing risks that can affect the
quality of the product. According to its principles, quality risk management should ensure that:
 The evaluation of the risk to quality is based on scientific knowledge, experience with the
process and ultimately links to the protection of the patient and users;
 The level of effort, formality, and documentation of the quality risk management process is
commensurate with the level of risk. c) The general quality risk management process and
integration into the product quality can be referred to in ICHQ9.

Sanitation and hygiene

Sanitation and hygiene are vital in every aspect of the manufacturing process. It covers
anything that can cause contamination such as personnel, the premises, equipment, containers,
and production materials. All potential sources of contamination should be identified and
eliminated with a comprehensive sanitation and hygiene program.

Building and facilities/premises

As a principle, the premises should be situated in an environment that is suitable for its
operations and one that is free from risks of contamination of materials and products. The
premises should also be designed to minimize errors in operations and should be easy to clean
and maintain.
Equipment
Same with the premises, equipment should be designed, located, and maintained to
function according to its intended use. Additionally, it should be cleaned and stored according to
procedures. In the event of a defect or malfunction, it should be removed or labeled as defective.

Raw materials
All materials used for production should be stored properly according to the appropriate
conditions which are set by the manufacturers. There should be a proper stock management
system implemented to ensure that all incoming materials are correct and of high quality.

Personnel
The success of GMP compliance heavily relies on the people implementing it. For this
reason, it is vital that all personnel are qualified and trained to do the job. They should be aware
of the principles of GMP and receive continued training, hygiene instructions, and other tools
relevant to their needs. Respective managers should be clear on job descriptions for each worker
to avoid misunderstandings and reduce the risk of issues like overlapping responsibilities.

Validation and qualification

Qualify systems, premises, and equipment if they are fit/ready for their intended use and
validate if processes and procedures can repeatedly produce high-quality products. Critical steps
in the manufacturing process should be verified to ensure that product quality is consistent and
maintained at a high level. According to the WHO (World Health Organization), qualification
and validation should establish and provide documentation stating that:

 the premises, supporting utilities, equipment, and processes have been designed in
accordance with the requirements for GMP (design qualification or DQ)
 the premises, supporting utilities, and equipment have been built and installed in compliance
with their design specifications (installation qualification or IQ);
 the premises, supporting utilities, and equipment operate in accordance with their design
specifications (operational qualification or OQ); and a specific process will consistently
produce a product meeting its predetermined specifications and quality attributes (process
validation or PV, also called performance qualification or PQ)

Complaints
Handling complaints is also part of GMP, therefore all manufacturing companies should
have a well-designed GMP complaint system. Ideal complaint handling should have a ready
solution to provide for all contingencies.

Documentation and record keeping - Audit

Good documentation and record keeping are an essential part of the quality assurance
system and are required in compliance with GMP requirements. Accurate recordkeeping can
help managers and supervisors keep track of the historical record of manufacturing procedures
and corrective measures implemented. Below are general requirements for documentation:

 Documents must be designed, prepared, reviewed, and distributed with care.

 Documents should be clear and legible.
 Documents must be approved, signed, and dated by appropriate and authorized personnel.
 Documents must have unambiguous contents such as title, nature, and purpose.
 Documents must be regularly reviewed and updated.
 Documents must not be handwritten.
 Any corrections made to a document or record must be signed or initialed and dated. The
reason for the correction should also be recorded (where appropriate).
 Record each action taken for traceable activities such as manufacturing and control of
products.

Inspections & quality audits

Inspections should be regularly performed to monitor if GMP is implemented and
complied with. Document what areas need more work and provide corrective measures for
continuous improvement. Quality audits are done to assess the quality systems implemented by
the manufacturing company. GMP audit checklists can help companies comply with GMP
guidelines set by regulatory authorities. By performing site visual walkthroughs and conducting
manufacturing evaluations, you can identify non-compliant processes and take immediate action
to address areas for improvement.

Quality Control of Microbiological Culture Media

Control of Media Preparation
QC laboratories acquire media in one of two ways, either purchasing the media pre-made
from a manufacturer, or making the media (either in whole or in part) in-house. These
preparation schemes must be considered separately.

If the media is purchased from the vendor there is little opportunity to control the
preparation beyond having confidence in the supplier.

Media prepared in-house offers several opportunities for quality control. The raw
materials (either the dehydrated complete media or the components) must be stored under
appropriate and controlled conditions and used within established expiry dates. The
compounding of the media must be controlled to ensure the media is prepared correctly. Agar
media must be pre-warmed to dissolve the agar prior to sterilization, but not heated so
extensively as to damage any heat-labile components. The sterilization procedure also must be
under control.

Growth Promotion Testing

There are some significant concerns as to the need for GP testing of standard media. It
can be argued that since all preparation conditions are under control and the physical parameters
of the finished media is checked, there is little additional information gathered by the labor-
intensive and time-consuming procedure of checking the growth promoting capabilities of the
media.

Growth Promotion Testing of Agar Media

A singular advantage of agar media tests is that they provide numbers – colony forming
units (CFU). To analyze CFU you must use statistical tools designed for the Poisson distribution
(Ilstrup 1990) or else convert the data to approximate the normal distribution. This data
conversion can be done by using its log10 values or by taking the square root of (n+1) (Ilstrup
1990). Once this is done, plate counts can be directly compared using ―Student‘s‖ T Test or other
tests of normally distributed data.

There are, of course, several less demanding tests for demonstration of equivalency
between two agars:

Spread Plates or Pour Plates

The compendia assume a GP test by comparison of CFU, with the cells plated in the
normal fashion for the lab. The compendia generally require that the colony counts derived from
growth on the current batch of media be no less than 50% (USP 2003b) or 70% (USP 2004) of a
previously qualified batch. This approach provides the advantages of colony counts and a large
area for the colonies to grow, but it is somewhat laborious and expensive in terms of material.

Miles-Misra (Drop Count) Technique

This technique involves dropping the cells in a 10 µL aliquot onto the surface of an agar
plate (Miles and Misra 1938). When used carefully, an entire 6-fold dilution scheme can be
plated in a single Petri dish and if read early, the individual drops can be used to yield estimates
of the number of CFU/mL in the challenge suspension. This method offers significant
advantages in terms of labor and material resources.

Ecometric Method
This method is a variation of streaking to extinction. A fresh suspension of the challenge
organism is taken into a calibrated loop and streaked in five parallel lines over four sections of an
agar plate in sequence, then once through the middle (image from Mossel 1980). These plates are
then incubated overnight for growth. The patterns of growth are interpreted to provide an
Absolute Growth Index (AGI):
 Growth AGI
All Streaks 5.0
All but middle streak 4.0
All in quadrants 1, 2, and 3 but half in quadrant 4 and none in middle streak 3.5
All in quadrants 1, 2, and 3 but no growth in quadrant 4 or middle streak 3.0
Growth scored on half quadrant scores to – 2.5, 2.0, 1.5 and so on.

This technique is somewhat operator-dependent and offers a lower precision than those
yielding CFU, but can be used to great effect with practice.

Growth Promotion Testing of Broth Media

Copious Growth
This is the current compendial method of choice. In this method, the challenge organism
is inoculated at a very low level (< 100 CFU per unit) and incubated at the prescribed
temperature for the prescribed period of time (3 days or 5 days). Growth in the batch of media is
then compared to a parallel sample from a previously qualified batch of the same media. The
growth is to be comparable between the two and copious. The advantage of this method is that it
does not require a great deal of labor, but the quality of the data for the comparison between the
growth promoting characteristics of the media is exceptionally poor. This can be described as a
crude end-point test with an ―n‖ of 1.

End-point Methods
In this approach to growth promotion testing, very low levels of inoculum are added to
multiple tubes of the two media being examined. Then the resultant growth frequency is
compared between the two media to determine equivalency. For example, comparing an old and
a new batch of Trypticase Soy Broth (Soy Bean Casein Digest Broth) might be performed by
taking 100 tubes of each media, and then inoculating all 200 tubes with <5 CFU of the challenge
organism Staphylococcus aureus. After incubation, the number of turbid tubes would be
compared – say 30/100 of the new media turbid vs. 46/100 of the old media. The statistical
comparison could be performed using the Chi Square Test or Fisher‘s Exact Test. This
evaluation would be performed separately for each challenge organism. The number of tubes
used can be decreased (or increased) at the expense of the statistical power of the method.

End-point methods to growth promotion of broth media are obviously very laborious and
technically demanding. It is not difficult to envision a design that would require more than a
thousand tubes and the need to accurately create an inoculum of <5 CFU of a variety of
challenge microorganisms.

MPN
The Most Probable Number method of enumerating microorganisms is most commonly
used in the QC lab as part of the Microbial Limits Test (USP 2003b) or in other situations where
the sample cannot be put into an appropriate suspension or be filtered (Aspinall and Kilsby
1979). In this technique, the unknown sample is prepared in a ten-fold dilution series and added
to nutrient broth in replicate tubes (normally either 3, 5 or 10 replicates are used). The tubes will
then either turn turbid (growth) or remain clear, and allow for an estimate of the most probable
number of microorganisms.

BIOBURDEN DETERMINATION

Bioburden is a term used to describe the microbial numbers on a surface (or complete
item) or inside a device or from a portion of liquid. In the lexicon of microbiology, the term
―bioburden‖ is somewhat misleading as ―burden‖ implies that the level of microorganisms is
automatically a problem or concern; whereas, in practice, the objective of qualitative or a
quantitative testing is to ascertain if the types and numbers of microorganisms present are
satisfactory or unsatisfactory when compared to predefined acceptance criteria.

Total microbial count

The term ―total microbial count‖ can refer to the total number of bacteria and fungi
present or to the total number of bacteria. This confusion with the term has been enhanced by a
pharmacopoeial chapter called the ―microbial limits test‖ (which is harmonized between the
European, US, and Japanese pharmacopeia). TMC referes to ―total aerobic microbial count‖
(TAMC), which refers to bacteria only, and a ―total yeast and mould count‖ (TYMC; which
refers to fungi only). The two aspects of the microbial count—TAMC and TYMC—involve
testing a sample on different agar and subjecting the tests to different incubation conditions.

Non-sterile products and microbial limits testing

The microbiological quality of the finished product is determined by the quality of the
starting materials; materials with a known low bioburden should be purchased whenever
possible. These are, in most cases, ―non-sterile‖ materials or products. Such materials are either
used to manufacture more complex pharmaceutical products (such as tablets, creams, or
ointments) or used in the preliminary stages of what will become sterile products.

The compendial microbial limits test is made up of two parts [14]:

● TAMC. This is an estimation of viable aerobic mesophilic microorganisms that can be derived
from a general purpose medium (the pharmacopeia recommends soya bean casein
digest medium).

● TYMC. This is an estimation of mesophilic aerobic fungi (yeast like and filamentous, and
those that are dimorphic). The test uses a general purpose fungal medium (the pharmacopeia
recommends Saboraud dextrose agar).
 The bioburden test is either one or both of the compendial TAMC or TYMC methods, or
an alternative. For the examination of the microbial count, there are four recommended methods.

Membrane filtration
This is the method of choice and should be applied to samples that contain antimicrobial
substances. With the method, the sample is passed through a membrane filter with a pore size of
0.45 μm or less. Filters about 50 mm across are recommended, but other sizes may be used.
Usually, the test measures two test fluids of 10 mL each, passing each sample through a separate
filter. It is important to dilute the pretreated test fluid if the bacteria concentration is high, so that
10–100 colonies can develop per filter. After filtration, each filter must be washed three times or
more with an appropriate liquid such as phosphate buffer, sodium chloride-peptone buffer or
fluid medium. The volume of the washings should be about 100 mL each. If the sample includes
lipid, polysorbate 80 or an appropriate emulsifier may be added to the washings.

After filtration, for bacteria detection, the two filters must be placed on a plate of
soybean–casein digest agar medium, and for fungi detection, an antibiotic is added to the
medium and placed onto a plate of one of the Sabouraud glucose agar. Plates are incubated for at
least for 5 days at 30–35 °C for bacteria detection and at 20–25 °C for fungi detection. At the end
of the incubation period, the number of colonies is counted.

Pour plate method

With the pour plate method, Petri dishes of 9–10 cm in diameter are used, with two agar
media used for each dilution. For the test:

● Take 1 mL of the test fluid or its dilution into each Petri dish aseptically, add to each dish 15–
20 mL of sterilized agar medium, previously melted and kept below 45 °C, and mix (45 °C is
just above the point of solidification to minimize heat-induced cell death).

● For bacteria detection, use soybean–casein digest agar medium and for fungi detection, use
Sabouraud glucose agar media, to which antibiotic has previously been added.

● After the agar solidifies, incubate at least for 5 days at 30–35 °C for bacteria detection and at
20–25 °C for fungi detection. If a large number of colonies develop, calculate viable counts
based on counts obtained from plates with not more than 300 colonies per plate for bacteria
detection and from plates with not more than 100 colonies per plate for fungi detection.

Spread plate method

With the spread plate method:
● Place 0.05–0.2 mL of the test fluid on the solidified and dried surface of the agar medium and
spread it uniformly using a spreader.
● Proceed under the same conditions as for the pour plate method, especially with regard to
Petri dishes, agar media, incubation temperature and time, and calculation method.
● A variant, not described in the pharmacopeia, is the drop-plate method (or Miles and Misra
 method), wherein a very small aliquot (usually about 10 μL) of sample from each dilution in
series is dropped onto a Petri dish.

Most probable number method

The MPN method (alternatively, the method of Poisson zeroes) is a method of obtaining
quantitative data on concentrations of discrete items from positive/negative (incidence) data. The
method involves taking the original solution or sample, and subdividing it by orders of
magnitude (frequently 10× or 2×) into culture broth, and assessing the presence/absence in
multiple subdivisions. The major weakness of MPN methods is the need for large numbers of
replicates at the appropriate dilution to narrow the confidence intervals. The MPN is only
effective of the examination of bacteria, as it does not provide reliable results for the
enumeration of fungi.
To determine the accuracy and sensitivity of the test methods used for microbial limit
testing, 10 g or 10 mL samples of the test material are examined. It is also important, when
conducting these tests, to ensure that the testing method does not either introduce bacteria into
the test sample or kill bacteria in the test sample. Furthermore, the dilution of microbial
challenges needs to be as precise as possible.

Method verification
Method verification is an important step. While the total count method is a ―compendia
test,‖ which means that, by convention, the test itself does not require validating, the suitability
of each material must be qualified to show that the test method is not inhibitory and that any
microorganisms present can be recovered. This assessment is particularly important for samples
that have antimicrobial activity.

When test samples have antimicrobial activity or when they include antimicrobial
substances, these antimicrobial properties must be eliminated by dilution, filtration,
neutralization, inactivation, or other appropriate means. The tests should be conducted for
samples prepared by mixing multiple portions randomly chosen from individual ingredients or
products. When samples are diluted with fluid medium, the tests must be conducted quickly. Due
attention must be paid to the effective quality control and the prevention of biohazard.

IN-PROCESS MATERIAL BIOBURDEN ASSESSMENT

The environmental and process bioburden should be monitored to ensure that they are
both within acceptable limits. With bioburden testing, an appropriate test method should be
selected. Here either membrane filtration (using a 100 mL sample) or the pour plate (using a 1
mL sample) are the most common methods. It is more common to use one general purpose agar,
such as soybean–casein digest medium, rather than two different agars and to incubate samples
between 20 and 35 °C. In establishing an in-process bioburden regime, appropriate limits should
be set (in the form of ―alert‖ and ―action‖ levels). The levels can be defined as:
  Alert level: a level that, when exceeded, indicates a process may have drifted from its
normal operating condition. Alert levels constitute a warning, but do not necessarily
warrant corrective action.
 Action level: a level that, when exceeded, indicates a process has drifted from its normal
operating range. A response to such an excursion should involve a documented
investigation and corrective action.

These limits should decrease as the process moves downstream. While limits will relate
to specific processes, a general guideline is 100 CFU for the start of the process and 10 CFU near
the end of the process (per 1 mL of per 100 mL).

PRESTERILIZATION BIOBURDEN ASSESSMENT

With terminally sterilized products, understanding the bioburden is necessary because the
extent of the treatment of a sterilization process is a factor of the typical bioburden on or in the
product; the resistance of the microorganisms that make up the bioburden; and the sterility
assurance level required [20]. The test is important because an under-estimation of the bioburden
population could result in a miscalculation of the sterilizing requirements for a given product; in
contrast, an overestimation could result in excessive exposure to the sterilizing agent, which in
turn could affect the quality of the product.

ASEPTICALLY FILLED PRODUCTS

With aseptic processing, one of the most important samples is taken from the bulk
material prior to transfer through a sterilizing grade filter in preparation for aseptic filling. The
filters used are generally of a pore size of 0.22 μm, configured in a series. The ―sterilizing filter‖
was defined in 1987 by the US Food and Drug Administration (FDA) on the basis of its retaining
a minimum of 1 × 107 CFUs of Brevundimonas diminuta per square centimeter of effective
filtration area (EFA).

MEDICAL DEVICES
A third area for bioburden determination prior to sterilization is in relation to medical
devices. As with terminally sterilized pharmaceutical products, the objective is to ensure that the
presterilization bioburden is below that used to qualify the sterilization process. Bioburden
testing for medical devices made or used in the United States is governed by Title 21 of the Code
of Federal Regulations and worldwide by the standard ISO 11737.
Bioburden control with medical devices, as described in ISO 11737, consists of five key
steps. These are:
1. Sample selection;
2. The process of removing any microorganisms from the sample;
3. Transfer of microorganisms to recovery solutions;
 4. Enumeration of microorganisms;
5. Data interpretation (with the application of correction factors, where necessary).

STERILITY TESTING OF STERILE PRODUCTS

This test is harmonized with the European Pharmacopoeia and the U. S. Pharmacopeia.

The test is applied to substances, preparations or articles which, according to the

Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no
contaminating micro-organism has been found in the sample examined in the conditions of the
test.

Precautions against microbial contamination

The test for sterility is carried out under aseptic conditions. In order to achieve such
conditions, the test environment has to be adapted to the way in which the sterility test is
performed. The precautions taken to avoid contamination are such that they do not affect any
micro-organisms which are to be revealed in the test. The working conditions in which the tests
are performed are monitored regularly by appropriate sampling of the working area and by
carrying out appropriate controls.

Culture media and incubation temperatures

Media for the test may be prepared as described below, or equivalent commercial media may be
used provided that they comply with the growth promotion test.

The following culture media have been found to be suitable for the test for sterility. Fluid
thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will
also detect aerobic bacteria. Soya-bean casein digest medium is suitable for the culture of both
fungi and aerobic bacteria.

Fluid thioglycollate medium

L-Cystine 0.5 g
Agar 0.75 g
Sodium chloride 2.5 g
Glucose monohydrate/anhydrous 5.5 / 5.0 g
Yeast extract (water-soluble) 5.0 g
Pancreatic digest of casein 15.0 g
Sodium thioglycollate or 0.5 g
Thioglycollic acid 0.3 mL
Resazurin sodium solution (1 in 1000), freshly prepared 1.0 mL
Water 1 000 mL
(pH after sterilization 7.1 ± 0.2)
Soya-bean casein digest medium
Pancreatic digest of casein 17.0 g
Papaic digest of soya-bean meal 3.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose monohydrate/anhydrous 2.5 / 2.3 g
Water 1000 mL
(pH after sterilization 7.3 ± 0.2)

SUITABILITY OF THE CULTURE MEDIUM

The media used comply with the following tests, carried out before or in parallel with the
test on the product to be examined.

Sterility
Incubate portions of the media for 14 days. No growth of micro-organisms occurs.

Growth promotion test of aerobes, anaerobes and fungi

Test each batch of ready-prepared medium and each batch of medium prepared either
from dehydrated medium or from ingredients. Suitable strains of micro-organisms are indicated
in Table 4.06 -1.
Inoculate portions of fluid thioglycollate medium with a small number (not more than
100 CFU) of the following micro-organisms, using a separate portion of medium for each of the
following species of micro-organism: Clostridium sporogenes, Pseudomonas aeruginosa,
Staphylococcus aureus.

Inoculate portions of soya-bean casein digest medium with a small number (not more
than 100 CFU) of the following micro-organisms, using a separate portion of medium for each of
the following species of micro-organism: Aspergillus niger, Bacillus subtilis, Candida albicans.
Incubate for not more than 3 days in the case of bacteria and not more than 5 days in the
case of fungi.

Strains of the test micro-organisms suitable for use in the Growth Promotion Test and the
Method suitability Test

Aerobic bacteria
Staphylococcus aureus - ATCC 6538, NBRC 13276, CIP 4.83, NCTC 10788,
NCIMB 9518
Bacillus subtilis - ATCC 6633, NBRC 3134, CIP 52.62, NCIMB 8054
Pseudomonas aeruginosa - ATCC 9027, NBRC 13275, NCIMB 8626, CIP 82.118
 Anaerobic bacterium

Clostridium sporogenes - ATCC 19404, NBRC 14293, CIP 79.3, NCTC 532，

ATCC 11437

Fungi
Candida albicans - ATCC 10231, NBRC 1594, IP 48.72, NCPF 3179
Aspergillus niger - ATCC 16404, NBRC 9455, IP 1431.83, IMI 149007

Method suitability test

Carry out a test as described below under Test for sterility of the product to be examined
using exactly the same methods except for the following modifications.

Membrane filtration
After transferring the content of the container or containers to be tested to the membrane
add an inoculum of a small number of viable micro-organisms (not more than 100 CFU) to the
final portion of sterile diluent used to rinse the filter.

Direct inoculation
After transferring the contents of the container or containers to be tested to the culture
medium add an inoculum of a small number of viable micro-organisms (not more than 100 CFU)
to the medium.

In both cases use the same micro-organisms as those described above under Growth
promotion test of aerobes, anaerobes and fungi. Perform a growth promotion test as a positive
control. Incubate all the containers containing medium for not more than 5 days.

If clearly visible growth of micro-organisms is obtained after the incubation, visually

comparable to that in the control vessel without product, either the product possesses no
antimicrobial activity under the conditions of the test or such activity has been satisfactorily
eliminated. The test for sterility may then be carried out without further modification.

If clearly visible growth is not obtained in the presence of the product to be tested,
visually comparable to that in the control vessels without product, the product possesses
antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test.
Modify the conditions in order to eliminate the antimicrobial activity and repeat the method
suitability test.
This method suitability is performed:
a) when the test for sterility has to be carried out on a new product;
 b) whenever there is a change in the experimental conditions of the test.

The method suitability may be performed simultaneously with the Test for sterility of the
product to be examined.

TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED

The test may be carried out using the technique of membrane filtration or by direct
inoculation of the culture media with the product to be examined. Appropriate negative controls
are included. The technique of membrane filtration is used whenever the nature of the product
permits, that is, for filterable aqueous preparations, for alcoholic or oily preparations and for
preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not
have an antimicrobial effect in the conditions of the test.

Aqueous solutions
If appropriate, transfer a small quantity of a suitable, sterile diluent such as a 1 g / L
neutral solution of meat or casein peptone pH 7.1 ± 0.2 onto the membrane in the apparatus and
filter. The diluent may contain suitable neutralizing substances and/or appropriate inactivating
substances for example in the case of antibiotics.

Transfer the contents of the container or containers to be tested to the membrane or membranes,
if necessary after diluting to the volume used in the method suitability test with the chosen sterile
diluent but in any case using not less than the quantities of the product to be examined prescribed
in Table:1. Filter immediately. If the product has antimicrobial properties, wash the membrane
not less than three times by filtering through it each time the volume of the chosen sterile diluent
used in the method suitability test. Do not exceed a washing cycle of 5 times 100 mL per filter,
even if during method suitability it has been demonstrated that such a cycle does not fully
eliminate the antimicrobial activity. Transfer the whole membrane to the culture medium or cut it
aseptically into two equal parts and transfer one half to each of two suitable media. Use the same
volume of each medium as in the method suitability test. Alternatively, transfer the medium onto
the membrane in the apparatus. Incubate the media for not less than 14 days.
Table:1 Minimum quantity to be used for each medium
Soluble solids
Use for each medium not less than the quantity prescribed in Table 4.06-2 of the product
dissolved in a suitable solvent such as the solvent provided with the preparation, water for
injection, saline or a 1 g / L neutral solution of meat or casein peptone and proceed with the test
as described above for aqueous solutions using a membrane appropriate to the chosen solvent.

Oils and oily solutions

Use for each medium not less than the quantity of the product prescribed in Table 4.06-2.
Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a
dry membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as
isopropyl myristate shown not to have antimicrobial activity in the conditions of the test. Allow
the oil to penetrate the membrane by its own weight then filter, applying the pressure or suction
gradually. Wash the membrane at least three times by filtering through it each time about 100
mL of a suitable sterile solution such as 1 g / L neutral meat or casein peptone containing a
suitable emulsifying agent at a concentration shown to be appropriate in the method suitability of
the test, for example polysorbate 80 at a concentration of 10 g / L. Transfer the membrane or
membranes to the culture medium or media or vice versa as described above for aqueous
solutions, and incubate at the same temperatures and for the same times.

Ointments and creams

Use for each medium not less than the quantities of the product prescribed in Table 4.06-
2. Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1 per cent in
isopropyl myristate as described above, by heating, if necessary, to not more than 40 °C. In
exceptional cases it may be necessary to heat to not more than 44 °C. Filter as rapidly as possible
and proceed as described above for oils and oily solutions.
Direct inoculation of the culture medium
 Transfer the quantity of the preparation to be examined prescribed in Table 4.06-2
directly into the culture medium so that the volume of the product is not more than 10 per cent of
the volume of the medium, unless otherwise prescribed. If the product to be examined has
antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing
substance or by dilution in a sufficient quantity of culture medium.
Oily liquids
Use media to which have been added a suitable emulsifying agent at a concentration
shown to be appropriate in the method suitability of the test, for example polysorbate 80 at a
concentration of 10 g / L.
Ointments and creams

Prepare by diluting to about 1 in 10 by emulsifying with the chosen emulsifying agent in

a suitable sterile diluent such as a 1 g / L neutral solution of meat or casein peptone. Transfer the
diluted product to a medium not containing an emulsifying agent.

Incubate the inoculated media for not less than 14 days. Observe the cultures several
times during the incubation period. Shake cultures containing oily products gently each day.
However when fluid thioglycollate medium is used for the detection of anaerobic micro-
organisms keep shaking or mixing to a minimum in order to maintain anaerobic conditions.
Application of the test to parenteral preparations, ophthalmic and other non-injectable
preparations required to comply with the test for sterility
When using the technique of membrane filtration, use, whenever possible, the whole
contents of the container, but not less than the quantities indicated in Table: 1, diluting where
necessary to about 100 mL with a suitable sterile solution, such as 1 g / L neutral meat or casein
peptone.
When using the technique of direct inoculation of media, use the quantities shown in
Table: 1, unless otherwise justified and authorized. The tests for bacterial and fungal sterility are
carried out on the same sample of the product to be examined. When the volume or the quantity
in a single container is insufficient to carry out the tests, the contents of two or more containers
are used to inoculate the different media.
MINIMUM NUMBER OF ITEMS TO BE TESTED
The minimum number of items to be tested in relation to the size of the batch is given in Table:3.
Table:3 Minimum number of items to be tested
Factors affecting the efficacy of sterilization

Factors Effect
Cleaning Failure to adequately clean instrument results in higher bioburden, protein
load, and salt concentration. These will decrease sterilization efficacy.
Bioburden The natural bioburden of used surgical devices is 100 to 103 organisms
(primarily vegetative bacteria), which is substantially below the 105-
106 spores used with biological indicators.
Pathogen type Spore-forming organisms are most resistant to sterilization and are the test
organisms required for FDA clearance. However, the contaminating
microflora on used surgical instruments consists mainly of vegetative
bacteria.
Protein Residual protein decreases efficacy of sterilization. However, cleaning
appears to rapidly remove protein load.
Salt Residual salt decreases efficacy of sterilization more than does protein load.
However, cleaning appears to rapidly remove salt load.
Biofilm Biofilm accumulation reduces efficacy of sterilization by impairing exposure
accumulation of the sterilant to the microbial cell.
Lumen length Increasing lumen length impairs sterilant penetration. May require forced
 flow through lumen to achieve sterilization.
Lumen diameter Decreasing lumen diameter impairs sterilant penetration. May require forced
flow through lumen to achieve sterilization.
Restricted flow Sterilant must come into contact with microorganisms. Device designs that
prevent or inhibit this contact (e.g., sharp bends, blind lumens) will decrease
sterilization efficacy.
Device design and Materials used in construction may affect compatibility with different
construction sterilization processes and affect sterilization efficacy. Design issues (e.g.,
screws, hinges) will also affect sterilization efficacy.

TEST FOR PYROGENS / ENDOTOXINS

PYROGEN: In Greek Pyro = fire, gen = beginning

A Pyrogen is a substance i.e. products of the growth of microorganisms or may be parts of dead
cells or metabolic products which cause febrile reactions like fever, chills, back pain etc.

Sources of Pyrogens and its elimination methods:

TEST FOR PYROGENS:

The Pyrogen test is designed to limit the risk of febrile reaction following parentral
administration of drugs. It includes both In vitro and In vivo tests.
1. In Vitro Test / LAL Test
2. In Vivo Test / Rabbit Test.

1. LAL Test: Limulus Amoebocyte Lysate Test. (In Vitro Test)

In Vitro assay used to detect the presence and concentration of bacterial endotoxins in
drugs and biological products.

Limulus Amoebocyte Lysate (LAL) is an aqueous extract of blood cells (amoebocytes)

from the horseshoe crab, Limulus polyphemus.

LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane

component of Gram negative bacteria and forms gel which is then used for the detection and
quantification of bacterial endotoxins.
Sample + LAL (mL) --------------------------------------- Gel formation (+)
Incubation at 37 °C for 1 hr.

Limitations of LAL Test:

1. Disturbed by endotoxin binding components like lipids, blood components etc.
2. Difficult to correlate with rabbit test.
3. False positive for cellulose and many herbal preparations.

2. Rabbit Test: SHAM TEST (In Vivo)

Principle:
The test involves the measurement of rise in body temperature of rabbits following IV
injection of sterile solution of substance being examined.

Animals used:
Select same variety of healthy mature rabbits weighing less than 1.5Kg and should
maintain balanced diet. They should not show any loss of body weight during the preceding
week of test.
Temperature measurement:
Accurate temperature sensing devices such as Clinical Thermometer graduated in 0.1°C /
Thermister / Probes are used to measure the temperature of rabbit. Insert the thermometer into
the rectum of rabbit to a depth of not less than 6 cm and record the temperature.

Materials used:
Glass ware and syringes must be washed with water for injection and heated to 250°C for
30 minutes / 200°C for 1 hour in hot air oven.

Procedure:
It includes
1. Preliminary test.
2. Main test.

Preliminary test:
Select fresh animals / Animals not been used during 2 previous weeks.

Conditioned them for 1 to 3 days.

With hold the food from animal before 2hours of starting the test and access
to water may be allowed.

Record the temperature of animals using thermometer.

After 90 min, give IV injection 10mL/Kg (Pyrogen free saline solution)

Record the temperature of animals after IV injection at an interval of 30min

and continued for 3 hrs after injection.
 Animals show temperature variance of 0.6°C should not be used for main test.

Note: It is carried out in room without disturbances and temperature variance must be ± 3°C.

Main test:
Preparation of Sample: Dissolve test substances in Pyrogen free saline water and warm the
liquid to 38°C before giving injection.

3 groups of selected rabbits (preliminary test passed rabbits)

With held food for 2 hrs before experiment and during the experiment

Record initial temperatures (mean of two measurements at an interval of 30

minutes)

Rabbits showing a temperature variance ≥0.2 °C between two successive

readings should not be used.

Use only those rabbits that do not deviate a temperature variance 1°C
between two successive readings.

Inject sample into the marginal vein of the ear of 3 rabbits

Not less than 0.5mL/ Kg and not greater than 10mL/Kg body wt.

Record the temperature of animals during a period of 3 hours at intervals of 30

minutes.

Interpretation of Results

Case: 1
No rabbit shows individual raise in temperature of 0.6 °C (or)
Sum of three rabbits raise in temperature does not exceed 1.4 °C
 Absence of Pyrogens in the test sample.

Case: 2
If 2 or 3 rabbits show increase in temperature ≥0.6 °C or
Sum of three rabbits raise in temperature exceeds 1.4 °C

Continue or Repeat the experiment using additional 5 rabbits.

Not more than 3 rabbits shows individual raise in temperature of 0.6 °C (or)

Sum of eight rabbits raise in temperature does not exceed 3.7 °C

Absence of Pyrogens in the test sample

CLEAN ROOM – CONTAMINATION AND CLASSIFICATION

Cleanroom is a controlled placement where different products are manufactured. And
concentration of airborne particles is controlled to specified limits. So we need to control process
of killing ultra-fines airborne contaminants. The contaminations are generated by people,
processes, facilities, and equipment. They must be continually removed from the air. The level of
air cleanliness in the room must be regulated by standards. The most frequently used standard is
the ISO 14644. It is a document that establishes standard classes of air cleanliness in terms of
airborne particulate levels in cleanrooms and clean zones.

The basic function of a cleanroom is to protect the manufactured product from

contamination. In the pharmaceutical production economical survival of the manufacturer
depends on the safety of the finished product. So, it is needed to know a potential source of
contamination, which could include the working environment itself.

Contamination sources:

There are several sources of contamination such as process equipment, personnel and
surfaces. Bacteria are the most important contaminant in a pharmaceutical cleanroom. Almost all
of these come from the people in the room. So we need to know the number of people who are
working in the rooms. As this will have a direct behavior on the quantity of air required to dilute
and remove the airborne dispersion of contamination from their bodies.

Temperature level of cleanroom ordinary is 20C with an RH (relative humidity) of 40%

± 5%. For moisture sensitive materials it is required lower RH 25% ± 5%. Also these levels
depend on geographical location, production and clothing worn. So dry bulb temperatures can
vary in the range of 18C to 22C.

Another significant source of particulate contamination is process equipment. Prevention

by removal of particles at source should be the first objective before a limitation is made for
removing it once it has entered the cleanroom space. This will ensure a more cost-effective
design.

Pharmaceutical cleanroom suites consist of different cleanrooms, where are made several
steps of production. Standards of environmental control increase step by step when product
materials and packaging components are carried out processes into different rooms. It is
continued until one reaches the moment of product filling, closing and sealing. There is required
the highest quality condition. Less environmental conditions are required when a sealed product
coming for labeling and inspection. Different standards of environmental control are reached by
various air supply rates and the usage of unidirectional flow units or isolators at the critical areas.

CLEANROOM CLASSIFICATION
Cleanrooms are divided into different classes in standards. The equivalence of classes
form different international standards is shown in table 1. For the manufacturing sterile products
there is certain classification (table 2) with grades A to D which are characterized to activity
category (tables 3 and 4).
Remarks: (а) In order to reach the B, C, and D air grades, the number of air changes should be
related to the size of the room and the equipment and personnel present in the room. The air
system should be provided with appropriate filters such as HEPA for grades A, B, and C.
(b) At rest should be received unmanned state after the 15-20 min ―clean up‖ period.

(c) Appropriate alert and action limits should be set for the results of particulate and
microbiological monitoring. If these limits are exceeded, operating procedures should prescribe
corrective action.

Demands for other parameters like temperature, relative humidity, etc. depend on product
and manufacturing operation nature. These parameters have no connections to purifying classes.

Remarks: After washing, components should be handled in at least a grade D environment.

Handling of sterile starting material should normally be done in a grade A environment with a
grade B background. The preparation of solutions which are to be sterile filtered during the
process should be done in a grade C environment; if not filtered, the preparation of materials and
products should be done in a grade A environment with a grade B background. The handling and
filling of aseptically prepared products should be done in a grade A environment with a grade B
background. The preparation and filling of sterile ointments, creams, suspensions and emulsions
should be done in a grade A environment, with a grade B background, when the product is
exposed and is not subsequently filtered.