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This complete overview of ISO cleanroom classification and risk assessment explains how particle limits define a cleanroom, how EU GMP Annex 1 grades relate to ISO classes, and how a risk assessment sets the monitoring plan.
ISO 14644-1 classifies cleanroom air by the maximum particles per m3 into ISO Classes 1 to 9. In pharma, EU GMP Annex 1 grades A to D map to ISO 5, 7 and 8, and a risk assessment under International Council for Harmonisation (ICH) Q9(R1) sets the monitoring plan.
Complete overview of ISO cleanroom classification and risk assessment
An ISO cleanroom classification is a standardised ranking of a room by the maximum concentration of airborne particles of a stated size. A cleanroom keeps particle and viable counts within set limits at all times. Sterile products are made in cleanrooms because contamination can harm the patient.
Rooms are categorised by the size of the particles and their quantity per cubic metre of air. Each cleanroom also has limits for microbial count and air changes.
Cleanroom classification systems: current and withdrawn
Four systems have been used in the pharmaceutical industry:
- ISO 14644-1 (current)
- EU GMP Annex 1 grades (current)
- FS (Federal Standard) 209E (withdrawn)
- BS (British Standard) 5295 (withdrawn)
Clarification of different cleanroom classifications
| |ISO Classes| |EU GMP| |F.S. Standred| |
| ISO 5 Grade A Class 100 |
| ISO 6 Grade B Class 1000 |
| ISO 7 Grade C Class 10,000 |
| ISO 8 Grade D Class 100,000 |
1. ISO 14644-1 cleanroom classification
ISO 14644-1 (International Organization for Standardization, 2nd edition 2015) classifies air cleanliness by particle concentration into ISO Classes 1 to 9. ISO Class 1 is the cleanest and Class 9 the least clean, and Classes 5 to 8 are the ones mostly used in pharmaceuticals. According to ISO 14644-1:2015, ISO Class 5 allows 3,520 particles per m3 at ≥0.5 µm, ISO 7 allows 352,000 and ISO 8 allows 3,520,000.
Formula for cleanroom classification
The maximum concentration for a class is calculated as:
Cn = 10^N × (0.1 / D)^2.08
where:
- Cn is the maximum concentration in particles/m3, rounded to no more than three significant figures.
- N is the ISO class, from 1 to 9.
- D is the particle size in µm, from 0.1 to 5.
- 0.1 is a constant in µm.
For ISO 5 at 0.5 µm, Cn = 10^5 × (0.1 / 0.5)^2.08, which gives 3,520 particles/m3.
2. EU GMP Annex 1 grades A to D
EU GMP Annex 1 (2022) lists Grade A as ISO 5 at rest and in operation, and the other grades are linked to ISO classes as shown below. The revised Annex 1 has applied since 25 August 2023, with point 8.123 (lyophiliser loading and unloading) applying from 25 August 2024.
| Grade | ISO class at rest | ISO class in operation | Viable limit in operation |
|---|---|---|---|
| A | ISO 5 | ISO 5 | No growth |
| B | ISO 5 | ISO 7 | 10 cfu/m3 air, 5 cfu/4 h settle plate, 5 cfu/plate contact, 5 cfu/glove |
| C | ISO 7 | ISO 8 | 100 cfu/m3 air, 50 cfu/4 h settle plate, 25 cfu/plate contact |
| D | ISO 8 | Not defined | 200 cfu/m3 air, 100 cfu/4 h settle plate, 50 cfu/plate contact |
ISO 5 and Grade A are therefore not identical, because Grade A also requires no microbial growth, compliance in operation and continuous monitoring.
Annex 1 (2022) gives 5 µm limits only for Grades C and D (29,300 particles/m3 in operation for C and at rest for D, and 2,930 at rest for C). For Grades A and B, no 5 µm classification limit is set, and the site must define it from the contamination control strategy and trend data. An isolated ≥5 µm count in Grade A may be a false count caused by electronic noise, but repeated counts may signal a contamination event and must be investigated.
3. FS (Federal Standard) 209E cleanroom classification
Federal Standard 209E described the classes by the particles per cubic foot at ≥0.5 µm:
- Class 100, equal to ISO 5
- Class 1,000, equal to ISO 6
- Class 10,000, equal to ISO 7
- Class 100,000, equal to ISO 8
Related: Air Change Per Hour (ACPH) Calculation
Note: FS 209E was cancelled on 29 November 2001 and superseded by ISO 14644-1, although some companies still use the old class names.
4. BS (British Standard) 5295
The BSI published BS 5295 to define and classify cleanrooms. Like FS 209E, it has been withdrawn and replaced by ISO 14644-1.
Classification, monitoring and qualification
Classification, routine monitoring and requalification are separate activities. Classification is done under ISO 14644-1. Monitoring is covered by ISO 14644-2, which sets annual retesting as the default and allows an extension through a documented risk assessment. A documented operations control programme for cleanroom operations is added by ISO 14644-5:2025, and ISO 14644-14 reached final draft stage in October 2025.
Requalification intervals under Annex 1 are 6 months for Grades A and B and 12 months for Grades C and D. Guidance values are an airflow of 0.36 to 0.54 m/s at the working position of unidirectional flow and a pressure differential of 10 to 15 Pa between adjacent grades. The air changes of the room are calculated as described in the ACPH post above. The supporting Heating, Ventilation and Air Conditioning (HVAC) system is qualified as described in the Air Handling Unit (AHU) Installation Qualification (IQ) protocol.
Contamination control strategy and monitoring
Annex 1 requires a documented contamination control strategy (CCS) that covers design, equipment, personnel, materials, cleaning, process controls and monitoring. The monitoring plan for all grades (A to D) is justified by a documented risk assessment as part of the CCS, so no fixed daily or weekly rule applies.
Grade A particle monitoring must be continuous during the whole critical operation, including equipment set-up. Monitoring should therefore cover the whole filling operation, including batch changeover and equipment set-up, because these activities can contaminate the room that is being prepared for filling.
The two states are defined as follows:
- At rest: the installation is complete and the equipment is running, but no personnel are present.
- In operation: the room is working in the defined operating mode with the specified personnel present.
Risk assessment for cleanrooms
Risk assessment is an essential part of cleanroom management, and the monitoring plan puts its result into practice. EU GMP Chapter 1 defines quality risk management as a systematic process to assess, control, communicate and review risks to product quality. It also requires effort and formality to match the level of risk, and ICH Q9(R1) gives the tools.
Steps of a cleanroom risk assessment
Map the process, the people flow and the material flow, so that the critical areas are understood.
Identify the critical locations where product or sterile surfaces are exposed.
Rank the risks with FMEA (failure mode and effects analysis) or FMECA, which give a numerical ranking, or with HACCP (hazard analysis and critical control points) or FTA (fault tree analysis), which give qualitative high, medium and low results.
Choose the sampling sites and the frequency, and justify both. Sampling locations should be where contamination is most likely, from people and also from equipment, materials and airflow. A poor choice can harm product quality and the patient, and it raises operating cost.
Set alert and action limits, using the viable limits of Annex 1 as the upper reference for each grade.
Trend the data and investigate every excursion through Quality Assurance (QA).
Important documents for cleanroom risk assessment
ICH Q9(R1), effective in the EU from 26 July 2023, is the framework for quality risk management, and it reduces subjectivity in risk assessments and scales formality to the level of risk. For general risk methods, see Risk Assessment Protocol in Pharmaceuticals.
Frequently Asked Questions
The particle limit of Grade A equals ISO 5, both at rest and in operation. However, Grade A also has a viable limit of no growth in operation. It requires continuous particle monitoring during the whole critical process and must comply in both states. The two terms are therefore not interchangeable.
No. Federal Standard 209E was cancelled on 29 November 2001 and superseded by ISO 14644-1. Its class names, such as Class 100, still appear in some documents. Each old class maps to an ISO class: Class 100 equals ISO 5, Class 1,000 equals ISO 6, Class 10,000 equals ISO 7 and Class 100,000 equals ISO 8.
The 2015 edition (the second edition of ISO 14644-1) is the edition in force. It replaced the 1999 edition. A revision is under discussion, and a move from the formula to table-based limits has been reported, but a new Part 1 edition has not been published
The revised EU GMP Annex 1 has applied since 25 August 2023. EU GMP Annex 1, which covers the loading and unloading of lyophilisers, was delayed and applied from 25 August 2024. The revision made the contamination control strategy, continuous Grade A monitoring and risk-based environmental monitoring central.
FMEA and FMECA suit a numerical ranking of risks, while HACCP and FTA give qualitative high, medium and low results. HACCP identifies critical control points and FTA finds root causes. ICH Q9 lists these tools, and the choice depends on whether a numerical or a qualitative result is needed.
References
- EU GMP Annex 1: Manufacture of Sterile Medicinal Products (2022), European Commission
- ICH Q9 Quality risk management, EMA
- Cleanroom classification: ISO 14644 and EU GMP grades, IntuitionLabs
- The EU GMP Annex 1 (2022) and the 5 µm Particles in Grade A and B, gmp-compliance.org
- Continuous monitoring in pharmaceutical cleanrooms, News-Medical
- DIN EN ISO 14644-5:2025, GMP Publishing
- EU GMP Volume 4, Chapter 1, European Commission, 2019
- Process validation, risk assessment methods, RAPS, 2001
- EU GMP Annex 1: 8.123
Satpal Singh — Quality Assurance & Regulatory Specialist
Satpal Singh is an experienced pharmaceutical Quality Assurance and Regulatory professional with expertise in GMP compliance, quality systems, technical documentation, and regulatory requirements. He reviews our pharmaceutical content to ensure accuracy, industry relevance, and alignment with current quality and regulatory standards.
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