Cleanroom grades A, B, C and D: EU GMP Annex 1 limits and ISO equivalents

What cleanroom grades A, B, C and D mean

EU GMP Annex 1 sets four grades of cleanroom or zone for making sterile medicines. Grade A is the critical zone for high-risk operations such as the filling zone, stopper bowl, open primary packaging and aseptic connections. Grade B is the background cleanroom for Grade A in aseptic work. Grades C and D are for less critical stages, as a background for isolators, or for preparing and filling terminally sterilised products.

The current text is the 2022 revision, published on 25 August 2022. It came into operation on 25 August 2023, except point 8.123 on lyophilisers, which applied from 25 August 2024. PIC/S adopted the same dates. The HPRA inspects Irish manufacturers against GMP, and Annex 1 is written to help national authorities like it apply EU law. Our Annex 1 article for Irish sites and sterile manufacturing knowledge hub summary cover the wider changes.

Cleanroom grades vs ISO classes: particle limits

Each grade has a maximum total particle count at rest and in operation. "At rest" means equipment installed but not operating and no personnel present. "In operation" means equipment running with the maximum number of people doing, or simulating, routine work.

Source: EU GMP Annex 1 (2022), Table 1, limits for classification. ISO column matched on the 0.5 µm limits in the FDA 2004 aseptic guidance, Table 1.

Annex 1 itself does not label grades with ISO classes. The equivalents above come from matching the 0.5 µm figures to the ISO 14644-1 values the FDA lists: ISO 5 is 3,520, ISO 7 is 352,000 and ISO 8 is 3,520,000 particles per m³. Annex 1 points to ISO 14644-1 for the number and position of sampling locations.

Points that catch people out

• For classification, 5 µm limits at Grade A, and at Grade B at rest, are "not specified". For routine monitoring (Annex 1 Table 5) they are 29 per m³.
• Grade D in-operation limits are set by the manufacturer from a risk assessment and routine data.
• The at-rest limits should be reached after a "clean up" period, with a guidance value of under 20 minutes.
• Requalification is due at least every 6 months for Grades A and B and every 12 months for Grades C and D.

Microbial action limits by grade

Particle counts are only half of Annex 1. Each grade also has maximum action limits for viable (living) contamination, and for Grade A the limit is no growth.

Source: EU GMP Annex 1 (2022), Table 6.

Any growth in Grade A should lead to an investigation, and organisms found in Grades A and B should be identified to species level. When an action limit is exceeded, Annex 1 asks for a root cause investigation, a product impact assessment and corrective and preventive actions. Our guide to CAPA vs RCA explains how that works.

Which operations happen in which grade

The grade follows the risk: the more exposed the sterile product, the cleaner the air must be.

Source: EU GMP Annex 1 (2022), Tables 3 and 4 and points 8.1 to 8.12.

Barrier technology changes the background. Open isolators generally need at least Grade C around them, closed isolators at least Grade D, and RABS at least Grade B. Irish investment is still going into this work: on 14 September 2026 Astellas announced a second aseptic filling line for its Tralee plant. See our report on the Astellas Tralee expansion and the Kerry pharma map.

How the FDA approach differs

The FDA uses ISO classes rather than grades, and classifies under dynamic conditions. Its 2004 aseptic processing guidance bases all classifications on data measured near exposed materials "during periods of activity", and says ISO 5 "approximately equals EU Grade A".

• Critical area, ISO 5 (Class 100): no more than 3,520 particles of 0.5 µm or larger per m³, counted normally not more than 1 foot from the work site during filling.
• Supporting area next to the line: at least ISO 7 (Class 10,000) under dynamic conditions, or ISO 6 or ISO 5 if the firm chooses.
• Less critical work, such as equipment cleaning: ISO 8 (Class 100,000).
• Microbial action levels: active air 1, 7, 10 and 100 CFU/m³ for ISO 5, 6, 7 and 8; settle plates 1, 3, 5 and 50 CFU per 4 hours. ISO 5 samples "should normally yield no microbiological contaminants".

Irish plants that ship to the US are inspected against both. Our FDA aseptic guidance summary has the detail.

Gowning differences by grade

Gowning gets stricter with each grade. Annex 1 describes typical clothing for each:

• Grade D: hair and beards covered, a general protective suit and disinfected shoes or overshoes.
• Grade C: hair and beards covered, a single or two-piece trouser suit gathered at the wrists with a high neck, and disinfected shoes or overshoes.
• Grades A and B: sterile headgear, sterile facemask and eye coverings, sterilised over-boots, and sleeves tucked into a second pair of sterile gloves. A clean, sterilised gown is put on at each entry.

The FDA guidance likewise recommends sterilised, non-shedding gowns for aseptic areas. Gowning qualification is a common starting point for new operators, and Irish courses such as the ATU Certificate in Aseptic Biopharmaceutical Operations cover aseptic work.