Dirty Equipment Hold Time Study Report: Injection Ampoule Section

In short: A dirty equipment hold time (DEHT) study proves the longest period that used equipment can wait before cleaning and still be cleaned to the same standard. This article explains how to plan and report one for an injection ampoule section, with an example report format. All numbers in the example are illustrative and must be replaced with your own validated data.

What is a dirty equipment hold time?

The dirty equipment hold time is the maximum time between the end of manufacture (or use) and the start of cleaning. During this wait, product residue can dry and harden, and micro-organisms can multiply, so cleaning becomes harder. The related clean equipment hold time is the maximum time cleaned equipment may be stored before it is used again.

Both are expected by the regulators. EU GMP Annex 15 says the time between manufacture and cleaning, and between cleaning and use, should be taken into account to define dirty and clean hold times for the cleaning process (Annex 15, clause 10.8). The WHO guideline on validation lists, as content of a cleaning validation protocol, the maximum period equipment may be left dirty and the time that should elapse after cleaning and before use (WHO TRS 1019, Annex 3, Appendix 3).

In an injection ampoule section the risk is higher than in oral dosage areas. The product is injected, so residue, bioburden and endotoxin carry-over matter directly to patient safety.

Equipment and products to include

Choose equipment that is representative of routine production. For an ampoule section this normally means:

the manufacturing vessel, transfer lines and filter housings;
the filling machine product path (filling needles, pump, manifold);
the ampoule-washing and sterilising equipment, where product contact occurs.

Group similar equipment and justify the worst case. The worst-case product is usually the one that is hardest to clean, least soluble or most potent, or that supports microbial growth most. Record each item with its type, location and unique ID.

Protocol design

Dirty hold time conditions

Complete a normal batch (or a worst-case simulation) and leave the equipment dirty without cleaning.
Hold the equipment in the normal storage condition of the area for the test periods, for example 0, 12, 24 and 48 hours (example in-house criterion; choose periods that cover your realistic delays, including weekends).
At each period, clean the equipment using the approved cleaning SOP exactly as in routine practice.
Sample and test after cleaning.

Clean hold time conditions

Clean and dry the equipment, protect it as in routine practice (covered, wrapped or stored in a classified area) and sample after defined periods. If sterilised equipment is used, the sterile hold time is studied separately.

Sampling

Select sampling points by risk: hard-to-clean locations, dead legs, valves, gaskets, filling needles and the lowest points where liquid collects. Use swab sampling for surfaces and rinse sampling for lines and closed parts. Use the recovery factors from the validated sampling method.

Tests

Test	Purpose
Visual inspection	No visible residue (necessary, but not sufficient on its own)
Active residue by validated method (e.g. HPLC, TOC)	Carry-over of the product
Cleaning agent residue (if used)	Detergent removal
Bioburden (TAMC / TYMC)	Microbial growth during hold
Bacterial endotoxin (LAL)	Endotoxin from microbial growth
Conductivity / pH of final rinse	Indicative cleanliness

Particulate contamination is mainly controlled by the cleaning procedure, filtration and the clean-area environment. It can be added as a visual or particle check on final rinse water where the process justifies it.

Acceptance criteria

Set the criteria before the study, with a scientific rationale. Residue limits come from your approved cleaning validation (for example a health-based exposure limit calculation). Bioburden and endotoxin limits must be consistent with the cleaning validation and the intended bioburden control of the product. Example in-house criteria used in the report below: no visible residue; residue below the approved limit; final rinse bioburden within the approved alert and action levels; endotoxin within the approved limit.

Critical parameters and risks

Equipment design and condition: crevices, worn gaskets and dead legs hold residue and organisms.
Cleaning procedure: the study is valid only when the routine cleaning SOP is followed unchanged.
Ambient conditions: temperature, humidity and area classification influence drying and microbial growth.
Product characteristics: solubility, viscosity and the ability to support microbial growth.
Microbial contamination and endotoxin: growth during a long dirty hold can create endotoxin that is not removed by later sterilisation.

Example report format

The table below shows how results can be presented. The data are illustrative only and are not real study results.

Item	Hold period	Visual	Residue vs limit	Bioburden (CFU)	Endotoxin	Result
Filling machine product path	0 h	Clean	Below limit	Within limit	Within limit	Pass
Filling machine product path	24 h	Clean	Below limit	Within limit	Within limit	Pass
Filling machine product path	48 h	Clean	Below limit	Within limit	Within limit	Pass

Report contents

Objective and scope
Reference documents (cleaning validation protocol, cleaning SOP, analytical methods)
Equipment list with IDs and worst-case rationale
Study design, sampling plan and acceptance criteria
Results for each hold period, with raw data references
Deviations and investigations
Conclusion: the approved dirty hold time (for example “up to 24 hours”) and clean hold time
Recommendations, such as the procedure when the hold time is exceeded (re-clean, extra sampling, or deviation)
Approval signatures

If any result fails, do not simply repeat sampling until it passes. Investigate the cause, correct the cleaning process or shorten the hold time, and repeat the study. Include the approved hold time in the batch record or cleaning SOP and monitor it routinely.

Records

Keep protocols, raw data, chromatograms and reports as GMP records for at least 1 year after the expiry date of the respective product batch (EU GMP Part I, Chapter 4, clause 4.11; 21 CFR 211.180(a)), longer if local law or the marketing authorisation requires.

Frequently asked questions

Is a hold time study a one-time activity? No. Repeat it after changes to the product, equipment, cleaning procedure or hold period, and review it as part of the periodic cleaning validation review.

Can the dirty hold time be the same for all products? Only if the worst-case product is studied and the rationale is documented. Otherwise define the time per product group.

What if the equipment is left dirty longer than the approved time? Raise a deviation, assess the risk, and re-clean with extra verification before use.

Is visual cleanliness enough? No. Annex 15 states that visual inspection cannot be the only acceptance criterion; combine it with analytical and microbiological results.

References

European Commission, EudraLex Volume 4, Annex 15: Qualification and Validation, clauses 10.1-10.8 (in operation since 1 October 2015).
WHO Technical Report Series No. 1019 (2019), Annex 3: Good manufacturing practices: guidelines on validation, Appendix 3: Cleaning validation.
EudraLex Volume 4, Part I, Chapter 4: Documentation, clause 4.11.
US FDA, 21 CFR 211.180(a): General requirements for records and reports.