Endotoxin Challenge in the Validation of Dry-Heat Sterilizers

The most controversial aspect of endotoxin challenge testing is how much endotoxin
challenge to use. The PDA [13] suggests using a level of endotoxin in
excess of the level expected in the item being subjected to the dry-heat cycle.
Simmons [22] suggested the use of 10,000 ng endotoxin. Akers et al. [26] used
only 10 ng endotoxin.
Papers by Ludwig et al. (27–30) expanded knowledge of dry heat depyrogenation
of glass surfaces using e. coli endotoxin challenges.
The step-by-step procedure for the endotoxin validation of a dry-heat process
may be as follows:
1. Inoculate commodity samples with a known amount of endotoxin
(e.g., 10–100 ng Escherichia coli lipopolysaccharide, obtainable from
several commercial sources). The endotoxin should be contained in a
volume of water equal to the residual water volume following the washing
procedure used prior to sterilization.
2. Thermocouples should be placed in commodities adjacent to those
containing endotoxin for temperature monitoring and correlation with
LAL test results.
3. Endotoxin destruction should be ascertained at the coolest location of
the load. Load configurations should be identical to those used in the
microbial validation studies.
4. Several endotoxin challenge samples should be done per cycle, and
the studies must be adequately replicated (3–5 repeats).
5. Following the dry-heat cycle, aseptically transfer the units containing
endotoxin to an aseptic area for extraction procedures, sampling, and
conducting the limulus amebocyte lysate (LAL) test.
6. F values required for endotoxin destruction at various temperatures
and/or cycle time–temperature variations can be determined using a
Z value of 54°C and the following equation:
Fendo. = Δt Σ 10(T−170)/54
This approach was used by Akers et al. [26].
When the validation studies described in this section have been completed,
all data are analyzed and a decision is made concerning their acceptability. If
acceptable, the entire validation procedure and all appropriate supporting data
are documented in a bound manual. If the studies are unacceptable because of
unsubstantiated claims of the process or a lack of reproducibility, further testing
must be performed or process variables changed followed by additional validation
studies.
The final document will be reviewed and approved by various plant disciplines
(engineering, microbiology, production, etc.) before the dry-heat sterilizer
is considered fully validated and released for use.