APR January/February 2022 - 44

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There are, indeed, a small number of pyrogens that possess a different
structure and that cannot be detected using the test for bacterial
endotoxins. Such pyrogenic substances are detected using the
procedures described in the general chapter Monocyte-activation test
(2.6.30). The monocyte-activation test is therefore an in vitro pyrogen
test that has the advantage not only of avoiding the use of laboratory
animals, but also of being able to detect any pyrogenic substance, i.e.
both endotoxin and non-endotoxin pyrogens.
Replacement of the Rabbit
Pyrogen Test
The Council of Europe's European Convention for the Protection
of Vertebrate Animals used for Experimental and Other Scientific
Purposes was opened for signature in 1986. Since that time, the Ph.
Eur. Commission and its experts have carried out a program of work
committed to Replacing, Reducing and Refining (3Rs) the use of animals
for test purposes. Achievements have been significant,1
but there are
still challenges ahead. The Convention is referred to in a number of Ph.
Eur. texts, including chapter 2.6.8: " In accordance with the provisions of
the European Convention for the Protection of Vertebrate Animals used
for Experimental and Other Scientific Purposes, tests must be carried out
in such a way as to use the minimum number of animals and to cause the
least pain, suffering, distress or lasting harm. Wherever possible and after
product-specific validation, the pyrogen test is replaced by the monocyteactivation
test (2.6.30). " In spite of this explicit instruction to replace
the rabbit pyrogen test (RPT) by its in vitro alternative, the animal test
continues to be widely used.
At its annual conference in 2018,2
the European Partnership for
Alternative Approaches to Animal Testing (EPAA) reported on a survey
performed among European companies and testing institutes that
still routinely perform the RPT and found that there is little incentive
to perform alternative testing when a pyrogen test is prescribed in a
monograph. The regulatory burden linked with the change to the in
vitro test was also mentioned.
Reading the Ph. Eur. texts only, users reported a potential discrepancy
between monographs and EU Directive 2010/63/EU:3
" Article 4
Principle of replacement, reduction and refinement
1. Member States shall ensure that, wherever possible, a
scientifically satisfactory method or testing strategy, not
entailing the use of live animals, shall be used instead of a
procedure. "
" Article 13
Choice of methods
1. Without prejudice to national legislation prohibiting
certain types of methods, Member States shall ensure that
a procedure is not carried out if another method or testing
strategy for obtaining the result sought, not entailing the
use of a live animal, is recognised under the legislation of the
Union. "
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| January/February 2022
According to Article 13 of the directive, the instruction given in chapter
2.6.8 - to use an alternative to the animal test - should be applied
systematically, but this is not done in practice.
In view of the situation, the complete removal of the RPT from the Ph.
Eur. is necessary if the aim is to move towards the exclusive use of in
vitro tests for the control of pyrogens.
Currently, chapter 2.6.8 is prescribed in 59 texts of the Ph. Eur.: three
general monographs (including 2034 Substances for pharmaceutical
use), three dosage form monographs (including 0520 Parenteral
preparations), three general chapters and 50 individual monographs,
covering such diverse products as antibiotics, human vaccines and
blood products. In June 2021, the Ph. Eur. Commission endorsed
the strategy for the replacement of 2.6.8 in all of these 59 texts.4
A
new general chapter 5.1.13. Pyrogenicity will be introduced in the
Ph. Eur., which will provide guidance to help users decide on their
own approach to pyrogenicity testing, based on a risk assessment:
depending on the potential presence of non-endotoxin pyrogens, the
user will have the choice between an in vitro pyrogen test or a test for
bacterial endotoxins. Suppressed from all texts of the Ph. Eur., chapter
2.6.8 will no longer be an option and will ultimately be suppressed
from the Ph. Eur. The whole exercise will take approximately 5 years and
stakeholders will be consulted via the usual channels with, in 2023, the
chance to consult all proposed revisions and the new general chapter
5.1.13 - currently under preparation - in Pharmeuropa online5
comment as necessary.
and to
Recombinant Factor C
The test for bacterial endotoxins uses, as its main reagent, the
amoebocyte lysate from an animal, the horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus). Discussions among Ph. Eur.
experts on the use of a synthetic alternative to this natural reagent,
recombinant factor C (rFC), have been ongoing since 2006. It took over
a decade to collect sufficient data for the method using the synthetic
reagent to be described in the Ph. Eur. A major breakthrough came
on July 1, 2020 with the publication of general chapter 2.6.32. Test for
bacterial endotoxins using recombinant factor C in the Ph. Eur.,6
giving
an official status to the procedure using the recombinant reagent.
In January 2021, the procedure entered official use as a Ph. Eur.
method. In April 2021, the EDQM broadcast a webinar on the bacterial
endotoxin test using rFC, explaining its current status as an alternative
to the bacterial endotoxin test using the amoebocyte lysate.7
General chapter 2.6.14. Bacterial endotoxins gives a choice of six
methods, A to F (gel-clot method: limit test, gel-clot method:
quantitative test, turbidimetric kinetic method, chromogenic kinetic
method, chromogenic end-point method, or turbidimetric end-point
method), the Ph. Eur.'s aim would be to add a seventh method, method
G, that could be used instead of any of the other methods. However,
because the chapter has undergone International Harmonisation
within the Pharmacopeial Discussion Group (PDG), no changes
can be made to the chapter without the agreement of the other
participating pharmacopoeias (United States Pharmacopeia and the
APR January/February 2022

Table of Contents for the Digital Edition of APR January/February 2022
