131
1: Pyrogens
VII.
Pyrogens
(Methods of Analysis V.2.1.4)
The test for pyrogens is stipulated by several mono-
graphs, and it will therefore be discussed in this chapter
independently from individual monographs.
VII.1 Requirement/practical application
The following Ph. Eur. monographs for human
immunoglobulins stipulate the test for pyrogens (V.2.1.4)
in rabbits:
Normal Human Immunoglobulin
(applies both to normal human immunoglobulins, of which
nine products were licensed in 1993 in Germany, and to
“special” human immunoglobulins of which 16 were
licensed for intramuscular use in 1993 in Germany)
Human Normal Immunoglobulin for Intravenous
Use
(new Ph. Eur. monograph operative since 01.07.1994; in
1993, 17 products were licensed in Germany)
Meningococcal Polysaccharide Vaccine
(in 1993, two products were licensed in Germany)
Pneumococcal Polysaccharide Vaccine
(in 1993, one product was licensed in Germany)
These monographs refer to the test described in the
Section Methods of analysis (V.2.1.4). Three rabbits are
required per test. Each test consists of a preliminary test,
in which the body temperature of the animals is observed
for three hours after intravenous injection of saline, and
the main test, in which the test preparation is injected into
the marginal ear vein of each rabbit. The injection volume
(0.5 to 10 ml/kg) depends on the product to be tested and
is stated in the relevant monograph. The temperature of
each rabbit is recorded, at intervals of not more than 30
min, for at least 3h after injection. The product passes the
test, if the sum of the individual temperature responses,
namely the difference between the initial temperature and
the maximum recorded for each rabbit, does not exceed a
value tabulated in the monograph. If this figure is exceed-
ed, the test may be repeated in a further three rabbits, pro-
vided that the summed response did not exceed a second
value. The test may be repeated no more than four times.
The first WHO recommendations for meningococcal
vaccines (WHO Tech Rep Ser 594, 1976) required the
pyrogenicity test to be conducted on the final lot, using a
dose of 0.0025 µg/kg per polysaccharide (PS) component.
This corresponds to 1/20,000 of the human dose (HD) per
kg of the rabbit weight, since one HD (0.5 ml) contains 50
µg of each PS component. The recommended rabbit dose
was increased by a factor of 10 to 0.025 µg/kg in the first
addendum to the recommendations (WHO Tech Rep Ser
610, 1976). The reason given for this change was that vac-
cine purification had so been improved that a ten-fold
higher dose would still be tolerable for the rabbits in the
pyrogenicity test. This recommendation is still valid and
has also been incorporated into the Ph. Eur. monograph
(rabbit dose: 1/2,000 HD/kg). The rabbit dose stipulated
Chapter VII.
for pneumococcal vaccines is markedly higher and corre-
sponds to 1/10 HD/kg.
The results of our data collection on the use in practice
of the pyrogenicity test for polysaccharide vaccines are
given in Table VII-1. The data refer to the two meningo-
coccal vaccines licensed in Germany. It should be noted
that one of the manufacturers uses a dose (0.25 µg/kg or
1/200 HD/kg) which is ten times higher than the dose stip-
ulated by the Ph. Eur., but which corresponds to the dose
required e.g. in the USA (CFR 21, 610.13 (6), 1986). Very
few tests were repeated (1/11 tests).
The two manufacturers producing meningococcal vac-
cines test for pyrogenicity already at the purified polysac-
charide stage of production. It is noteworthy that one of
them uses the LAL test (test for bacterial endotoxins, see
also VII.3 Alternatives) instead of the pyrogenicity test in
rabbits, which he conducts only on the finished product as
required by the Ph. Eur. monograph. More rabbits (45)
were used for in-process testing than for finished product
testing (33 rabbits, Table VII-1). According to data sub-
mitted by the only manufacturer of a pneumococcal vac-
cine licensed in Germany, 27 rabbits were used to test nine
batches in 1991-1993. No tests had to be repeated and no
testing was carried out during the production process.
Sixty rabbits were required in total to test all batches of
polysaccharide vaccines produced in Germany in 1991-
1993, and a further 45 rabbits were used for in-process
testing.
The number of rabbits needed by the manufacturers for
the batch control of human immunoglobulins in 1991-1993
could only be roughly estimated:
From available data on the number of batches released
in 1992, it was calculated that 1550 batches were released
in total in 1991-1993 (988 batches of human immunoglob-
ulins for intravenous use and 562 batches of human
immunoglobulins for intramuscular use). Assuming that
each test required at least three rabbits and that no test
was repeated, a minimum of 4650 rabbits must have been
used for this purpose.
VII.2 Evaluation of the Method
The pyrogenicity test in rabbits is a functional test which
is able to detect reliably all substances/contaminants that
are pyrogenic in this species. Pyrogens are a very hetero-
geneous group of substances and pyrogenicity is the only
property that they have in common.
The use of the in vivo test can therefore still be justified
in cases where the pyrogenicity cannot be defined, e.g.
where it cannot be excluded that it is caused by pyrogens
other than bacterial endotoxins (lipopolysaccharide, LPS).
However, in the cases under discussion it may be ques-
tioned whether a pyrogenicity test is justifiable, necessary
or relevant.
All immunobiologicals for humans listed in the Ph. Eur.
are for parenteral use, however the monograph
Preparations for Parenteral Use does not necessarily apply
to immunobiologicals. The requirements for pyrogenicity
of immunobiologicals are not harmonised, but are defined
separately in each specific monograph. The monograph
Preparations for Parenteral Use requires preparations to
Chapter VII.2: Pyrogens 132

Table VII-1: Use in practice of the pyrogenicity test for meningococcal polysaccharide vaccines
(includes all vaccine batches released by the PEI in 1991-93)

manufacturer/tester
req. by
A B total mean from to Ph. Eur.

testing for n different products 1(M) 1(M) —

number of polysaccharide types 2 4 6 —
tested final lots 91-93# 5 5 10 1x
repeated tests 91-93# 1 0 1 —
repetitions/test (%)# 20% 0% 10% 0% 20% —
total number of tests 91-93+ 6 5 11 —

species rabbits rabbits rabbits rabbits

amount of polysaccharide/0.5ml (=HD) 50µg 50µg

rabbit dose/ml/kg 0.025µg each PS 0.025µg each PS 0.025µg each PS
corresponds to an equivalent of HD/kg 1/2,000 1/200 1/2,000

animals used per test 3 3 3.00 3

animals required/final lot+ 4 3 3.30
animals required 91-93+ 18 15 33

additional tests in-process yes yes —

stage of production purified PS + purified PS
diluent
method used pyrogenicity LAL test
test
number of additional tests 91-93# 15 —
additional animals 91-93 45 — 45

PS = polysaccharide; HD = human dose; # = data derived from batch release protocols or extrapolated from spot
checks; + = value is calculated from above data (includes frequency of repeat testing).

be free of pyrogens only if volumes > 15 ml are to be inject-
ed. This monograph would therefore not be applicable to
any vaccine and to most sera, because these are used at
much smaller doses.
The monograph Vaccines for Human Use does not
include a general requirement that preparations be free of
pyrogens. Many old vaccines would probably not satisfy
such a requirement, because they are considered to be
potentially pyrogenic, and indeed their pyrogenicity is
seen as contributing to their efficacy. The testing of poly-
saccharide vaccines for pyrogenicity has therefore been an
exception, although some new and recently revised mono-
graphs do now include the pyrogenicity test. For example,
this test is required by the revised monograph for rabies
vaccines for human use (see note in chapter XI), while the
monographs for two new vaccines (influenza vaccine and
inactivated hepatitis A vaccine) prescribe the use of the
LAL test for endotoxins, since this test has been validated
for this purpose.
Polysaccharide vaccines contain purified capsid polysac-
charides. In the case of Gram-negative meningococcae, it is
plausible that large amounts of endotoxin (lipopolysaccha-
ride from the bacterial cell membrane) could contaminate
the vaccine and make it strongly pyrogenic. This also
accounts for the need to use high dilutions of these vac-
cines (1/2,000 HD) for pyrogenicity testing.
When compared on a per kilogram basis, humans and
rabbits are equally sensitive to pyrogens [1]. Injection of a
dose less than 1/70 HD/kg rabbit is therefore insufficient
to exclude pyrogenic activity in humans. The expert opin-
ion is that only doses > 1/10 HD/kg rabbit are relevant [2].
The dose (1/2,000 HD/ml and kg rabbit) prescribed for the
testing of meningococcal vaccines therefore cannot ensure
the absence of pyrogens in the vaccine. In this case, a valid
pyrogenicity test serves only to limit the pyrogen content
(maximum pyrogenic activity) to a maximum equivalent to
2,000 ng E. coli endotoxin/HD since the detection limit in
rabbits is approximately 1 ng E. coli endotoxin/ml [1].
Pyrogenic activity in vaccinated individuals can be exclud-
ed if 1 HD contains not more than 35 ng endotoxin.
If it is not possible to produce pyrogen-free vaccines, at
the very least it is necessary to maximise test sensitivity, in
order to maximise vaccine safety, although these vaccines
appear to be very well tolerated, with only a few cases of
mild fever reported in clinical trials.
Therefore, it should be investigated whether the
improved conditions of production would permit the dose
given to rabbits to be increased again (see VII.1). Table
VII-1 shows that one of the manufacturer involved in our
survey already uses a ten-fold higher dose (0.25 µg/PS =
133
1/200 HD). Since this higher dose is stipulated in the USA,
it would be feasible to introduce such a modification into
the Ph. Eur.
Tester A already tests the purified polysaccharide bulk
for pyrogenicity (Table VII-1), which results in 2-4 addi-
tional tests being conducted on each vaccine batch. This is
not recommended by the WHO and should be opposed.
However, this very test strategy is included in the draft for
revision of the monograph for meningococcal vaccines
(Pharmeuropa, Vol. 6, No. 2/94).
In contrast to meningococcal vaccines, pneumococcal
vaccines might become contaminated by exogenous pyro-
gens. This is the reason why a much higher dose is admin-
istered to rabbits (1/10 HD/kg rabbit). The dose used is
sufficiently high to guarantee the absence of pyrogens in
the vaccine. The monograph requires pyrogenicity testing
to be conducted only on the final lot, as was and should
continue to be the case with meningococcal vaccines.
In contrast to the monographs for vaccines for human
use, the monographs for human immunoglobulins (for
intravenous and for intramuscular administration)
require the products to be tested for pyrogenicity. This dif-
ference is unlikely to be due to the size of the doses used,
since these are small, at least, with respect to
immunoglobulins for intramuscular administration, but
rather because it is now possible to produce pyrogen-free
preparations. The pyrogen test is therefore useful and nec-
essary. However, the test is not stipulated for animal-
derived immunosera, such as the classical horse antisera
against diphtheria, gas-gangrene, etc. (monograph for
immunosera for human use). These preparations are of
low purity and thus would probably fail the pyrogenicity
test. The potential fever-inducing capacity of these (old)
products was accepted on account of their life-saving prop-
erties.
(On the other hand, the WHO recommends pyrogenici-
ty testing, e.g. of snake venom antisera, when the final
bulks are to be sold for marketing to other companies [3].)
CONCLUSIONS:
With respect to pneumococcal vaccines and immunoglobu-
lins, the pyrogenicity test in rabbits is necessary and justi-
fiable. The test ensures that the preparations will not be
pyrogenic when used in humans.
With respect to meningococcal vaccines, the pyrogenici-
ty test can only ensure that the vaccine contains less than
a certain quantity of pyrogens. It might be feasible to
increase the dose administered to rabbits and thus
increase test sensitivity and decrease the maximum limit.
Furthermore, it should be possible to replace the pyro-
genicity test in rabbits with the endotoxin (LAL) test
(VII.3, Alternatives) since, in this case, the detected pyro-
gens are endotoxins.
VII.3 Alternatives
The currently discussed alternatives to the pyrogen test
are shown in Table VII-2.
The best known alternative method is the Limulus
amoebocyte lysate test (LAL test) which is included in
the Ph. Eur. monograph Test for Bacterial Endotoxins
(V.2.1.9). The LAL test is based on the fact that the amoe-
bocyte lysate obtained from Limulus polyphemus reacts
with bacterial endotoxins to form a gel. The LAL test is
very sensitive and can detect approximately 3 pg E. coli
endotoxin, which corresponds to 0.03 International Units
(IU) of endotoxin/ml (detection limit of the pyrogenicity
test in rabbits is 1 ng E. coli endotoxin/ml). However, var-
Chapter VII.3: Pyrogens
ious factors can interfere with the test and a certain level
of experience is required.
In November 1992, the German Health Institute and
the Paul Ehrlich Institute published an official statement
(in Bundesanzeiger) that all manufacturers of prepara-
tions for parenteral use could replace the pyrogenicity test
in rabbits with the LAL test, provided that the require-
ments for validation (given in the appendix) had been met
and the replacement was officially announced.
The requirements for validation (published in Pharm
Ind 54, Nr. 10, 1992) are: firstly, confirmation of the
absence of non-endotoxin pyrogens (comparative testing of
not less than three batches with the pyrogenicity test in
rabbits and the LAL test); secondly, definition of the max-
imum permitted concentration of endotoxin in the product
(usually 5 IU endotoxin per kg body mass per hour for
Preparations for Parenteral Use, which is equivalent to 350
IU endotoxin for an 70 kg adult), and thirdly, validation of
the LAL test procedure (interfering factors which should
be eliminated, determination of the maximum valid dilu-
tion).
To date, the LAL test has only been fully validated and
accepted for the pyrogenicity testing of influenza vaccine.
The test is now included in the monograph, and the maxi-
mum permitted endotoxin concentration is 100 IU endo-
toxin/HD, which is lower than for Preparations for
Parenteral Use). It is also intended to incorporate the LAL
test into the monograph for Hepatitis A vaccines, for
which the maximum permitted endotoxin concentration
will be 2 IU endotoxin/HD.
It is not possible to exclude the presence of non-endo-
toxin pyrogens in pneumococcal vaccines and human
immunoglobulins, therefore pyrogenicity testing in rabbits
continues to be required.
It will most probably be possible to validate the LAL test
for the pyrogenicity testing of meningococcal vaccines.
There are no theoretical objections to this, because the
pyrogenicity of these products is due to bacterial endotox-
ins (deriving from the meningococcae). As can be seen in
Table VII-1, tester B already uses the LAL test for in-
process control. He has also stated that he would immedi-
ately replace the rabbit pyrogenicity test on the finished
product with the LAL test, if the Ph. Eur. monograph
would permit this. This highlights the general problem
that, although manufacturers could themselves validate
alternatives, they prefer to achieve this by the more com-
fortable (and mandatory) way, through modifications to
the monographs.
The principal factor determining the suitability of the
LAL test is the maximum dilution that can be used, i.e.
whether the LAL test is capable of detecting the maximum
permitted concentration in products for parenteral use of
5 IU endotoxin/kg (350 IU endotoxin/HD or 700 IU endo-
toxin/ml, since 1 HD = 0.5 ml of vaccine). Assuming a
detection limit of 0.1 IU endotoxin/ml (the usual limit
being 0.03-0.05 IU endotoxin/ml), the test product could be
diluted down to 1/7,000 without any risk that the maxi-
mum permitted concentration would not be detected.
Therefore, the objection that test reproducibility may be
affected by the very high dilutions at which meningococcal
vaccines have to be evaluated in the LAL test (personal
communication, RIVM, Bilthoven, NL) is not convincing.
Moreover, tester B obtains a negative result in the LAL
test already at a dilution of 1/400, and this should be repro-
ducible.
Animals are still required to obtain the amoebocyte
lysate for the LAL test and annual bleeding increases the
natural mortality rate of the horseshoe crabs (Limulus
polyphemus) by 10% (comment in the German
Pharmacopoeia (DAB 10)). Efforts were therefore made to
culture/replicate amoebocytes in vitro. First attempts in
both liquid medium and a bioreactor seem promising, how-
Table VII-2: Methods for pyrogenicity testing

detection
limit know-
test (E. coli animal how/
method source specificity principle endotoxin) species number distress duration cost equipment advantages disadvantages stage of validation
Chapter VII.3: Pyrogens

rabbit pyrogen Ph. Eur. pyrogens functional c. 1 ng/ml rabbits 3 to 12 slight 1 day ++ experience in direct measurements animal test, duration, good correlation with
test V.2.1.4 in vivo (with pre- animal testing of biological effect; costs, biological fever in humans
(systemic) liminary test is based on long variability, some
(fever) test 2-3 experience products cannot be
days) evaluated

LAL test Ph. Eur. endotoxins of Gram- functional max. 2.5 Limulus natural slight 1 hour + great accuracy short duration, non-bacterial pyrogens not validated for many product
V.2.1.9 negative bacteria in vitro pg/ml poly- mortality (heart and experience sensitive, IU can be detected (false negatives); groups; routinely used to
(gel form- phemus rate inclines punction) required determined, cheap other substances can cause detect bacterial endotoxins
ation) blood by 10% false-positive or false- and for screening; individual
negative results; dilution validation for each product
often required; requires since various factors affect
animals the test

IL-6 release from Taktak et al., substances which functional max. 2.5 — — — 2-3 days ++ cell culture and lab- animal-free; similar to LAL non-pyrogenic used in laboratories world-
monocytes 1991 cause IL-6 release in vitro pg/ml oratory know-how test in sensitivity; may substances can lead to wide with good results, but
from human mono- necessary; cell line possibly detect pyrogenicity false-positive or false- cell line not freely available
cytes (eg MONO MAC 6); which cannot be detected in negative reactions; more
experience with the rabbit test (poor sensit- costly than the LAL test
ELISA ivity) or in the LAL test
(specificity), simple and
cheap

cytokine release Hartung and substances which functional ? — — — <1 day + experience with animal-free; similar to LAL standardisation?; blood method not yet established,
from whole Wendel, 1995 cause cytokine in vitro ELISA test in sensitivity; may donors required; non- but showing promise in
blood release from human possibly detect pyrogenicity specific cytokine preliminary studies; to be
monocytes which cannot be detected in release/interaction evaluated in BMBF-funded
the rabbit test (poor sensit- project at the PEI
ivity) or in the LAL test
(specificity), simple and
cheap
134
135
ever, problems might arise in trying to ensure that the cul-
ture system is totally free of pyrogens, as is required for
the LAL test [4]. One target could be the establishment of
a standardised cell line which has not lost the capacity to
agglutinate in the presence of endotoxins.
Another possibility could be the development of a very
specific endotoxin ELISA, using monoclonal antibodies
against lipopolysaccharide. Such monoclonal antibodies
are now available and are currently being tested in clinical
studies on sepsis therapy.
Apart from the desire to replace animal tests, there are
also other reasons for developing alternatives to the rabbit
pyrogenicity test and the LAL test. There are constant
reports of products found to be pyrogenic in humans,
which were positive only in the rabbit test and not in the
LAL test [5], or which were negative in both tests [6]. In
the latter case, it was shown that incubation of the test
sample with human peripheral blood monocytes resulted
in a culture supernatant that was pyrogenic in rabbits [6].
There is evidence that the pyrogenic activity of micro-
organisms results from the release of various cytokines
(IL-1α + β, TNFα, IL-6 and interferons) by peripheral
blood monocytes (endogenous pyrogens).
Attempts have been made to use cytokine release from
human peripheral blood monocytes as an endpoint for
endotoxin/pyrogenic activity [7, 8]. Most of the work on
the development of a cytokine release test has been carried
out at the NIBSC in Great Britain. A recently published
paper [9] shows IL-6 to be the most suitable for such a test
(IL-6 release test), since very small concentrations of
endotoxin can already induce the release of detectable lev-
els of IL-6. In addition, of two human monocyte cell lines
(MONO MAC 6 and THP1) which were evaluated, the
MONO MAC 6 cell line proved to be more suitable.
The preparation of blood monocytes is time-intensive
and therefore inappropriate for routine testing. This
would be avoided by the use of cell lines. IL-6 levels could
be quantified using ELISA, for which a commercial kit
could be made available as already done for other inter-
leukins. The sensitivity of the IL-6 determination is com-
parable to the sensitivity of the LAL test (c. 2.5 pg E. coli
endotoxin/ml). It is necessary to dilute the sample because
various factors could otherwise interfere with the test.
Dilution could, however, reduce test sensitivity.
Comparative studies, which so far have only been con-
ducted by the NIBSC group, gave very promising results,
since it was possible to identify batches with pyrogenic
activity using the IL-6 release test, but not by means of the
rabbit pyrogenicity test or the LAL test [9]. Therefore, it is
likely that the IL-6 release test detects pyrogens which
cannot be detected in the rabbit pyrogenicity test, due to
lack of sensitivity, nor in the LAL test, due to lack of speci-
ficity.
According to Dr Poole (NIBSC, GB), attempts to vali-
date an IL-6 release test in MONO MAC 6 cells are hin-
dered mainly because the originator of the MONO MAC 6
cell line does not permit larger-scale use of these cells
without payment. Until another, freely available cell line is
developed, it will not be possible to establish an IL-6
release test. Dr Poole himself would like to promote the
development of a suitable cell line, however, this will
depends on future financial and personnel resources (per-
sonal communication, 1994).
Hartung and Wendel [10] avoided the need to isolate
blood cells or to develop a cell line by measuring directly
cytokine release from monocytes in whole blood. Apart
from the simplicity and low costs of this model, a further
strong argument for a whole cell model is the fact that the
blood cells remain in their natural environment. After
incubation with small amounts of pyrogens, it was possible
to measure a significant increase in IL-1 levels. It is pro-
posed to evaluate this method, in collaboration with the
Chapter VII.3: Pyrogens
Paul Ehrlich Institute, as a BMBF-funded project.
The main problem of this model is the possibility of
false-positive or false-negative results resulting from com-
plex interactions. For example, non-specific immunostim-
ulants (PHA, etc) may also induce the release of cytokines
[10].
Much research is still required before the rabbit pyro-
genicity test or the LAL test can be replaced by animal-
free test methods.
LITERATURE:
1. Gommer AM, Animal Usage in Quality Control of
Pharmaceutical Products. (RIVM, Ed.), Bilthoven,
1991.
2. Dressel H, Falschpositive und falschnegative
Resultate bei der Prüfung auf pyrogene
Verunreinigungen. Pharmazie 46:582-586 (1991).
3. Requirements for snake antivenins. WHO Tech Rep
Series 463: (1971).
4. Friberg JA, Weathers PJ, and Gibson DG, Culture of
amebocytes in a nutrient mist bioreactor. In Vitro Cell
Dev Biol 28A:215-217 (1992).
5. Cradock JC, Vishnuvajjala BR, Chin TF, Hochstein
HD, and Ackerman SK, Uridine-induced hyperther-
mia in the rabbit. J Pharm Pharmacol 38:226-229
(1986).
6. Dinarello CA, O’Connor JV, Lopreste G, and Swift RL,
Human leukocytic pyrogen test for detection of pyro-
genic material in growth hormone produced by recom-
binant Escherichia coli. J Clin Microbiol 20:323-329
(1984).
7. Poole S, Thorpe R, Meager A, and Gearing AJH, Assay
of pyrogenic contamination in pharmaceuticals by
cytokine release from monocytes. Dev Biol Stand
69:121-123 (1988).
8. Hansen EW, and Christensen JD, Comparison of cul-
tured human mononuclear cells, limulus amebocyte
lysate and rabbits in the detection of pyrogens. J Clin
Pharm Ther 15:425-433 (1990).
9. Taktak YS, Selkirk S, Bristow AF, Carpenter A, Ball C,
Rafferty B, and Poole S, Assay of pyrogens by inter-
leukin-6 release from monocytic cell lines. J Pharm
Pharmacol 43:578-582 (1991).
10. Hartung T und Wendel A, Die Erfassung von
Pyrogenen in einem humanen Vollblutmodell. ALTEX
12:70-75 (1995).
SUMMARY OF RECOMMENDATIONS
With respect to pneumococcal vaccines and
immunoglobulins for human use, the pyrogenicity
test in rabbits continues to be both necessary and justifi-
able. This test ensures the absence of pyrogens in such
products for human use.
With respect to meningococcal vaccines, the pyro-
genicity test can only confirm the absence of pyrogens up
to a certain threshold. It is necessary to investigate
whether improvements in vaccine manufacturing practice
could enable the rabbit dose to increase to 0.25 µg poly-
saccharide/kg (as already stipulated in the USA). This
modification would serve to decrease the threshold value
and thus increase vaccine safety with respect to pyro-
genicity.
It should also be investigated whether the Test for
Bacterial Endotoxins (V.2.1.9) (LAL test) would be suit-
able for the testing of these vaccines, since the pyrogens
that are potentially present are mainly endotoxins deriv-
ing from meningococcae. At the very least, the monograph
should note the possibility of replacing the rabbit pyro-
Chapter VII.3: Pyrogens 136

gencity test with a validated LAL test. This would encour-

age the manufacturers to validate the LAL test for this
purpose.
A single run of the pyrogen/endotoxin test on the final
lot should suffice. The additional test on the purified poly-
saccharide included in the draft for revision of the mono-
graph for meningococcal vaccines should be deleted.
Efforts should be directed at the establishment of an
amebocyte cell line, which would enable the LAL test to be
a purely in vitro, non-animal method.
The development of in vitro cytokine-release assays in
isolated monocytes or whole blood should be encouraged,
because these tests detect all pyrogens, not just bacterial
endotoxins.