Brazilian Journal of Microbiology (2004) 35:48-53

ISSN 1517-8382

COMPARATIVE EVALUATION OF PYROGENS TESTS IN PHARMACEUTICAL PRODUCTS

Rosimar L. Silveira; Simone S. Andrade; Cleber A. Schmidt; Renata G. Casali; Sérgio L. Dalmora*

Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria,
RS, Brasil.

Submitted: January 06, 2003; Returned to authors: July 25, 2003; Approved: March 04, 2004.

ABSTRACT

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The
rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard
for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce
a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the
pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and
chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good
agreement. The use of β-glucan-reactive and non-reactive LAL reagents identified some products with false-
positive results. The interference test was carried out and the specifications validated for some new products
as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended
for the evaluation of purity and quality control of parenteral medicinal products, improving the existing
methodologies in the context of reduction and replacement in the use of animal models.

Key words: bacterial endotoxin, Limulus amoebocyte lysate, pyrogens, rabbit pyrogen test

INTRODUCTION endogenous pyrogens, i.e. pyrogens generated by the host.

Endogenous pyrogens have potent pyrogenic and inflammatory
Pharmaceutical products intended for parenteral use must activities and include interleukin 1-α (IL-1α), interleukin-1β (IL-
be free of pyrogens, which can originate from Gram-negative or 1β), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)
Gram-positive bacteria, viruses and fungi. Endotoxins (10,21).
(Lipopolysaccharides, LPS) from Gram-negative bacteria are The rabbit pyrogen test based on the intravenous injection
commonly found in parenteral pharmaceuticals and medical of a sterile solution was adopted for many years for the quality
devices and are of particular concern to the pharmaceutical control of parenteral preparations (14). Alternatives for the
industry. Endotoxins are large molecular weight complexes (~106 refinement of the test, including the comparison of rabbit
Da) associated with, and shed from, the outer membranes of responses to two E. coli endotoxin preparations, suggested
Gram-negative bacteria (8,14). Endotoxins consist of three that the temperature cut-off of 0.6ºC should be decreased to
distinct chemical regions: a lipid moiety (lipid A) which is linked 0.5ºC, as the criterion for a positive result (9,20). However, the
to a polysaccharide core that is, in turn, linked to O-antigenic rabbit pyrogen test has several drawbacks including low
side-chains (8,18). Each endotoxin presents a composition and sensitivity, absence of quantitation, unsuitability for some
a variable structure that affects its function and biological product categories and the involvement of animals (1,6).
activity; endotoxin functions include the induction of fever and The observation that the blood (haemolymph) of the
acute phase proteins, headache and severe hypotensive shock. horseshoe crab became clotted in the presence of the bacterial
There is good evidence that the fever response to various endotoxins gave rise to the Limulus amoebocyte lysate test
exogenous pyrogens (e.g. endotoxin) is mediated by (LAL), which is specific and sensitive for endotoxins from Gram-

*Corresponding author. Mailing address. Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria.
97105-900, Santa Maria, RS, Brasil. Tel/Fax: (+5555) 2208805. E-mail: [email protected]

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 Pyrogens tests in pharmaceutical products

negative bacteria (11,20). However, the test can give false- containing any detectable endotoxins. The geometric mean
negative and false-positive results and, being a test for Gram- endpoint concentration of the solutions was determined using
negative LPS, it does not detect Gram-positive exotoxins, viruses the equation M = antilog (∑e/f ), where: ∑e is the sum of the log
and fungi (7,13,16). In the LAL test, the LPS-induced reaction endpoint concentrations of the dilution series used, and f is the
can be measured using various approaches, which were adopted number of replicate test tubes. If the sensitivity of the lysate
together with the rabbit pyrogen test in the collaborative studies determined in the presence of the sample solution under test is
that established the 1st and 2nd International Standard for not less than 0.5 λ and not greater than 2 λ, the sample solution
bacterial endotoxins (15,17). However, despite the specification does not contain factors which interfere under the experimental
of the LAL test for pharmaceutical products, there remain a conditions.
number of complex preparations, such as biologicals, without
specifications or that cannot be tested by the LAL test (6). Gel-clot assay
In the context of the development of alternatives for the The assays were performed in Petri dishes (2,3,20), adjusting
refinement, replacement and reduction of biological tests, the the volumes and maintaining constant the relationship between
LAL test and the rabbit pyrogen test are also used for the them. Equal volumes of lysate and test solution or standard
validation of novel in vitro assays under investigation, based (usually 10 µL) were added to Petri dishes. The reaction mixture
on the use of cell lines, peripheral blood monocytes and whole was incubated at 37ºC for 1 h. A number of two-fold serial
blood (5,7,13,16). dilutions were tested and the gel-clot endpoint was determined
The aims of the present study were to validate the by adding 1 µL of a 0.2% new methylene blue solution, observing
specifications for the bacterial endotoxins test in parenteral the mixing (negative reaction) or distribution on the surface of
pharmaceutical products, to refine the rabbit pyrogen test, to the gel (positive reaction). The endotoxin concentration was
evaluate the responses of different LAL reagents, and to calculated by multiplying the reciprocal of the greatest dilution
correlate the results of the two methodologies, thus contributing of the test solution that gave a positive endpoint by the
to the quality control of medicines. sensitivity (to endotoxin) of the lysate preparation; the results
were expressed in EU/mL.
MATERIALS AND METHODS
Chromogenic assay
Reference standard and reagents The assay was performed in a microplate at 37 ±1ºC (2,20).
The 2nd International Standard for bacterial endotoxins, Fifty µL of the standard or samples were dispensed into the
10,000 EU/vial (WHO 94/580), was kindly donated by the appropriate microplate wells. Then, 50 µL of the Limulus
National Institute for Biological Standards and Control (NIBSC), amoebocyte lysate solution were added and the microplate was
Herts, UK. Limulus amoebocyte lysate, 0.06 EU/mL was obtained incubated for 10 min at 37 ±1ºC. One hundred µL of the substrate
from Endosafe (Charleston; SC, USA), Biowhittaker solution were pipetted and the reaction was stopped after 6
(Walkersville; MD, USA) and Cape Code (Cape Code; MA, minutes by adding 100 µL of 25% acetic acid. The absorbance
USA). New methylene blue was purchased from Sigma (St. was read at 405 nm in a microplate reader and a standard curve
Louis; MO, USA). A number of parenteral pharmaceutical plotted. The results were expressed in EU/mL.
products were used, in some cases different batches of the
same product, all within their period of validity. Other reagents RESULTS
and plasticware were purchased as sterile and pyrogen-free
and glassware was baked at 250ºC for 1 h prior to use. Rabbit pyrogen test
The dose-response curve of the 2nd International Standard
Rabbit pyrogen test for bacterial endotoxins was obtained by recording the rise in
This test was carried out according to the literature (3,20), temperature at 15 minutes intervals for three hours. The
using the New Zealand white rabbit strain. regression line was calculated and the concentrations that
produced a temperature rise of 0.5ºC and 0.6ºC were calculated
Limulus amoebocyte lysate test (LAL) as 13.81 EU/mL/kg and 18.57 EU/mL/kg, respectively (Fig. 1).
The bacterial endotoxin limits for the parenteral
pharmaceutical products without specifications were calculated Limulus amoebocyte lysate test (LAL), gel-clot
as the maximum valid dilution (MVD) (2,20). The labeled reagent LAL sensitivity of 0.06 EU/mL was
confirmed before carrying out the assays. The interfering factors
Interfering factors test test was performed for the products without specifications
The inhibition/enhancement test was performed (2,20) on (Table 1), at dilutions not exceeding the MVD spiked with the
the sample solutions at a dilution less than the MVD, not 2nd International Standard, and the samples returned positive

49
S.L. Dalmora et al.

results, thereby enabling the determination of the minimum valid The comparative evaluation of pyrogens by the qualitative
dilution of the sample free of interference and the endotoxin rabbit pyrogen test and the semi-quantitative LAL test showed
content in EU/mL. a contamination level that, in most cases, was lower than the
The comparative results of LAL reagents using the gel-clot sensitivity of the lysate used (Table 4); furthermore, there was
endpoint revealed interference with the β-glucan-reactive LAL good correlation within the two assays.
reagent for ampicillin samples and erythropoietin C, which gave
false-positive results (Table 2). DISCUSSION
The LAL gel-clot test, more widely used as a qualitative or
semi-quantitative test, was compared to the chromogenic, The rabbit pyrogen test was studied in the context of the
quantitative assay (Table 3) and produced comparable results importance of alternatives that could contribute towards its
for all samples with the exception of methylprednisolone, which refinement. A dose of 13.81 EU/mL/kg of the 2nd International
gave a lower value for the chromogenic method. Standard for bacterial endotoxins was identified as that which

Table 1. Inhibition/enhancement test of pharmaceutical products, by the Limulus amoebocyte lysate (LAL) assay, with a sensitivity
of 0.06 EU/mL.

Endotoxins limit Maximum Sample Minimum Geometric

Products
calculated valid dilution test valid dilution mean EU/mL
Ciprofloxacin 2 mg/mL 0.87 EU/mg 1:29 -- 1:16 0.06
Ketoprofen 100 mg/2 mL 3.50 EU/mg 1:2917 -- 1:32 0.03
Diclofenac 75 mg/3 mL 4.70 EU/mg 1:1958 -- 1:64 0.03
Dipyrone 500 mg/mL 0.70 EU/mg 1:5833 -- 1:10 0.03
Erythropoietin 2000 IU/vial 2.50 EU/2000 IU 1: 42 -- 1:2 0.03
recG-CSF 300 mcg/vial 2 EU/mL 1:33 -- 1:8 0.06
Calcium folinate 50 mg/5 mL 0.60 EU/mg 1:100 -- 1:1 0.04
Fluconazol 2 mg/mL 2.33 EU/mg 1:78 -- 1:2 0.06
Mesna 0.87 EU/mg 1:1458 -- 1:32 0.06
Midazolam 50 mg/10 mL 35 EU/mg 1:2917 -- 1:64 0.06
Pantoprazol 40 mg/10 mL 8.75 EU/mg 1:583 -- 1:256 0.03
Tenoxican 40 mg/mL 8.75 EU/mg 1:2917 -- 1:640 0.03

(-) Negative response.

Table 2. Results of pharmaceutical products by the LAL gel-clot test with different reagents.

Endotoxin limit
Products Reagent Limulus amoebocyte lysate (LAL)
EU/mL
Ia IIa IIIb
EU/mL EU/mL EU/mL
Ampicillin 1000 mg/5 mL A 30 153.60 – 307.20 76.80 – 153.60 <0.06
Ampicillin 1000 mg/5 mL B 30 30.72 – 61.44 15.36 – 30.72 <0.06
Insulin 100 U/mL 80 <0.06 <0.06 <0.06
Erythropoietin 2000 IU/vial A 2.50 491.50 – 983 491.50 – 983 245.75 – 491.50
Erythropoietin 4000 IU/vial C 5 3.84 –7.68 7.68 –15.36 <0.06
Gentamicin 80 mg/2 mL 68 <0.06 <0.06 <0.06
Oxacillin 500 mg/5 mL 20 <0.06 <0.06 <0.06
Heparin 5000 IU/mL 150 <0.06 <0.06 <0.06
a
β-glucans-reactive; b β-glucans non-reactive.

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 Pyrogens tests in pharmaceutical products

Table 3. Results of pharmaceutical products by the LAL gel-clot and produced a 0.5ºC rise in temperature (Fig. 1); this
Chromogenic tests. value, although variable according to the strain of
the animals used, is recommended as a criterion for
LAL test positive responses (9,17). Despite its shortcomings,
Products Results
Gel-clot Chromogenic the test is recommended by the Pharmacopoeias
EU/mL EU/mL (2,3,20), and it is important for the validation of new
in vitro assays under development (4,7,16).
Cytarabine 100 mg/5 mL <0.06 0.06 Pass
Diclofenac 75 mg/3 mL <0.06 <0.06 Pass The Limulus amoebocyte lysate test (LAL) is
Ranitidine 25 mg/mL 1.20 – 2.40 1.37 Pass recommended (2,3,20) for the quality control of
Heparin 5000 IU/mL <0.06 <0.06 Pass medicines, but the specifications need to be
Erythropoietin 4000 IU/vial B 0.48 – 0.96 0.84 Pass validated for new biological medicines, which are
Furosemide 10 mg/mL <0.06 <0.06 Pass produced mainly through recombinant DNA
Methylprednisolone 500 mg/5 mL 245.75 – 491.50 146 Fail technology (Table 1). The validation studies
Insulin 100 U/mL <0.06 <0.06 Pass indicated geometric means between ≥ 0.5λ and ≤
Vancomycin 500 mg/5 mL 2.40 – 4.80 3.20 Pass 2λ, with recovery between 50 and 200% (2,20). This
Dexamethasone 4 mg/mL <0.06 <0.06 Pass
test is important in order to eliminate false-negative
Oxacillin 500 mg/5 mL <0.06 <0.06 Pass
Gentamicin 80 mg/mL <0.06 <0.06 Pass and false-positive results (13,14,16) caused by the
Metoclopramide 10 mg/mL <0.06 <0.06 Pass interference of the active substance or components
recG-CSF 300 mcg/vial <0.06 <0.06 Pass of the final product formulation. Thus, the
Vitamin K 10 mg/mL <0.06 <0.06 Pass establishment and validation of the specifications
for the quality control of the medicinal products
tested is recommended.
The evaluation of pharmaceutical products
Table 4. Results of the parenteral pharmaceutical products by the rabbit using different LAL reagents (Table 2) showed that
pyrogen test and Limulus amoebocyte lysate test. some batches of ampicillin and human recombinant
erythropoietin, which gave positive results with
Endotoxin limit Rabbit
Products LAL test the β-glucan-reactive reagents, gave negative
EU/mL test
results when analyzed by the β-glucan non-
Amikacin 500 mg/2 mL 0.33 EU/mg <0.06 Pass reactive reagent, thus demonstrating interference.
Ampicillin 1000 mg/5 mL A 0.15 EU/mg <0.06 Pass
These observations are important for quality
Ampicillin 1000 mg/5 mL B 0.15 EU/mg <0.06 Pass
Ketoprofen 100 mg/2 mL 3.50 EU/mg* <0.06 Pass control, assisting in the selection of the LAL
Ciprofloxacin 2 mg/mL 0.87 EU/mg* <0.06 Pass reagent and the evaluation of the results obtained
Cytarabine 100 mg/5 mL 0.07 EU/mg <0.06 Pass for the samples (12,19). It should be recalled that
Dexamethasone 4 mg/mL 31.30 EU/mg <0.06 Pass this difference between the reagents could result
Diclofenac 75 mg/3 mL 4.70 EU/mg* <0.06 Pass in the incorrect rejection of batches, mainly when
Dipyrone 500 mg/mL 0.70 EU/mg* <0.06 Pass
Enoxaparin 100 mg/mL 0.01 EU/U anti-Xa <0.06 Pass the response levels are near to the maximum valid
Erythropoietin 2000 IU/vial A 2.50 EU/2000 IU* 245.75 – 491.50 Fail dilution specified for test compliance.
Erythropoietin 4000 IU/vial B 5 EU/4000 IU* 0.48 – 0.96 Pass The comparative tests of pharmaceutical
Erythropoietin 4000 IU/vial C 5 EU/4000 IU* <0.06 Pass products using the LAL test with chromogenic
recG-CSF 300 mcg/vial 2 EU/mL* <0.06 Pass and gel-clot endpoints gave similar results (Table
Fluconazol 2 mg/mL 2.33 EU/mg* <0.06 Pass
Calcium folinate 50 mg/5 mL 0.60 EU/mg* <0.06 Pass 3). These experiments are important considering
Gentamicin 80 mg/2 mL 1.7 EU/mg <0.06 Pass that the gel-clot method, being less expensive and
Mesna 0.87 EU/mg* <0.06 Pass easier to perform, has been used routinely for the
Methylprednisolone 500 mg/5 mL 0.17 EU/mg 245.75 – 491.50 Fail quality control of medicines with limits declared
Midazolam 50 mg/10 mL 35 EU/mg* <0.06 Pass as endotoxin units (2,3,20). In the present study, it
Oxacillin 500 mg/5 mL 0.20 EU/mg <0.06 Pass
was also used for comparison with the rabbit
Pantoprazol 40 mg/10 mL 8.75 EU/mg* <0.06 Pass
Ranitidine 25 mg/mL 7 EU/mg 1.20 – 2.40 Pass pyrogen test, showing good agreement (Table 4)
Saline solution 0.9% 0.50 EU/mL 48 – 96 Fail and demonstrating the importance of both
Glucose 0.5% 0.50 EU/mL 1920 – 3840 Fail methodologies for the development and validation
Tenoxican 40 mg/2 mL 8.75 EU/mg* <0.06 Pass of new pyrogens tests.
Vancomycin 500 mg/5 mL 0.33 EU/mg 2.40 – 4.80 Pass
Our results show that the rabbit pyrogen test,
*Endotoxin limits calculated – DFI/UFSM. which is being gradually replaced by the LAL test,

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S.L. Dalmora et al.

produtos farmacêuticos obtendo-se resultados concordantes.

Avaliaram-se as respostas de reagentes LAL reativos e não-
reativos a β-glicanos, observando diferenças que poderiam
reprovar amostras com base em resultados falso-positivos.
Executou-se o teste de interferências, validou-se o procedimento
e estabeleceu-se a máxima diluição válida para produtos
farmacêuticos sem especificações farmacopéicas. Os resultados
enfatizam a importância e as limitações dos ensaios preconizados
para avaliação da pureza e controle da qualidade de produtos
farmacêuticos parenterais, contribuindo para aprimorar as
metodologias existentes no contexto da redução e substituição
dos modelos animais.

Palavras-chave: endotoxinas bacterianas, lisado do amebócito

do Limulus, pirogênios, hipertermia em coelhos

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