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The

Bacterial
Endotoxins
Test
Limulus Amebocyte Lysate
Test
A Reference Guide
By Bio Solutions.
B-14, Kanika Co-Op. Society, Sarvapally Radhakrishnan Marg, Off Nagardas Road,
Near Bhuta High School, Andheri (East),Mumbai – 400069. TELEFAX: 0091-22-26824494.
Phone: 9819973583. mail@biosolutions.co.in, biosolutions@rediffmail.com
Introduction

History,
Horseshoe
crab
&
Endotoxins
INTRODUCTION
The most significant application of the Limulus Amebocyte Lysate (LAL) test is evaluation
of parenteral drugs and medical devices for endotoxins [Lipopolysaccharides (LPS)] content.
The use of LAL to detect and control the presence of pyrogenic substances in
pharmaceuticals and medical devices is a relatively recent development.

Pyrogens, endotoxins and lipopolysaccharides are words that are interchangeably used when
referring to pyrogen testing. The word pyrogen applies to any material which induces fever.
It describes an in vivo characteristic. A solution that contains a fever producing substance is
said to be pyrogenic. The word endotoxin refers to a specific pyrogen associated with the
membrane of gram negative bacteria. Endotoxins are the most prevalent pyrogen found in
aqueous solutions. Lipopolysaccharides LPS describes the biochemical structure of
endotoxin. Endotoxins contain a fatty acid portion termed Lipid A and a long chain of
repeating sugars or polysaccharides. LPS are purified endotoxins and are used as standards
for in vivo pyrogen tests or in vitro assays.

Biochemistry
The biochemical basis for the use of Limulus amebocyte lysate in the detection of
endotoxin lies entirely in the coagulation reaction inherent in Limulus blood. The ability of
Limulus blood to from a gelatinous clot was initially described by Howell, Lobe, and
Blanchard. These early investigations showed that coagulation was induced by foreign
substance and that the circulating amebocyte (= granulocytes) was involved in the reaction.
The association between endotoxin and coagulation began in 1956, when Bang reported that
infecting the horseshoe crab, Limulus polyphemus, with Vibrio, a Gram-negative bacteria,
caused a fatal intravascular coagulation. By 1964, Levin and Bang had demonstrated the
extracts of the circulating amebocytes would gel in the presence of Gram-negative bacterial
endotoxin.

Limulus blood has always held a certain fascination because of its bluish appearance.
The blue color is due to hemocyanin, a copper-based oxygen acceptor. The hemocyanin
while produced by the cyanocytes, is extra-corporeal and remains in the plasma after
centrifugation of whole blood. The amebocyte is the only cell present in Limulus blood. It
represents a homogeneous population of nucleated, disk-shaped granular cells. The
amebocyte contains all the components of the entire coagulation systems. Cell-free
hemolymph does not gel in the presence of endotoxin and does not enhance the endotoxin-
mediated reaction associated with amebocyte extracts. Lysates from washed amebocytes
have been prepared by lysis in hypotonic solutions by physical disruption using mechanical
shear, or by freeze-thaw cycles. The sensitivity of amebocyte lysate to endotoxin can be
increased by subsequent extraction with organic solvents.

A simplified description of the Limulus coagulation reaction sequence was presented


by Young et al. They fractionated amebocyte lysate into tow active fractions using gel
filtration chromatography. Fraction I, the column void, contained a heat-labile component
and represented material > 75 kDa. Fraction II represented a heat-stable component of lower
molecular weight. A mixture of both fractions was necessary for the regeneration of the
reactivity to endotoxin seen in unfractionated lysate. Incubation of endotoxin with fraction I
material prior to the addition of material from Fraction II greatly accelerated the time course
of coagulation. This supported the two-step mechanism originally proposed by Levin and
Bang. Namely, proenzyme (Fraction I) in the lysis is activated by the presence of endotoxin.
This active enzyme subsequently catalyzed the conversion of a clotting protein (Fraction II)
into an insoluble gel. The rate-limiting reaction is that of endotoxin and the proclotting
enzyme with the concentration of active clotting enzyme formed being proportional to the
initial concentration of endotoxin.

The coagulation reaction is not as simple as originally proposed. It exists as a


multicomponent cascade, which is initiated by endotoxin and terminates with gelation. The
majority of the biochemical analysis has been performed by Japanese workers using lysate
from another species of horseshoe crab. Tachypleus tridentatus, but their observations are
thought to apply to Limulus as well. The first coagulation factor, Factor C directly interacts
with endotoxin to from Factor C. An anti LPS factor, found in both Limulus and Tachypleus,
inhibits the interaction of Factor C and endotoxin. Factor C possesses enzymatic activity,
which activates Factor B. Factor B, the active form of Factor B, is then responsible for
activating the proclotting enzyme. Factors B and C have not been directly demonstrated in
Limulus polyphemus: however Nakamura and Levin have described and activator of the
proclotting enzyme and protease N in Limulus. The exact relationship of these Limulus
proteins to Factor B and C of Tachypleus is unknown it present.

The clotting protein, coagulogen, is the last component of the coagulation cascade. It
comprises almost half of total protein present in amebocyte lysate. The active clotting
enzyme is a serine protease and hydrolyzes a single peptide bond within the coagulogen to
from a shorter peptide, coagulin. Once generated, the coagulin self-associates, forming a
three-dimensional lattice structure and eventually gels.

The catalytic nature of each activated component in the coagulation cascade serves to
amplify the next step in turn. This amplification most probably results in the extreme
sensitivity of Limulus amebocyte lysate to endotoxin. All commercial lysates are capable of
detecting picogram (pg) quantities (10 -12) of endotoxin.

In addition to the endotoxin-mediated cascade which activates the proclotting


enzyme, another protein component. Factor G appears to represent an alternative coagulation
pathway. Factor G is present in both Tachypleus and Limulus and is activated by B-1,3-D-
glucans. Factor G directly activates the proclotting enzyme, which in turn causes the gelation
of coagulogen.
ACTIVATION OF ENZYMATIC PATHWAY
BY ENDOTOXINS

Endotoxins

Factor C Factor C (active)

Factor B Factor B (active)

Proclotting Enzyme Clotting Enzyme

Coagulogen Coagulin
This section explains how an MBL scientist
discovered some amazing properties in
horseshoe crab blood that enable has
revolutionized ways to detect potentially Enter Limulus and an MBL scientist named Fred
lethal bacterial toxins and spawned a Bang.
multi-million dollar industry.

Bang was studying the circulation of blood using horseshoe


crabs when he found that one of his crabs died as a result of a
Vibrio bacterial infection. The infection caused a strange
disease in which almost the entire blood volume of the crab
clotted into a semi-solid mass. Other bacteria had not
produced this sort of reaction at all. Bang began to
investigate further and found that only gram-negative
bacteria produced this reaction. Furthermore, heat-treated
bacteria (dead bacteria) continued to produce the reaction so
it wasn't a pathological disease but something different.

Bang noted that the reaction he was observing was very


similar to a well-known endotoxin reaction in mammals, the Schwartzman
reaction. Back at Johns Hopkins University, he pushed to have this new
phenomenon researched more intensively. Jack Levin, a hematologist, joined Dr.
Bangs laboratory. What they eventually found was the "fire alarm" system that
could be used to detect, with exquisite sensitivity, the fever-producing
endotoxins that are so dangerous to people.

Limulus lives in an aquatic world; the sea. The sea is almost literally awash in
gram-negative bacteria. Millions can be found in a single gram of sediment.
Bacteria that are both harmless as well as pathogenic (disease-causing).
Why is horseshoe crab blood blue? Limulus is an
arthropod, a close
relative to spiders.
In fact more closely
related to spiders
than to true crabs.
Arthropods possess
a semi-closed
circulatory system.
We mammals have
literally thousands of miles of blood vessels that
The oxygen-carrying pigment in carry blood to our tissues through vast networks
horseshoe brab blood is a protein called
hemocyanin. It is very similar to the of capillaries. Bacteria entering our bodies
hemoglobinmolecule we have in our through these capillaries are initially limited in the
blood. Hemoglobin gets it's red color area they can infect, having to fight their way into
(which makes our blood red) from the
iron molecule in the center of the
the body through these narrow channels, all the
protein. Hemocyanin contains a copper while in contact with the white blood cells that are
molecule which results in a blue color. our first line of defense.

The circulatory system of Limulus is far more open. Large sinuses exist that
allow blood direct contact with tissues. There are many wide open spaces and
bacteria entering a crack in the shell of a horseshoe crab have easy access to
large internal areas of the crab, a potentially deadly scenario. Over the course of
it's hundreds of millions of years of interacting with the bacterial swarms it
coexists with, Limulus, like us, has developed exquisitely sensitive means for
detecting the presence of bacteria through the LPS they shed into their
environment.

Limulus is cold-blooded. It can't raise it's body temperature to kill off an


infection. Nor does it have the vast confusing network of blood vessels to
contain an infection. It needs to act quickly, and sometimes even rashly. The
soldiers of the immune system in Limulus are it's single type of blood cell, the
amoebocyte. As it's name implies it is an amoeboid cell (it has motility). The cell
itself is often obloid in the blood stream and perform most of the normal
functions associated with blood cells, engulfing foreign or dead cells, transport
and storage of digested materials, repair of wound sites, etc. The cells appear
oval when seen inside a living crab and they are packed with small granules.
These granules contain clotting factors that are released outside the cell when it
detects the bacterial endotoxin. When the hemocyte is in the presence of
endotoxin it changes dramatically, so much so that it was originally believed
Limulus had several types of blood cells. The compact shape changes to an
irregular amoeboid shape with numerous cytoplasmic processes streaming in all
directions. The cell discharges the granules of coagulogen which empty the cell.

It's a very sensible system. Imagine a horseshoe crab has sustained a small
injury. Seawater comes into contact with the tissue and bacteria come into
contact with the blood and begin to enter (ie infect) the body of the crab. Small
bits of the cell wall slough off as the bacteria propel itself through the blood. A
Limulus blood cells detects this tiny fragment and responds by releasing the
contents of the granules into the surround medium. These granules contain a
clotting factor, called coagulogen. The thought is that by clotting the
immediate surroundings very quickly, the invading
bacteria can become enmeshed and therefore
stopped! Larger clots may not only stop enmeshed
bacteria but serve as a barrier to the outside
environment in the case of a severed limb or large
incision. Bang found these clots to be very stable and
prevented even Brownian motion in trapped bacteria.

The bottom line to all this is that Limulus contains an exceeding sensitive means
to detect the presence of bacterial endotoxins that can be detected by the
formation of a gel-like clot. This may not strike one as significant until one
understands the impact of endotoxins on our health and healthcare systems.

Anything that goes into your body during surgery, by injection, or for therapy,
has to be free of bacteria. If not, the recipient will get an infection. Not only
must this material be sterile (meaning no living bacteria are present) but it must
be pyrogen-free.! As was demonstrated long ago, our bodies, like the bodies of
horseshoe crabs , respond to the presence or endotoxin, not just the bacteria.
The industry of ensuring that injectable drugs, irrigation fluids, surgical tubing,
etc are free of bacterial endotoxins is a big business. In the past, companies
maintained large rabbit colonies. Rabbits, like use, are sensitive to endotoxin
and if a suspect sample of saline injected into a rabbit caused a fever then it
was contaminated. No fever, no contamination. This method was not only
expensive (it isn't cheap to keep thousands of rabbits) it is also slow. A rabbit
test might require 48 hours to obtain a result. A Limulus amoebocyte lysate
(LAL) assay can take as little as 45 minutes. A suspect sample is mixed with
reconstituted LAL and allowed to sit in a small tube. After 45 minutes the tube is
inverted and if a clot has formed it will stick to the top of the inverted tube.

LAL is a multi-million dollar business. It received FDA approval in the 1970's for
use in the testing of drugs, blood products, intravenous fluids, and disposable
pharmaceutical devices and in 1983 was registered in the U.S. Pharmacopeia.
The lysate is produced by extracting blood from the crab. This is done using a
non-lethal method where blood is taken from a large dorsal blood sinus, the
pericardium. The crabs are returned to the water within 24 hours and
completely recover.
The blood is a milky-blue color due to the copper-
pigmented hemocyanin molecule which turns blue upon
contact with oxygen the same way our blood turns red.
The blood is a mixture of liquid serum and suspended
amoebocytes. Of course, the conditions for extracting
blood must be sterile and pyrogen-free else the
amoebocytes would immediately do their job and form a
clot. If these conditions are met, the blood can be
centrifuged and the result is a separation of blood cells
from the serum. A small whitish pellet forms at the
bottom of the tube. Technicians pour off the serum and
rinse the pellet with saline. It is then resuspended and
added to the collection of amoebocytes. Eventually
pyrogen-free, distilled water is mixed with the Jack Levin
suspension of blood cells. This causes the cells to absorb demonstrates the
fresh water and balloon until they eventually burst (or removal of blood from a
"lyse" - hence "lysate"). This releases the coagulogen Limulus. This
into solution. procedure does not
permanently harm the
The resultant solution is filtered to remove cellular debris animal.
and then freeze-dried to form a white powder of the
lysate. This lysate is then packaged and sold to be reconstituted as the assay
described above.

A few decades ago, there was a bounty on Limulus as it was perceived to be a


threat to the shellfish industry. The work of Bang and the resultant market
developed by Watson and others has turned this animal into a valued resource.
Not only is this commodity renewable and sustainable but the methods are non-
lethal to the animal as well. This is a good example or basic research providing
additional leverage in the conservation of Limulus and the aquatic environments
it inhabits. Who knows where the next discoveries may lead. The line between
basic research and applied science is indistinct and quite often it is the
unexpected discoveries that are the most rewarding.
References used in this section

1. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of


Horseshoe Crabs, p.334

2. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of


Horseshoe Crabs , p.334

3. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of


Horseshoe Crabs , p.338

4. Mürer, E.H., Levin. J. and Holm, R., 1975. Isolation and studies of the granules of the
ameobocytes of Limulus polyphemus, the horseshoe crab. J. Cell Physiol., 86: 533-542

5. Armstrong, P.B. 1979, Motility of the Limulus Amebocyte, Biomedical Applications of the
Horseshoe Cran (Limulidae), 73-92.

Quigley, J.P., Corcoran, G., Armstrong, P.B., A Hemolytic Activity Secreted by the
Endotoxin-Challenged Horseshoe Crab: A Novel Immune System Operating at the Surface
of the Carapace. , Biological Bulletin, 193: 273 (October 1997)

6. Milne, Edwards, H., Historie naturelle des Crustacea., Paris, 1834-40

7. Milne, Edwards, H., L'Anatomie des Limules, 1873

8. Sargent, William., The Year of the Crab., W.W. Norton & Company 1987
THE HORSESHOE CRAB, Limulus polyphemus
A “living fossil”

Not a true crab, the horseshoe crab is actually more closely related to spiders,
scorpions, and ticks than to other crustaceans. This "living fossil" has been
around since well before the dinosaurs. Its large eyes and its unique blood have
made it uniquely valuable for biomedical research.

Spring high tides trigger a massive migration of the Atlantic genus, Limulus
polyphemus onto sheltered Delaware and New Jersey beaches. The sand can be
piled deep with spawning crabs, and females can lay as many as 88,000 eggs
each season. Migratory shorebirds rely on these abundant eggs to fuel their
journey from South America to the Arctic Circle.

However, horseshoe crabs have become valuable as bait for commercial


fishermen, who can catch tens of thousands in one day. Since 1990, the
horseshoe crab population of the Delaware Bay has been reduced by half, and
the number of shore birds has also greatly declined. Scientists, biologists, and
fishermen are now working to restore a balance that has been in place for
millions of years.

A Very Ancient History

Not a true crab, the horseshoe crab is actually more closely related to spiders,
scorpions, and ticks than to other crustaceans. This large marine arthropod gets
its common name from its shell, or carapace, which is U-shaped. Brownish-gray
in color, the carapace provides camouflage against the muddy or sandy seabed
where the horseshoe crab lives.

They haven't changed much since the Devonian era, some 360 million years
ago, well before dinosaurs. Fossil data suggests that there were never more
than twenty species of horseshoe crabs. Today only four species, grouped into
three genera, remain.

Two genera are found along the coast of Southeast Asia and nearby countries
such as Japan. The third is found along the entire Atlantic coast of the United
States and along the Gulf of Mexico as far as the Yucatan. Believed to have once
been far more widely distributed, Limulus is the only surviving species in its
genus, which indicates that its lineage is very ancient.

The scientific name for the Atlantic coast crab is Limulus polyphemus, after the
one-eyed giant of Greek myth.

Because its basic body design has changed so little over the ages, the horseshoe
crab is often referred to as a "living fossil.” They have as much genetic variation
as many other species, but are so well adapted to their ecological niche that
large-scale species radiation simply hasn't occurred. As biologist Mark Botton of
Fordham University puts it, "They seem to have hit upon a body plan that
worked a long time ago, and hasn't really changed."

A Curious Body

The body of this extraordinarily well-adapted animal has highly unusual


features. Though named after a one-eyed character, Limulus (meaning
"sidelong") actually has ten eyes: one oval lateral eye on each side of its shell,
two small ones in the center, five light-receptive organs beneath its shell, and
one in its tail. The larger eyes are compound, like those of insects, allowing the
animal to see in all directions.

Unlike true crabs, which have five pairs of legs, horseshoe crabs have four. First
come a pair of appendages called "chelicerae," then a pair of "pedipalps,"
followed by the four pairs of legs. These eight legs have pincers, and are used
for walking and for grinding and manipulating food. To find its favorite foods--
worms, mollusks, and dead fish--the horseshoe crab crawls along the bay
bottom using its pedipalps as feelers to detect prey. When it comes upon a
worm or clam, the small claws pick it up and move it to the bristly areas near
the base of the walking legs where the mouth is located. The horseshoe crab
has no jaws and uses the bristles to crush the food as it moves its legs.

Behind the horseshoe crab’s mouth and the walking legs is its abdomen, which
is connected to the rest of the carapace by a flexible joint that allows it to move
up and down. The gills, which are called book gills and are also found in spiders,
are attached to the underside of the abdomen. Made up of about one hundred
thin leaves or plates, these respiratory organs enable the animal to get oxygen
from the water, and also from the air (if the crab is on the beach) as long as the
gills are wet. Last is a long tail, or telson, which helps the animal flip over if
turned upside down. The tail has no stinger and is not poisonous. The tail will
not regenerate if broken off, so a horseshoe crab should always be picked up by
the shell. Though they may look dangerous, horseshoe crabs are completely
harmless.

Young horseshoe crabs can swim upside down. To propel themselves, they flap
their gills and move their abdomens up and down.

Breeding Crabs Once Covered Beaches Meters Deep

During the cold months, Limulus lies buried in the bottom of the Delaware Bay
and the Atlantic Ocean. In the spring, a signal synchronized in some way with
spring high tides triggers a massive migration. By late May, millions of these
ancient creatures begin crawling shoreward in the Delaware Bay, seeking out
sandy beaches that are protected from the waves.

At peak times, beaches can be piled deep with crabs and the air filled with the
sound of shells clacking against each other. Peak spawning usually coincides
with the high tides that accompany the full and new moons in May and June,
and generally takes place at night. Water temperature and weather conditions
such as heavy surf can prevent spawning.
Males come in to shore attached to females, or alone, and accumulate at the
base of the beach. Studies have shown that males use their eyes to find mates.

The males, which are smaller than the females, have special pincers at the end
of their first pair of walking legs with which they attempt to hook onto the
female's abdomen. Sometimes a chain of several crabs is formed, as one male
clasps onto another behind a female in a tenacious embrace called "amplexus."
Once a male is in tow, the female slowly makes her way to the edge of the
water, where she scoops out a nest six to eight inches (about fifteen to twenty
centimeters) deep in the sand. There she deposits thousands of BB-sized eggs,
while the male passes over and fertilizes the eggs. As many as a dozen males
may jostle around the mating pair.

This form of external fertilization makes horseshoe crabs unique among


arthropods. Several nests may be dug during a single beach trip, and females
may make additional trips on subsequent tides. Studies in Delaware found that
females laid an average of 3,650 eggs per nest, and can lay as many as 88,000
eggs per season.

If nests are numerous, female crabs may disturb earlier nests when digging new
ones. The disturbed eggs accumulate on the surface of the beach, providing a
feast for shore birds, which cannot reach the buried eggs.

By spawning at full and new moons, when tides are highest, the female protects
her eggs from being washed away. Nests are usually located close enough to the
water to stay damp, but high enough for the sand to contain adequate oxygen.
These factors, along with the temperature, determine how long it takes for the
eggs to develop. Eggs usually hatch in about a month, and out come tiny
horseshoe crabs--minus tails--about three millimeters long. They're called
trilobite larvae because they look so much like ancient, extinct trilobites.

Molting

When a horseshoe crab outgrows its shell, it has to molt: leave the old shell and
grow a new one. The old shell splits around the front edge and the crab crawls
out. The new shell, which is about 25 percent larger, soon hardens. Shells found
on the beach are typically these outgrown ones.

Horseshoe crabs molt five times in their first year of life, two or three times
during their second year, twice in the third year, and then once a year until they
mature at nine or ten years of age.

For their first two years, juveniles tend to stay in the intertidal flats near where
they hatched, then gradually move to deeper waters. Feeble swimmers, adult
horseshoe crabs walk on the ocean floor. For most of the year they crawl along
the bottom of bays and along the continental shelf. Once they reach maturity,
they will crawl back to shore each year to spawn.
They have few natural predators, though loggerhead turtles sometimes tear out
the flapping gills and pieces of the horseshoe crab have been found inside
sharks. No one really knows how long horseshoe crabs can live. A few have been
kept in aquaria for as long as fifteen years.

Useful Horseshoe Crab

Useful in many ways, the horseshoe crab has proven uniquely valuable to basic
biomedical science.

Crab Blood Could Save Your Life

The blood (hemolymph) of Limulus turns blue when exposed to oxygen, and
turns out to be perhaps the most valuable part of this ancient creature, from a
human point of view. When a crab receives a wound, cells form a clot, and kill
certain kinds of bacteria which are also harmful to humans. This process was
discovered in the early 1950s by a scientist named Frederick Bang, who was
able to separate the chemical that caused the bacteria-sensitive clots to form.
Later, the extract was named LAL (Limulus Amoebocyte Lysate), and it is used
to detect whether things that go into the human body--including injectable
drugs, needles, and heart valves--are free of dangerous endotoxin producing
bacteria. Using LAL is more accurate, simpler, and less expensive than similar
tests for bacteria.

Blood is collected from horseshoe crabs taken out of the shallow waters off the
Atlantic coast, during the summer months.

They're checked for health and then bled through a stainless steel tube. It takes
around five minutes to extract about 20 percent of the animal's blood, after
which it is returned to the ocean. About 90 percent of horseshoe crabs survive
this procedure. Approximately two hundred thousand crabs are bled each year
for LAL.

Educational Eyes

For over fifty years, the eyes of horseshoe crabs have been used in eye
research. The crab's lateral compound eye has shown scientists a great deal
about how the human eye functions. It's easy to study because both the
horseshoe crab's eye and its optic nerve (which transmits signals from the eye
to the brain) are large, and because its organization is much simpler than a
human's. This has enabled scientists to analyze the electric signals that send
visual information to the brain, and therefore to understand many underlying
principles of all visual systems--how the human eye perceives lines, borders,
and contrasts, for example. Research on horseshoe crabs has also helped
researchers understand diseases such as retinitis pigmentosa, which causes
tunnel vision and can lead to blindness. In fact, the horseshoe crab is the only
animal for which we now understand the complete neural code for vision, which
should help us understand more complex visual systems in the future.
An Age-Old Survivor

The horseshoe crab, Limulus polyphemus, has been around for a very long time.
Fossils from British Columbia (situated at the time in warm, shallow tropical
seas) show that relatives lived in North America some 520 million years ago.
The horseshoe crab went on to survive several mass extinctions, including ones
that wiped out many other marine species. The habitat of this genus of the
ancient animal is now restricted to a little peninsula on the Eastern shore of the
United States, where it has been spawning since time immemorial in sequence
with the phases of the moon. It now faces arguably the most serious crisis ever,
one that is man-made.

. . . Until Now?

Between the 1880s and the 1920s about a million horseshoe crabs were
harvested each year for use as fertilizer and in hog fodder. Chemical
supplements replaced them, but fifty years passed before the crab population
rebounded.

Horseshoe crabs have again become a valuable commodity, this time as bait.
Traditionally fishermen picked crabs off the beaches by hand, for free, and the
harvest was small. However, the domestic and international market for eels and
whelks (also called conch) has been growing rapidly. Female, egg-bearing
horseshoe crabs make particularly good bait, so the demand for Limulus has
greatly increased as well. With blue crabs scarce and other fisheries in a slump,
horseshoe crabs have been selling for between eighty-five cents and a dollar a
piece, a price that has made it even more attractive as bait to commercial
fishermen and others. Some fishermen converted their boats to trawlers, which
drag nets across the ocean floor and can catch tens of thousands of horseshoe
crabs in one day.

A solution might lie in a synthetic horseshoe crab "scent" that would replace the
need to use actual animals as bait, which scientists are working on.

Since 1985, the commercial harvest of horseshoe crabs along the Atlantic coast
has greatly expanded. From 1990 to 1994, the National Marine Fisheries Service
reported the Delaware, Maryland, and New Jersey harvest increasing from
685,648 pounds (311,000 kilograms) to 1,386,367 (628,846 kilograms), which
translates to an annual haul estimated at half a million crabs. Dr. Mark. L.
Botton, associate professor of biology at Fordham University, cites a much
higher figure: "It's now becoming clear that the number taken is approaching a
million a year." Botton, who is studying the New Jersey crab population, notes
that "on the one hand, some of the watermen argue that there's absolutely no
indication that the numbers are going down. On the other hand, some of the
environmental spokespeople are talking about the population on the verge of
extinction. Neither of these extremes is correct, and we're trying to provide the
data that allow for proper management of the population."

This species matures slowly, and doesn't begin to reproduce until around age
ten. It's also long-lived. These factors lessen the effect of a single or several
poor spawning years, so horseshoe crab populations tend to be fairly stable. It
also means that serious, long-term impacts on the population will take a decade
to become fully apparent.

Habitat Degradation

It is the absence of inlets or other breaks in the Delaware Bay shorefront that
makes it an ideal habitat for horseshoe crabs. Environmentalists and researchers
now believe that much of the recent population decline can be attributed to
severe habitat degradation on the New Jersey side of the Delaware Bay, in the
form of man-made inlets and sandbars. These artificial breaks reduce beach
sand deposits, and large areas of mud flats form behind the breached
shorefronts. Huge numbers of horseshoe crabs--three hundred thousand by
some estimates--have been observed entrapped in these back-marsh mud
areas. The adult crabs are stranded and die, and their eggs do not develop in
the mud.

Ninety percent of the horseshoe crab stock is located between Virginia and New
Jersey, and only five years ago prime beaches would have been literally piled
meters deep with spawning horseshoe crabs. Spawning surveys are not always
reliable, but the effect of habitat degradation is becoming more and more
obvious. Four separate scientific studies conducted in Delaware Bay since 1990
have estimated that the horseshoe crab population has declined by more than
50 percent. By any count it is apparent that the Delaware Bay population of
Limulus polyphemus is swiftly declining.

Fewer Horseshoe Crabs Means Less For Other Species

Like all species, the horseshoe crab is part of a web of life, the disruption of
which affects many other kinds of animals:

• Migratory shorebirds rely on abundant crab eggs to fuel their northward


journey. These include red knots, sanderlings, ruddy turnstones, and
sandpipers. Good stopover sites for these migrating birds are few, and no
substitute exists for Delaware Bay.

It could take years for the crab numbers to rebound, during which time the bird
populations will be highly vulnerable.

• The highly nutritious eggs are also eaten by raccoons, foxes, diamondback
terrapins, moles, and even mollusks.

• Fish feed on juvenile horseshoe crabs and on recent molts.

A steep decline in the horseshoe crab population will have a significant impact
on the fishing and medical industries, as well as on ecotourism. Birdwatchers
who flock to the Delaware Bay each spring to see the feeding shorebirds bring in
millions of dollars to local businesses.
What's Being Done

In 1995, a large and unusual coalition of environmental groups and businesses


called for emergency regulations on horseshoe crab fishing. In response, the
New Jersey Department of Environmental Protection restricted hand-harvesting
to two nights a week.

Trawling--dragging nets across the ocean floor--continued, however, until the


Governor of New Jersey, Christine Whitman, issued a temporary ban on all
trawling and hand-harvesting of horseshoe crabs in May 1997. The ban was
subsequently struck down, then reversed in an out-of-court settlement with the
New Jersey Audubon Society and the American Littoral Society and reinstated.

In Maryland, trawling and dredging are prohibited between April 1 and June 30
within Chesapeake Bay, coastal bays, and within one mile (1.6 kilometers) of
the Atlantic Ocean. Hand collection is also limited during this period. Virginia has
banned trawling with state waters and within three miles (4.8 kilometers) of the
coast, and requires commercial fishermen to report any crabs harvested as
bycatch.

New Jersey has dedicated $80,000 to research the population size of the local
horseshoe crab population, and also that of migrating shorebirds.

Maryland and Virginia are also taking steps to learn more about the species and
to protect it, especially during spawning. Maryland began a spawning survey in
1994, and also tags horseshoe crabs. The hope is that a balance--a sensible
harvest--can be arrived at. "While I'm concerned that the trends are downward,
I don't think it's cause for panic yet," says Dr. Botton. "What I do think is called
for is some prudent management strategy to conserve enough crabs to keep the
shorebirds fed, but yet permit the watermen to have a livelihood."

Efforts in Japan

Another genus of horseshoe crabs is native to Japan. Along with over a dozen
other aquatic species, it has been declared endangered. Land reclamation and
water pollution are the culprits. The animal used to thrive in large areas around
the Seto Inland Sea and northern Hyushu, in particular on a tidal flat along
Kasaoka Bay. Although it was designated a protected area, in 1966 the
Japanese government began a huge land reclamation project here. Completed in
1990, the project has completely wiped out the local crab population.

In 1975. the Japanese government established the Horseshoe Crab Protection


Center. When the drastic effect of the reclamation project became apparent--the
estimate is that only two thousand to four thousand Japanese horseshoe crabs
are left--the center was turned into the Kasaoka City Horseshoe Crab Museum.
In 1993, the museum launched a five-year project to raise horseshoe crabs from
eggs for release into the bay.
BACTERIAL ENDOTOXINS
Endotoxins are part of the outer cell wall of bacteria. Endotoxins are invariably
associated with Gram-negative bacteria as constituents of the outer membrane
of the cell wall. Although the term endotoxin is occasionally used to refer to
any "cell-associated" bacterial toxin, it should be reserved for the
lipopolysaccharide complex associated with the outer envelope of Gram-negative
bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria,
Haemophilus, and other leading pathogens.

The biological activity of endotoxin is associated with the lipopolysaccharide


(LPS). Toxicity is associated with the lipid component (Lipid A) and
immunogenicity is associated with the polysaccharide components. The cell
wall antigens (O antigens) of Gram-negative bacteria are components of LPS.
LPS elicits a variety of inflammatory responses in an animal. Because it activates
complement by the alternative (properdin) pathway, it is often part of the
pathology of Gram-negative bacterial infections.

The relationship of endotoxins to the bacterial cell surface is illustrated in Figure


1 below.

Figure 1. Structure of the cell surface of a Gram-negative bacterium

Gram-negative bacteria probably release minute amounts of endotoxin while


growing. For example, it is known, that small amounts of endotoxin may be
released in a soluble form, especially by young cultures. However, for the most
part, endotoxins remain associated with the cell wall until disintegration of the
bacteria. In vivo , this results from autolysis of the bacteria, external lysis
mediated by complement and lysozyme, and phagocytic digestion of bacterial
cells.
Compared to the classic exotoxins of bacteria, endotoxins are less potent and
less specific in their action, since they do not act enzymatically. Endotoxins are
heat stable (boiling for 30 minutes does not destabilize endotoxin), but certain
powerful oxidizing agents such as superoxide, peroxide and hypochlorite,
degrade them. Endotoxins, although strongly antigenic, cannot be converted to
toxoids. A comparison of the properties of bacterial endotoxins and classic
exotoxins is shown in Table 1.

Table 1. Characteristics of bacterial endotoxins and classic exotoxins.


PROPERTY ENDOTOXIN EXOTOXIN
Lipopolysaccharide(mw = Protein (mw = 50-
CHEMICAL NATURE
10kDa) 1000kDa)
RELATIONSHIP TO
Part of outer membrane Extracellular, diffusible
CELL
DENATURED BY
No Usually
BOILING
ANTIGENIC Yes Yes
FORM TOXOID No Yes
POTENCY Relatively low (>100ug) Relatively high (1 ug)
SPECIFICITY Low degree High degree
ENZYMATIC ACTIVITY No Usually
PYROGENICITY Yes Occasionally

Lipopolysaccharides participate in a number of outer membrane functions that


are essential for bacterial growth and survival, especially within the context of a
host-parasite interaction. An intact outer membrane exerts several vital
functions in Gram-negative bacteria:

1. It is a permeability barrier that is permeable only to low molecular weight,


hydrophilic molecules. In the Enterobacteriaceae, the ompF and ompC
porins exclude passage of all hydrophobic molecules and any hydrophilic
molecules greater than a molecular weight of about 700 daltons. This
prevents penetration of the bacteria by bile salts and other toxic
molecules from the GI tract. It also retains periplasmic components.
2. It impedes destruction of the bacterial cells by serum components and
phagocytic cells.

3. It plays an important role as a surface structure in the interaction of the


pathogen with its host. For example, LPS may be involved in adherence
(colonization), or resistance to phagocytosis, or antigenic shifts that
determine the course and outcome of an infection.
Chemical Nature of Endotoxin.

Most of the work on the chemical structure of endotoxin has been done with
species of Salmonella and E. coli. LPS can be extracted from whole cells by
treatment with 45% phenol at 90o. Mild hydrolysis of LPS yields Lipid A plus
polysaccharide.

Lipopolysaccharides are complex amphiphilic molecules with a mw of about


10kDa, that vary widely in chemical composition both between and among
bacterial species The general architecture of LPS is shown in Figure 2. The
general structure of Salmonella LPS is shown in Figure 3 and the complete
structure of Salmonella lipid A is illustrated in Figure 4.

Figure 2. General architecture of Lipopolysaccharide

Figure 3. General Structure of Salmonella LPS

Glc = glucose; GlcNac = N-acetyl- glucosamine; Gal = galactose; Hep =


heptose; P = phosphate; Etn = ethanolamine; R1 and R2 =
phoshoethanolamine or aminoarabinose. Ra to Re indicate incomplete forms of
LPS. The Rd2 phenotype (not shown) would have only a single heptose unit. The
Rc, Rd2, and Rd1 mutants lack the phosphate group attached to Hep.
Figure 4. Complete structure of the Lipid A Moiety of LPS of S.
typhimurium, S. minnesota, and E. coli

LPS consists of three components or regions:

• Region I. Lipid A
• Region II. Core (R) antigen
• Region III. Somatic (O) antigen or O polysaccharide

1. Lipid A is the lipid component of LPS. It contains the hydrophobic,


membrane-anchoring region of LPS. Lipid A consists of a phosphorylated
N-acetylglucosamine (NAG) dimer with 6 or 7 fatty acids (FA) attached.
Usually 6 FA are found. All FA in Lipid A are saturated. Some FA are
attached directly to the NAG dimer and others are esterified to the 3-
hydroxy fatty acids that are characteristically present. The structure of
Lipid A is highly conserved among Gram-negative bacteria. Among
Enterobacteriaceae Lipid A is virtually constant.

2. Core (R) polysaccharide is attached to the 6 position of one NAG. The


R antigen consists of a short chain of sugars. For example:KDO - Hep -
Hep - Glu - Gal - Glu - GluNAc -

Two unusual sugars are usually present, heptose and 2-keto-3-


deoxyoctonoic acid (KDO), in the core polysaccharide. KDO is unique and
invariably present in LPS and so has been an indicator in assays for LPS
(endotoxin).

With minor variations, the core polysaccharide is common to all members


of a bacterial genus (e.g. Salmonella), but it is structurally distinct in
other genera of Gram-negative bacteria. Salmonella, Shigella and
Escherichia have similar but not identical cores.

3. The O antigen or O side chain is attached to the core polysaccharide. It


consists of repeating oligosaccharide subunits made up of 3 - 5 sugars.
The individual chains vary in length ranging up to 40 repeat units. The O
polysaccharide is much longer than the core polysaccharide and it
maintains the hydrophilic domain of the LPS molecule. A major antigenic
determinant (antibody-combining site) of the Gram-negative cell wall
resides in the O polysaccharide. Great variation occurs in the composition
of the sugars in the O side chain between species and even strains of
Gram-negative bacteria. At least 20 different sugars are known to occur
and many of these sugars are characteristically unique dideoxyhexoses,
which occur in nature only in Gram-negative cell walls. Variations in sugar
content of the O polysaccharide contribute to the wide variety of antigenic
types of Salmonella and E. coli and presumably other strains of Gram-
negative species. Particular sugars in the structure, especially the
terminal ones, confer immunological specificity of the O antigen, in
addition to "smoothness" (colony morphology) of the strain. Loss of the O
specific region by mutation results in the strain becoming a "rough"
(colony morphology) or R strain.

The structure of LPS in Salmonella typhimurium and E. coli is seen in Figure 3).
The elucidation of the structure of LPS relied heavily on the availability of
mutants each blocked at a particular step in LPS synthesis. The biosynthesis of
LPS is strictly sequential. The core sugars are added sequentially to Lipid A by
successive additions, and the O side chain is added last, one preassembled
subunit at a time. The properties of mutants producing incomplete LPS
molecules suggests the nature and biological functions performed by various
parts of the LPS molecule:

1. Loss of the O antigen results in loss of virulence suggesting that this


portion is important during a host-parasite interaction. It is known that
such "rough" mutants are more susceptible to phagocytosis and serum
bactericidal reactions.
2. Loss of the more proximal parts of the core, as in "deep rough" mutants
(i.e. in Rd1, Rd2, and Re mutants) makes the strains sensitive to a range
of hydrophobic compounds, including antibiotics, detergents, bile salts
and mutagens. This area contains a large number of charged groups and
is thought to be important in maintaining the permeability properties of
the outer membrane.
3. Mutants in the assembly of Lipid A cannot be isolated except as
conditional lethal mutants and this region must therefore be essential for
cell viability. The innermost region of LPS, consisting of Lipid A and three
residues of KDO, appears to be essential for viability, presumably for
assembling the outer membrane.

LPS and virulence of Gram-negative bacteria

Both Lipid A (the toxic component of LPS) and the polysaccharide side chains
(the nontoxic but immunogenic portion of LPS) act as determinants of virulence
in Gram-negative bacteria. Virulence and the property of "smoothness"
(associated with an intact O polysaccharide) are regularly associated in many
bacterial infections. The polysaccharide chain must also be important for
virulence as shown by the fact that small changes in the sugar sequences in the
side chains of LPS, result in major changes in virulence. How are the
polysaccharide side chains involved in the expression of virulence? There are a
number of possibilities:
a. Smooth antigens could allow organisms to adhere specifically to certain
tissues, especially epithelial tissues.

b. Smooth antigens probably allow resistance to phagocytes, since rough


mutants are more readily engulfed and destroyed by phagocytes.

c. The hydrophilic O polysaccharides could act as water-solubilizing carriers for


toxic Lipid A. It is known that the exact structure of the polysaccharide can
greatly influence water binding capacity at the cell surface.

d. The O antigens could provide protection from damaging reactions with


antibody and complement. Rough strains of Gram-negative bacteria derived
from virulent strains are generally non virulent. Smooth strains have
polysaccharide "whiskers" which bear O antigens projecting from the cell
surface. The O antigens are the key targets for the action of host antibody and
complement, but when the reaction takes place at the tips of the polysaccharide
chains, a significant distance external to the general bacterial cell surface,
complement fails to have its normal lytic effect. Such bacteria are virulent
because of this resistance to immune forces of the host. If the projecting
polysaccharide chains are shortened or removed, antibody reacts with antigens
on the general bacterial surface, or very close to it, and complement can lyse
the bacteria (Thus, "rough" colonial strains are non virulent.).

Biological Properties of Endotoxins

Endotoxins are toxic to most mammals. Even though endotoxins are strong
antigens, they seldom elicit immune responses which gives full protection to the
animal against secondary challenge with the endotoxin. They cannot be
toxoided. Regardless of the bacterial source, all endotoxins produce the same
range of biological effects in the animal host.

Most of our knowledge of the biological activities of endotoxins derives not from
the study of natural disease but by challenge of experimental animals.

The injection of living or killed Gram-negative cells, or purified LPS, into


experimental animals causes a wide spectrum of nonspecific pathophysiological
reactions such as:

1. fever
2. changes in white blood cell counts
3. disseminated intravascular coagulation
4. tumor necrosis
5. hypotension
6. shock
7. lethality

Injection of large doses of endotoxin results in death in most mammals. The


sequence of events follows a regular pattern: (1) latent period; (2) physiological
distress (diarrhea, prostration, shock); (3) death. How soon death occurs varies
on the dose of the endotoxin, route of administration, and species of animal.
Animals vary in their susceptibility to endotoxin
Since Lipid A is embedded in the outer membrane of bacterial cells, it probably
only exerts its toxic effects when released from multiplying cells in a soluble
form, or when the bacteria are lysed as a result of autolysis, complement and
the membrane attack complex (MAC), ingestion and killing by phagocytes, or
killing with certain types of antibiotics. It is thought that LPS released into the
bloodstream by lysing Gram-negative bacteria is first bound by certain plasma
proteins identified as LPS-binding proteins. The LPS-binding protein complex
interacts with CD14 receptors on monocytes and macrophages and other types
of receptors on endothelial cells. In monocytes and macrophages three types of
events are triggered during their interaction with LPS (See also Handout 11
Figure 5):

1. Production of cytokines, including IL-1, IL-6, IL-8, TNFalpha and platelet-


activating factor. These in turn stimulate production of prostaglandins and
leukotrienes. These are powerful mediators of inflammation and septic
shock that accompanies endotoxin toxemia.
2. Activation of the complement cascade.
3. Activation of the coagulation cascade. During infectious disease
caused by Gram-negative bacteria, endotoxins released from, or part
of, multiplying cells have similar effects on animals and significantly
contribute to the symptoms and pathology encountered.

The range of inflammatory effects caused by LPS during Gram-negative


bacteremia or septicemia are outlined below.

1. Complement activation: C3a and C5a cause histamine release (leading to


vasodilation) and effect neutrophil chemotaxis and accumulation. The
result is inflammation.
2. Initial activation of Hageman factor (blood-clotting Factor XII), which,
in turn, can activate several humoral systems (See Handout 11 Figure 6)
resulting in

a. coagulation: a blood clotting cascade that leads to coagulation,


thrombosis, acute disseminated intravascular coagulation, which depletes
platelets and various clotting factors resulting in internal bleeding.

b. activation of the complement alternative pathway (as above, which


leads to inflammation)

c. plasmin activation which leads to fibrinolysis and hemorrhaging.

d. kinin activation releases bradykinins and other vasoactive peptides


which causes hypotension. The net effect of LPS is to induce
inflammation, intravascular coagulation, hemorrhage and shock.

3. LPS acts as a B cell mitogen stimulating the polyclonal differentiation and


multiplication of B-cells and the secretion of immunoglobulins, especially
IgG and IgM.
4. LPS activates macrophages to enhanced phagocytosis and cytotoxicity.
Macrophages are stimulated to produce and release lysosomal enzymes,
IL-1 ("endogenous pyrogen"), and tumor necrosis factor (TNFalpha), as
well as other cytokines and mediators.

These physiological activities of endotoxins are mediated mainly by the Lipid A


component of LPS. The primary structure of Lipid A has been elucidated and
Lipid A has been chemically synthesized. Its biological activity appears to
depend on a peculiar conformation that is determined by the glucosamine
disaccharide, the PO4 groups, the acyl chains, and also the KDO-containing
inner core. Thus Lipid A is a powerful biological response modifier that can
stimulate the mammalian immune system.
The BACTERIAL ENDOTOXINS TEST

Procedure:

37°C ± 1°C,
60 ± 2 min

10X75 mm tubes

Negative Test
100mL Sample Positive Test Viscous/ Cloudy,
+ 100mL LAL Gel formation No gel

Important criteria

• Temperature should be 37 ± 1˚C.


• Time 60 minutes ± 2 minutes
• Test tube size 10 X 75 mm.
Reagents
• Lyophilized LAL Reagent, Sensitivity ( λ EU/mL)
• Control Standard Endotoxin (CSE) with CoA
• Lysate Reagent Water (LRW), LAL Reagent Water (LRW), BET Water.

Accessories

• Heating Block
• Vortex Mixer
• Depyrogenated glass test tubes for assay (10X75mm)
• Depyrogenated glass test tubes for dilutions
• Micro Pipette – 10 - 100µL, 20 - 200µL, 1000µL
• Sterile Micropipette Tips
• Timer
• Depyrogenated glass pipettes - 5mL, 2mL
REGULATORY INITIATIVES

LAL Reagent Licensure


The Bureau of Biologics (now the Centre for Biologics Evaluation and Research,
CBER) elected to regulate LAL reagents as an in vitro biologic because they were a blood
product with potential to become a human diagnostic test and a replacement for the Pyrogen
Test (U.S. Public Health Service 1977; Weary 1984). LAL reagents were first marketed in
1977 as a licensed biological product, but their use was restricted at that time to in-process
testing of parenterals. CBER exerts close scrutiny over the LAL industry through its
manufacturing compliance program.

LAL Test Guideline

After seven years of review, the FDA and the LAL industry agreed on the LAL TEST
Guideline (FDA 1987). A pharmaceutical or medical device producer could switch from
rabbit to LAL testing if the LAL Test Guideline were followed. The FDA approved LAL
because of concern about the relative insensitivity and unreliability of the rabbit test. There
was less concern about the remote possibility of missing non-endotoxin pyrogens or
materials-mediated pyrogens have not materialized.

The FDA’s LAL Test Guideline was the most influential document during the rapid
growth of LAL testing in the past two decades. It introduced the concept of the Endotoxin
Limits (EL) and provided formulas for dilution (MVD, Maximum Valid Dilution) and
concentration limits (MVC, Minimum Valid Concentration). The LAL Test Guideline has
detailed sections for testing parenteral drugs and medical devices. Three types of testing are
described. First, Initial QC is a set of assays designed to qualify analysts and confirm the
label-claim sensitivity of new LAL reagents with calibrated standards. These infrequent tests
require assaying one vial of reagent with four replicates from each endotoxin standard
dilution, 2λ through 1/4λ, which brackets the expected endpoint of the LAL reagent. For the
other two tests, the same standard dilution series (SDS) is required in quadruplicate for
validation and in duplicate for routine LAL test. In other words, the test for label claim is
accomplished with the SDS

Harmonized BET
A BET became effective in January 2001 in the 2nd supplement to USP 24 and the
European Pharmacopiea, which was produced by the International Conference of
Harmonization. The LAL Test Guideline and the new BET are now very similar. The new
text includes simplified procedures and inclusion of all LAL methods. In the unlikely case of
dispute, the gel-clot method is the referee test.
THE BACTERIAL ENDOTOXINS TEST

INTRODUCTION
The Bacterial Endotoxins Test can be performed using lysate extracted form the
Amoebocytes of Limulus polyphemus (LAL), Tachypleus tridentatus, Tachypleus gigas
(TAL), Carcinoscropius rotundicauda - CAL. USP 29 recognizes LAL and TAL from
T. tridentatus spp. The IP 2000 addendum recognizes all LAL, TAL and CAL.

BACTERIAL ENDOTOXINS
The test for bacterial endotoxins is used to detect or quantify endotoxins of gram-
negative bacterial origin using amoebocyte lysate from horseshoe crab (Limulus polyphemus
or Tachypleus tridentatus). There are 3 techniques for this test: the gel-clot technique, which
is based on gel formation; the turbidimetric technique, based on the development of turbidity
after cleavage of an endogenous substrate; and the chromogenic technique, based on the
development of colour after cleavage of a synthetic peptide-chromogen complex.

The following 6 methods are described in the present chapter:

Method A. Gel-clot method: limit test


Method B. Gel-clot method: semi-quantitative test
Method C. Turbidimetric Kinetic method
Method D. Chromogenic Kinetic method
Method E. Chromogenic end-point method
Method F. Turbidimetric end-point method

Proceed by any of the 6 methods for the test. In the event of doubt or dispute, the final
decision is made based upon method A unless otherwise indicated in the monograph.

The test is carried out in a manner that avoids endotoxin contamination.

Apparatus
Depyrogenate all glassware and other heat-stable apparatus in a hot-air oven using a
validated process. A commonly used minimum time and temperature is 30 minutes at 250 ºC.
If employing plastic apparatus, such as microtitre plates and pipette tips for automatic
pipetters, use apparatus shown to be free of detectable endotoxin and of interfering effects for
the test.

NOTE: In this chapter, the term ‘tube’ includes all type of receptacles, for example microtitre
plate well.
Preparation of the standard endotoxin stock solution
The standard endotoxin stock solution is prepared from an endotoxin reference
standard that has been calibrated against the International Standard, for example endotoxin
standard Biological Reference Preparation (BRP). (EP) or US Reference Standard (USP)

Endotoxin is expressed in International Units (IU). The equivalence in IU of the


International Standard is stated by the World Health Organisation.

NOTE: One International Unit (IU) of endotoxin is equal to one Endotoxin Unit (E.U.)

Follow the specifications in the package leaflet and on the label for preparation and
storage of the standard endotoxin stock solution.

Preparation of the standard endotoxin solutions


After vigorously mixing the standard endotoxin stock solution, prepare appropriate
serial dilutions of this solution using water for bacterial endotoxin test (water for BET).

Use the solutions as soon as possible to avoid loss of activity by adsorption.

Preparation of the test solutions


Prepare the test solutions by dissolving or diluting active substances or medicinal
products using water for BET. Some substances or preparations may be more appropriately
dissolved or diluted in other aqueous solutions. If necessary, adjust the pH of the test solution
(or dilution thereof) so that the pH of the mixture of the lysate and test solution falls within
the pH range specified by the lysate manufacturer. This usually applies to a product with a
pH in the range of 6.0 to 8.0. The pH may be adjusted by the use of acid, base or a suitable
buffer, as recommended by the lysate manufacturer. Acids and bases may be prepared for
concentrates or solids with water for BET in containers free of detectable endotoxin. Buffers
must be validated to be free of detectable endotoxin and interfering factors.

Determination of the Maximum Valid Dilution

The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample
at which the endotoxin limit can be determined. Determine the MVD using the following
formulae:

Endotoxin Limit x Concentration of test solution


MVD = ---------------------------------------------------------------
λ
Endotoxin limit

The Endotoxin Limit for active substances administered parenterally, defined on the basis of
dose, is equal to:

Endotoxin Limit = K / M

K= Threshold pyrogenic dose of endotoxin per kilogram of body mass in a single


hour period. 5 EU/Kg Body weight for parenteral drugs except those
administered intrathecally. 0.2 EU/kg for intrathecal drugs.

The limit formula for radio pharmaceuticals is 175/V except for intrathecally
administered products. 14/V for intrathecal drugs.
V equals the maximum recommended dose, in mL, at the expiration date or time.

For drugs administered on a per Square Meter of Body Surface:


5 EU/ [(dose * 1.8 sq.. m.)/ 70 Kg]

M= Maximum recommended dose of product per kilogram of body mass in a single


hour period.

The endotoxin limit for active substances administered parenterally is specified in


units such as IU/mL, IU/mg. IU/Unit of biological activity, etc., in monographs.

Concentration of test solution:

- in mg/mL if the endotoxin limit is specified by mass (IU/mg),


- in Units/mL if the endotoxin limit is specified by unit of biological activity (IU/Unit),
- in mL/mL if the endotoxin limit is specified by volume (IU/mL)

λ = the labeled lysate sensitivity in the gel-clot technique (IU/mL) or the lowest point
used in the standard curve of the turbidimetric or chromogenic techniques.
GEL-CLOT TECHNIQUE (METHODS A AND B)

The gel-clot technique allows detection or quantification of endotoxins and is based


on clotting of the lysate in the presence of endotoxin. The concentration of endotoxins
required to cause the lysate to clot under standard conditions is the labeled lysate sensitivity.
To ensure both the precision and validity of the test, confirm the labeled lysate sensitivity and
perform the test for interfering factor as described under 1. Preparatory testing.

1. PREPARATORY TESTING

(i) Confirmation of the labeled lysate sensitivity

Confirm in 4 replicates the labeled sensitivity λ, expressed in IU/mL, of the lysate


solution prior to use in the test. Confirmation of the lysate sensitivity is carried out when a
new batch of lysate is used or when there is any change in the experimental conditions which
may affect the outcome of the test.

Prepare standard solutions of at least 4 concentrations equivalent to 2λ,λ , 0.5λ and


0.25λ by diluting the standard endotoxin stock solution with water for BET.

Mix a volume of the lysate solution with an equal volume of 1 of the standard
solutions (such as 0.1 mL aliquots) in each tube. When single test vials or ampoules
containing lyophilized lysate are employed, add solutions directly to the vial or ampoule.
Incubate the reaction mixture for a constant period according to the recommendations of the
lysate manufacturer (usually at 37 +1 ºC for 60 + 2 min), avoiding vibration. Test the
integrity of the gel: for tubes, take each tube in turn directly from the incubator and invert it
through approximately 180º in one smooth motion. If a firm gel has formed that remain in
place upon inversion, record the result as positive. A result is negative if an intact gel is not
formed.

The test is not valid unless the lowest concentration of the standard solutions shows a
negative result in all replicate tests.

The end-point is the last positive result in the series of decreasing concentrations of
endotoxin. Calculate the mean value of the logarithms of the end-point concentrations and
then the antilogarithm of the mean value using the following expression:

Geometric Mean end-point concentration = antilog ∑ e / ƒ

∑ e = sum of the log end-point concentrations of the dilution series used,

ƒ = number of replicates.

The Geometric Mean end-point concentration is the measured sensitivity of the


lysate solution (IU/mL). If this is not less than 0.5λ and more than 2λ, the labeled
sensitivity is confirmed and is used in the tests performed with this lysate.
Procedure:
I. Preparation of Endotoxin dilutions from Control Standard Endotoxin (CSE) for
control curve & for positive product control:

Refer to Certificate of Analysis (COA) for matched CSE vial.

Steps:

1. Reconstitute the Lyophilized material with required quantity of LRW (mentioned in the
CoA) to obtain mentioned concentration of EU/mL with the help of pyrogen free
pipette.

2. Dilute CSE to 1 EU/mL using LRW and Vortex for 1 min

3. From 1 EU/mL prepare CSE of 4λ, 2λ,λ, 0.5λ,0.25λ dilutions. Where λ = Labelled
lysate sensitivity. e.g. λ = 0.125 EU/mL .Vortex each dilution for at least 1 min.

Control Standard Endotoxin Dilution Scheme: Concentration = __ EU/mL as per CoA

LAL Reagent Control Standard


Tube No. CSE Concentration
Water Endotoxin
I 8λ 1 EU/mL __ mL ___ mL of __ EU/mL
II 4λ 0.5 EU/mL 2 mL 2 mL of 1 EU/mL
III 2λ 0.25 EU/mL 2 mL 2 mL of 0.5 EU/mL
IV λ 0.125 EU/mL 2 mL 2 mL of 0.25 EU/mL
V ½ λ 0.0625 EU/mL 2 mL 2 mL of 0.125 EU/mL
VI ¼ λ 0.03125 EU/mL 2 mL 2 mL of 0.0625 EU/mL
Dilution Scheme

Labeled Claim sensitivity of Lysate (λ) is 0.125EU/mL. Following will be the dilutions one
needs to prepare for Confirmation of the labeled lysate sensitivity. From the Control Standard
Endotoxin vial prepare 1 EU/mL.

1EU/mL 0.5EU/mL 0.25EU/mL 0.125EU/mL 0.0625EU/mL 0.03125EU/mL

LAL Test procedure:

Control curve

Test carried out in clean depyrogenated 10 x 75 mm tubes only. Each dilution to be tested
in quadruplicate.

Total
LRW CSE Dilution in
Tube No. Dilution Volume
(µL) µL µL
1,2 Negative 100 - 100
Control
3,4,5,6 2λ - 100 ( of 2 λ ) 100
7,8,9,10 λ - 100 ( of λ ) 100
11,12,13,14 0.5 λ - 100 ( of 0.5 λ ) 100
15,16,17,18 0.25 λ - 100 ( of 0.25λ ) 100

To each tube, add 100 µl of LAL reagent. Mix gently & incubate in Heating Block at
37ºC ± 1ºC for 60±2 min. After incubation, remove the tubes gently from the Heating Block
& slowly invert through 180 º & scroll the result.

+ ve Æ gel that holds its integrity when tube is inverted 180º.

- ve Æ clear or viscous liquid which flows when tube is inverted.


Interpretation :

Correct or acceptable results:

I II III
NEG Control –– –– ––
2λ ++++ ++++ ++++
λ ++++ –––– ++++
0.5 λ –––– –––– ++++
0.25λ –––– –––– ––––
End point at λ 2λ 1/2λ
Ideal Within + one twofold dilution

Void or incorrect results:

IV V VI
NEG Control –– –– ++
2λ –––– ++++ ++++
λ –––– ++++ ++++
0.5 λ –––– ++++ ++++
0.25λ –––– ++++ ++++
CSE Storage (?) Dilutions (?) Accessories
Dilutions (?) LRW (?)
Geometric Mean Calculation- Example

Tube Blank 2λ λ 0.5 λ 0.25λ End point


1 – + + – – 0.125 EU/mL
2 – + + + – 0.0625 EU/mL
3 – + + – – 0.125 EU/mL
4 – + + + – 0.0625 EU/mL

Formula:

GM end point Concentration = antilog (∑e/ƒ)


Where,
∑e = Sum of log of Endpoint Concentrations
ƒ = Number of Replicates

Calculation:

GM = log (0.125) + log (0.0625) + log (0.125) + log (0.0625)


4
= Anti[(-0.9030) + (-1.2041) + (-0.9030) + (-1.2041)]
4

= -4.214
4
= Antilog [(-1.0536)]

= 0.0883 EU/mL

Label Sensitivity is confirmed if GM endpoint is between 2λ and ½ λ .Since


0.883EU/mL is in between 0.25EU/mL and 0.06EU/mL, Label Sensitivity is confirmed.
Inhibition / Enhancement Test and Product Validation

(ii) TEST FOR INTERFERING FACTORS

Prepare solutions A, B, C and D as shown in Table 2.6.14-1, and use the test solutions
at a dilution less than the MVD, not containing and detectable endotoxins, operating as
described under 1. Preparatory testing, (i) Confirmation of the labeled lysate sensitivity.

Solution A = solution of the preparation being examined that is free of detectable


endotoxins.
Solution B = test for interference.
Solution C = control of the labelled lysate sensitivity.
Solution D = negative control (water for BET).

The geometric mean end-point concentrations of solutions B and C are determined


using the expression described in 1.Preparatory testing, (i) Confirmation of the labelled lysate
sensitivity.

The test for interfering factors is repeated when any changes are made to the
experimental conditions that are likely to influence the result of the test.

The test is not valid unless all replicates of solutions A and D show no reaction and
the result of solution C confirms the labelled lysate sensitivity.

If the sensitivity of the lysate determined with solution B is not less than 0.5λ and not
greater than 2λ, the test solution does not contain interfering factors under the experimental
conditions used. Otherwise, the solution interferes with the test.

If the preparation being examined interferes with the test at a dilution less than the
MVD, repeat the test for interfering factors using a greater dilution, not exceeding the MVD.
The use of a more sensitive lysate permits a greater dilution of the preparation being
examined and this may contribute to the elimination of interference.

Interference may be overcome by suitable treatment, such as filtration, neutralization,


dialysis or heat treatment. To establish that the treatment chosen effectively eliminates
interfering factor using the preparation being examined to which the standard endotoxin has
been added and which has then been submitted to the chosen treatment.
INHIBITION / ENHANCEMENT TEST

Solution Endotoxin Conc. / Dilution Initial No. of


Diluent
Solution to which Factor Endotoxin Replicates
Endotoxin is added Conc.
A None/sample solution --- --- --- 4
B 2λ / sample solution Sample 1 2λ 4
solution
2 1λ 4
4 0.5λ 4
8 0.25λ 4
C 2λ / water for BET LAL 1 2λ 2
Reagent
Water
2 1λ 2
4 0.5λ 2
8 0.25λ 2
D None / LRW --- --- --- 2

Solution A: a sample solution of the preparation under test that is free of detectable
endotoxins.
Solution B: test for interference.
Solution C: control for labeled LAL Reagent sensitivity.
Solution D: negative control of LAL Reagent Water
SCREENING FOR INTERFERENCE:
Step I

1. Calculate the MVD for your product.


2. Prepare 1/32 MVD, 1/16 MVD, ⅛ MVD, ¼ MVD, ½ MVD, dilutions of your
product.
3. Test at 1/16 MVD, ⅛ MVD, ¼ MVD, ½ MVD, MVD.
4. The lowest dilution / highest concentration at which PPC is positive is the
Non Interfering Dilution. (NID) and None Interfering Conc. (NIC).
5. Choose any dilution / conc. between NID / NIC to MVD and carry out
product validation.

Test as follows – in duplicate

LRW Sample CSE LAL


Tube
(µL) (µL) (µL) (µL)
NPC ( 1/16MVD ) 50 1
50 ( /32 MVD) – 100
PPC – 50 ( 1/32 MVD) 50(4λ) 100
NPC ( ⅛ MVD ) 50 50 ( 1/16 MVD) – 100
PPC – 50 ( 1/16 MVD) 50(4λ) 100
NPC ( ¼ MVD ) 50 50 ( ⅛ MVD ) – 100
PPC – 50 ( ⅛ MVD ) 50(4λ) 100
NPC ( ½ MVD ) 50 50 ( ¼ MVD ) – 100
PPC – 50 ( ¼ MVD ) 50(4λ) 100
NPC ( MVD ) 50 50 ( ½ MVD ) – 100
PPC – 50 ( ½ MVD ) 50(4λ) 100

The Least DILUTION / Highest CONC. of product for which PPC is Positive
and NPC is Negative is the Non Interfering Dilution (NID)/ Non Interfering
Concentration (NIC).
EXAMPLE
Drug : Cefotaxime Sodium Sterile 1000mg/4mL
Endotoxin Limit : NMT 0.2 EU/mg
Lysate sensitivity : 0.125 EU/mL
MVD = Potency x E.L
λ
= 250 mg/ mL X 0.2 EU/mg
0.125EU/mL
MVD = 400
If you get the following results –

Dilutions /16 MVD


1
⅛ MVD ¼ MVD ½ MVD MVD
(1:25) (1:50) (1:100) (1:200) (1:400)
Conc. in mg/mL 10 5 2.5 1.25 0.625
NPC –– –– –– –– ––
PPC –– ++ ++ ++ ++

Then the Non-Interfering Dilution is ⅛ MVD=1:50,


Non-Interfering Concentration is 5 mg/mL
PRODUCT VALIDATION:

Since the Non- interfering dilution is ⅛ MVD (1:50). Product can be validated
between ¼ MVD and MVD.

Validation at ½ MVD (1:200). Lysate sensitivity (λ) is 0.125EU/mL.

Sample
Test Sample LRW CSE LAL
(1:100)
Negative Water Control 100 µL − − 100 µL

2 λ(0.25EU/mL) − − 100 µL of 2 λ 100 µL


λ(0.125EU/mL) − − 100 µL of λ 100 µL
0.5 λ (0.06EU/mL) − − 100 µL of 0.5 λ 100 µL
0.25 λ (0.03EU/mL) − − 100 µL of 0.25 λ 100 µL

Negative Product Control 50 µL 50 µL − 100 µL

Positive product control 2λ − 50 µL 50 µL of 4λ 100 µL

Positive product control λ − 50 µL 50 µL of 2λ 100 µL

Positive product control 0.5 λ − 50 µL 50 µL of λ 100 µL

Positive product control 0.25 λ − 50 µL 50 µL of 0.5 λ 100 µL


RESULTS:

Endotoxin Concentration Negative


Test Negative Test Endpt.
EU/mL. Product
Control EU/mL
2λ λ 0.5 λ 0.25 λ Control
+ + – – – 0.125
CSE
+ + – – – 0.125
Water
+ + – – 0.125
Control
+ + – – 0.125
+ + + – – 0.0625
Positive
+ + + – – 0.0625
Product
+ + – – – 0.125
Control
+ + – – – 0.125

GEOMETRIC MEAN CALCULATION

GM end point Concentration = antilog ( ∑ e / ƒ )

GM CSE in Water = log (0.125) + log (0.125) + log (0.125) + log( 0.125)
4
= Anti[(-0.9030) + (-0.9030) +(-0.9030) + (-0.9030)]
4
= Antilog [(-0.9030)]

= 0.125 EU/mL

GM CSE in Product = log (0.0625) + log (0.0625) + log (0.125) + log( 0.125)
4
= Anti[(-1.2041)+ (-1.2041)]+(-0.9030) + (-9030)]
4
= Antilog [(-1.0536)]

= 0.0883 EU/mL

Since Geometric mean of CSE in water and CSE in product is between 2λ (0.25EU/mL) and
½ λ (0.0625EU/mL) product is validated at ½ MVD (1:200).
2. LIMIT TEST (METHOD A)

(i) PROCEDURE

Prepare solution A, B, C and D as shown in Table 2.6.14 and perform the test on
these solutions following the procedure described under 1. Preparatory testing,
(i) Confirmation of the labelled lysate sensitivity.

Prepare solution A and solution B (positive product control) using a dilution not
greater than the MVD and treatments as described in 1. Preparatory testing, (ii) Test for
interfering factors. Solutions B and C (positive controls) contain the standard endotoxin at a
concentration corresponding to twice the labelled lysate sensitivity. Solution D (negative
control) consists of water for BET.

Endotoxin Conc/ Solution to


Solution No. of replicates
which Endotoxin is added
None / Diluted sample
A 2
solution
2λ / diluted sample solution
B 2
2λ / LAL Reagent Water
C 2
None / LAL Reagent Water
D 2

(ii) Interpretation

The test is not valid unless both replicates of the 2 positive control solutions B and C
are positive and those of the negative control solution D are negative.

The preparation being examined complies with the test when a negative result is
found for both replicates of solution A.

When a positive result is found for both replicates of solution A:

- if the preparation being examined is diluted to the MVD, it does not comply with the
test,
- if the preparation being examined is diluted to a dilution less than the MVD, the test
is repeated at a dilution not greater than the MVD.

Repeat the test if a positive result is found for one replicate of solution A and a negative
result is found for the other. The preparation being examined complies with the test if a
negative result is found for both replicates of solution A in the repeat test.
Example :

Drug : Cefotaxime Sodium Sterile 1000 mg/ 4mL


Endotoxin Limit : NMT 0.2 EU/mg
Lysate sensitivity : 0.125 EU/mL

MVD = Potency x E.L


λ
= 250mg/ mL X 0.2 EU/mg
0.125 EU/mL

MVD = 400

Limit Test (Routine Test) Using 50-50 Method.


If you want to test sample at MVD then prepare sample at ½ MVD and CSE concentration
4λ. In above case MVD is 1:400 hence sample is prepared at ½ MVD (1:200) and CSE
concentration at 4λ i e. 0.5EU/mL (λ = 0.125EU/mL).

Sample CSE
LRW LAL
(1:200) (4λ)
NWC 100 µL − − 100 µL
PWC 50 µL − 50 µL 100 µL
NPC 50 µL 50 µL − 100 µL
PPC − 50 µL 50 µL 100 µL

Limit Test (Routine Test) Using ‘HOT SPIKE’ Method.

In this method sample is directly prepared and tested at same dilution and CSE dilution is
prepared at 20λ = 20 x 0.125EU/mL = 2.5EU/mL

Sample CSE
LRW LAL
(1:400) (20λ)
NWC 100 µL − − 100 µL
PWC 100 µL − 10 µL 100 µL
NPC − 100 µL − 100 µL
PPC − 100 µL 10 µL 100 µL
Expected Results
NWC ––
PWC ++

Sample results
NPC –– ++ –– –+
PPC ++ ++ –– ++
Pass Fail Interference Repeat

Interference related problems are addressed separately later in this manual. Please refer to the
technical literature section also for a publication on Resolving LAL Test interferences and
The Impact of Non- Endotoxin LAL Reactive Material on LAL analyses.
3. SEMI – QUANTITATIVE TEST (METHOD B)
(i) Procedure
The test quantifies bacterial endotoxins in the test solution by titration to an end-point.
Prepare solution A, B, C and D as shown Table 2.6.14.-3, and test these solutions according to the
procedure described under 1. Preparatory testing, (i) confirmation of the labelled lysate sensitivity.

Solution Endotoxin Diluent Dilution Initial Number of


concentration / Factor Endotoxin replicates
solution to which concentration
Endotoxin is
added
A None / Test Water for BET 1 – 2
solutions
2 – 2
4 – 2
8 – 2
B 2λ / Test solution 1 2
C 2λ / Water for 1 2
BET
2 2
4 2
8 2
D None/ Water for – 2
BET

(ii) Calculation and interpretation


The test is not valid unless the following 3 conditions are met:

(a) both replicates of solution D (negative control) are negative,


(b) both replicates of solution B (positive product control) are positive,
(c) the geometric mean end-point concentration of solution C is the range of 0.5λ,
to 2λ,

To determine the endotoxin concentration of solution A, calculate the end-point concentration


for each replicate series of dilutions by multiplying each end-point dilution factor by λ.
The endotoxin concentration in the test solution is the geometric mean end-point
concentration of the replicates (see the expression given under 1. Preparatory testing,
(i) Confirmation of the labeled Lysate sensitivity). If the test is conducted with a diluted test solution,
calculate the concentration of endotoxin in the original solution by multiplying the result by the
dilution factor.

If none of the dilutions of the test solution is positive in a valid test, record the endotoxin
concentration as less than λ (or if a diluted sample was tested as less than λ x the lowest dilution
factor of the sample). If all dilutions are positive, the endotoxin concentration is recorded as equal to
or greater than the greatest dilution factor multiplied by λ .

The preparation meets the requirements of the test if the endotoxin concentration is less than
that specified in the individual monograph.
Example

Drug : Cefotaxime Sodium Sterile 1000 mg/ 4mL


Endotoxin Limit : NMT 0.2 EU/mg
Lysate sensitivity : 0.125 EU/mL

MVD = Potency x E.L


λ
= 250mg/ mL X 0.2 EU/mg
0.125 EU/mL

MVD = 400

1
/16MVD ⅛MVD ¼ MVD ½ MVD MVD
(1:25) (1:50) (1:100) (1:200) (1:400)
NPC –– ++ –– –– ––
PPC –– ++ ++ ++ ++

λ is Lysate sensitivity = 0.125EU/mL

Then Endotoxin content is = Dilution factor of maximum dilution at which


NPC is positive x λ

= 50 x 0.125EU/mL

= 6.25 EU/mL
250 mg/mL

= 0.025 EU/mg
REFERENCE STANDARD ENDOTOXIN

USP Endotoxin Reference Standard (RSE)


The variability of early endotoxin standard led to recognition that a reference standard
endotoxin (RSE) was needed to confirm the sensitivity of LAL reagents. The search for a
standard was complicated because endotoxin potency varied with method of purification,
bacterial origin, and formulation. Rudbach and associates (1976) were engaged by the USP
and the FDA to resolve this issue. The objective was selection and preparation of a stable
reference endotoxin that was free of biologically active proteins, had average endotoxic
activity, and could be chemically characterized. A 30g batch of reference LPS that met these
criteria was prepared from Escherichia coli 0113:H10:K(-) (Rudbach, Akiya and Elin 1976).
The FDA specified a means for standardizing other purified endotoxins against this reference
standard. An international World Health Organization (WHO) reference endotoxin was
prepared under the direction of people to harmonize the ELs so that 1 international unit (IU)
of endotoxin equal 1 endotoxin unit (EU) (Poole, Dawson and Gaines Das 1997). The FDA
has a sublot of the WHO reference endotoxin, known in the FDA as EC-6, which LAL
suppliers use for sensitivity assays (determination of lambda,λ) of their LAL reagents. USP
Endotoxin Lot G contains 10000 Units (EU) per vial. This is commercially available

Functions of Reference Standard Endotoxin


The RSE has three critical functions. Through its licensing process, the FDA requires
all LAL producers to determine the sensitivity of LAL by an assay of a two-fold dilution
series of RSE made from 1 EU/mL. Second, the RSE is the primary standard for
standardizing the potency of control standard endotoxin (CSE) with a specific lot of LAL, as
required by the LAL-Test Guideline (FDA 1987). Finally, RSE is used when a referee test is
needed to resolve a disagreement over test results.

RSE preparation to determine the sensitivity of LAL:


Method
Tubes RSE Concentrations. End Geometric G.M. = antilog ( Σ e/ f)
Replicates EU/mL point Mean
0.25 0.125 0.0625 0.03125 G.M. = antilog ( Σ (-0.9030)
1 + + – – 0.125 -0.9030 + (-0.9030) + (-0.9030) +
2 + + – – 0.125 -0.9030 ( -0.9030) /4
3 + + – – 0.125 -0.9030 G.M. = antilog ( Σ -3.612/ 4)
G.M. = antilog ( -0.9030)
4 + + – – 0.125 -0.9030
= 0.125 EU/mL

Hence Label Claim Sensitivity of this batch of lysate is 0.125 EU/mL.

Sensitivity is denoted by λ. ( Lambda )


CONTROL STANDARD ENDOTOXIN (CSE)

Purpose of Control Standard Endotoxin


Because the RSE is expensive and exhaustible, LAL reagent suppliers produce CSE for
routine tests. The sole purpose of CSE is to serve as a surrogate for RSE during routine endotoxin
testing. The roles of CSE are to (1) confirm LAL test validity by recovery of positive product controls
(PPC) and (2) verify control of test parameters (e.g., reagents, accessories, analyst proficiency)
through recovery of label-claim sensitivity (λ) during routine BET assays. In the Pharmacopoeial
Forum (USP 2000), the USP stated its recognition of equivalence for RSE and CSE preparations .The
USP does not mention the use of CSE and only refers to the RSE. The Preamble, Bacterial
Endotoxins Test, Pharmacopoeial Forum 26: 218 – 19, 2000 allows “The use of in-house standards
shown to be equivalent to USP Reference Standards is permitted under the requirements for
alternative methods in the General Notices.”

CSE is the in-house standard. The Standard Testing Procedure and Standard Operating Procedure is
in compliance if it requires. 1. Acceptance of CoA from the CSE vendor
2. Completion of a label claim verification.

If need for USP reference to use CSE then one can refer to Pg. 1696 of USP 23 which gives the most
recent description of RSE / CSE ratio determination for Gel Clot Methods.

USP requires ALL CSE to have a ratio between 2 EU/ng and 50 EU/ng.

Every Manufacturer of LAL provides a Control Standard Endotoxin with a certificate of analysis that
contains the RSE to CSE ratio for specific LAL lot. All RSE to CSE ratios are Lot specific. Hence
before using LAL one should check whether they are using the correct combination of LAL and CSE
which is mentioned on the CoA.
Preparation of CoA for RSE/CSE:

USP requires ALL Control Standard Endotoxins (CSE) to have a ratio between
2 EU/ng and 50 EU/ng.

Variations in RSE/ CSE ratio is due to the INHERENT + 2 FOLD VARIATION IN


THE LAL SENSITIVITY.

Hence with different batches of LAL of same sensitivity with same lot of CSE you will have
different RSE /CSE ratios. We can get different results which will result in different
RSE/CSE ratios. Some examples of the results and their calculations are listed below

Each CSE contains 100ng/vial. Each is assayed as follows. From each vial 0.025, 0.0125,
0.00625, 0.003125ng/mL is prepared and 0.1mL LAL is added to each tube. Assay is
performed in quadruplicate.

RSE to CSE ratio

Tubes RSE Concs. End Geometric G.M. = antilog ( Σ e/ f)


EU/mL point Mean
0.25 0.125 0.0625 0.03125 G.M. = antilog ( Σ -0.9030 +-
1 + + – – 0.125 -0.9030 0.9030 +-0.9030 + -0.9030)
2 + + – – 0.125 -0.9030 /4
3 + + – – 0.125 -0.9030 G.M. = antilog ( Σ -3.612/ 4)
4 + + – – 0.125 -0.9030 G.M. = antilog ( -0.9030)
= 0.125 EU/mL

CSE contains 100ng /vial and is diluted to the below mentioned concentrations.

Tubes CSE Concs. End Geometric G.M. = antilog ( Σ e/ f)


ng/mL point Mean
0.025 0.0125 0.00625 0.003125 G.M. = antilog ( Σ -1.9030
1 + + – – 0.0125 -1.9030 + -1.9030 + -1.9030 + -
2 + + – – 0.0125 -1.9030 1.9030) /4
3 + + – – 0.0125 -1.9030 G.M. = antilog ( Σ -7.612/
4 + + – – 0.0125 -1.9030 4)
G.M. = antilog ( -0.9030)
= 0.0125 ng/mL

RSE/CSE Ratio = Geometric Mean End point of RSE concentration


Geometric Mean End point of CSE concentration

= 0.125 EU/mL
0.0125 ng/mL

= 10 EU/ng
Result 2
CSE Concentrations

Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point
1 + + + − 0.00625
2 + + − − 0.0125
3 + + − − 0.0125
4 + + − − 0.0125

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points


f = No.of replicates.

G.M. = Antilog [log (0.00625) + log (0.0125) + log (0.0125) + log(0.0125)]


4
= Antilog [(-2.2041) + (-1.9030) + (- 1.9030) + (-1.9030) ]
4
= Antilog [- 7.91312]
4
= Antilog [- 1.978]

= 0.0105 ng/mL.

RSE/CSE = 0.125 EU/mL = 11.9 = 12 EU/ng.


0.0105ng/mL

Result 3 CSE Concentrations


Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point
1 + + + − 0.00625
2 + + + − 0.00625
3 + + − − 0.0125
4 + + − − 0.0125

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points


f = No.of replicates.

G.M. = log [ log (0.00625) + log (0.00625) + log (0.0125) + log(0.0125) ]


4
= Antilog [ (-2.2041) + (-2.2041) + (- 1.9030) + (-1.9030) ]
4
= Antilog [ - 8.2142 ]
4
= Antilog [ - 2.05355 ]

= 0.0088 ng/mL.

RSE/CSE = 0.125 EU/mL = 15 EU/ng.


0.008 ng/mL
Result 4
CSE Concentrations
Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point
1 + + + − 0.00625
2 + + + − 0.00625
3 + + + − 0.00625
4 + + − − 0.0125

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points


f = No.of replicates.
G.M. = Antilog [ log (0.00625) + log (0.00625) + log (0.00625) + log (0.0125) ]
4
= Antilog [ (-2.2041) + (-2.2041) + (-2.2041) + (-1.9030) ]
4
= Antilog [ - 8.515 ]
4
= Antilog [ - 2.128 ]

= 0.0074 ng/mL.

RSE/CSE = 0.125 EU/mL = 17 EU/ng.


0.007 ng/mL

Result 5
CSE Concentrations
Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point
1 + − − − 0.025
2 + − − − 0.025
3 + − − − 0.025
4 + − − − 0.025

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points


f = No.of replicates.
G.M. = Antilog [log (0.025) +log (0.025)+ log (0.025+ log (0.025)
4
= Antilog [(-1.6020) + (-1.6020) + (-1.6020) + (-1.6020) ]
4
= Antilog [- 6.4080 ]
4
= Antilog [- 1.6020]

= 0.025 ng/mL.

RSE/CSE = 0.125 EU/mL = 5EU/ng.


0.025 ng/mL

Hence the CoA contains one of these ratios as calculated above.


Reconstitution of LAL Reagent LIMUSATE using BET WATER

Reconstitute LAL after preparing and adding sample, CSE to the test tubes.

Steps :

1. Remove aluminum seal of the Lysate vial without opening rubber stopper.

2. Carefully remove the stopper. Keep the stopper in a clean surface without touching the
inner portion of stopper.

3. Rehydrate the lyophilized powder using required quantity of LRW with the help of
depyrogenated pipette.
With 1 mL LRW (for Limusate-1mL 10 test vial)
With 5 mL LRW (for Limusate-5 mL 50 test vial)

4. Stopper or use parafilm to seal the reconstituted vial. Do not shake. Mix gently without
formation of any air bubbles. AVOID TOUCHING OF LYSATE TO THE STOPPER.
DO NOT VORTEX.

Storage:

Store the reconstituted LAL reagent vial at 2 to 8° C for 8 hours. Reconstituted Lysate should
be preferably stored in the freezer below 0°C when it is to be used intermittently.
Freeze it & use on the next day. If not, can be frozen below - 20°C.up to 2 weeks, to be
frozen & thawed only once.
TESTING OF WATER FOR INJECTION USING HOT SPIKE
METHOD AND 50-50 METHOD.

Endotoxin Limit : 0.25 EU/mL


LAL sensitivity : 0.125
MVD = 2

Testing at 0.125EU/mL using Hot spike.

LRW WFI CSE LAL


(20λ)
NWC 100 µL − − 100 µL
PWC 100 µL − 10 µL 100 µL
NPC − 100 µL − 100 µL
PPC − 100 µL 10 µL 100 µL

Testing at 0.25EU/mL using 50-50 method

LRW WFI CSE LAL


(4λ)
NWC 100 µL − − 100 µL
PWC 100 µL − 50 µL 100 µL
NPC 50 µL 50 µL − 100 µL
PPC − 50 µL 50 µL 100 µL
QUANTIFYING ENDOTOXIN IN WATER

TESTING OF WATER FOR INJECTION FOR DIFFERENT LEVELS OF


ENDOTOXIN USING HOT SPIKE METHOD USING LAL λ =0.03 EU/mL.

Test directly at 0.03 EU/mL


LRW WFI CSE (20λ) LAL
NWC 100 µL − − 100 µL
PWC 100 µL − 10 µL 100 µL
NPC − 100 µL − 100 µL
PPC − 100 µL 10 µL 100 µL

If NPC −ve, PPC +ve report WFI contains less than 0.03 EU/mL.
If NPC +ve, PPC +ve report WFI contains 0.03 EU/mL

Prepare 1:2 and test at 0.06 EU/mL


LRW WFI CSE (20λ) LAL
NWC 100 µL − − 100 µL
PWC 100 µL − 10 µL 100 µL
NPC − 100 µL − 100 µL
PPC − 100 µL 10 µL 100 µL
If NPC −ve, PPC +ve report WFI contains less than 0.06 EU/mL
If NPC +ve, PPC +ve report WFI contains 0.06 EU/mL

Prepare 1:4 and test at 0.125 EU/mL


LRW WFI CSE (20λ) LAL
NWC 100 µL − − 100 µL
PWC 100 µL − 10 µL 100 µL
NPC − 100 µL − 100 µL
PPC − 100 µL 10 µL 100 µL
If NPC −ve, PPC +ve report WFI contains less than 0.125 EU/mL
If NPC +ve, PPC +ve report WFI contains 0.125 EU/mL

Prepare 1:8 and test at 0.25 EU/mL


LRW WFI CSE (20λ) LAL
NWC 100 µL − − 100 µL
PWC 100 µL − 10 µL 100 µL
NPC − 100 µL − 100 µL
PPC − 100 µL 10 µL 100 µL
If NPC −ve, PPC +ve report WFI contains less than 0.25 EU/mL
If NPC +ve, PPC +ve report WFI contains 0.25 EU/mL and report failure.
NUCLEAR MEDICINE

Product : Fludeoxyglucose F18 Injection


EL : 25 EU/mL
LAL sensitivity : 0.03EU/mL (for all calculations sensitivity = 0.03125EU/mL)
MVD : 800.
Prepare 1:800 dilution & test as follows.
From CSE = 20EU/mL, prepare 20 λ = 20 x 0.03125 = 0.625EU/mL

Tube Concentration LRW(µL) Sample(µL) CSE(µL) LAL Reading


no. (µL)
1,2,3 NPC - 100(1:800) -- 100 1 tube at 20
mins & 2
tubes at 60
mins.
4,5 PPC-2λ - 100(1:800) 10µL of 20λ 100 Both tubes
at 60 mins
6 PPC-160λ - 100(1:800) 25µL (20EU/mL) 100 At 20 mins.
7,8 PWC - - 100(2λ) 100 At 60 mins.
9,10 NWC 100 - - 100 At 60 mins.
DRY HEAT STERILISATION

The USP requires you to show a 3 log reduction in Endotoxin


The Validation procedure here assumes that a loaded oven in the maximum configuration
would serve as a ‘worst case’ model.

Time and Temperature Profile

z Depends upon ovens/tunnels specifications and type


z As temperature is increased, time required is less for approximately the same amount
of thermal destruction
z 250°C for 1 hour for DHS
z 350°C for 4-6minutes for Tunnel
z Revalidation if profile is changed

Temperature Profile Unloaded Vs. Loaded Oven

z Check proper installation


z Operator’s manual
z Utility connections
z Structural dimensions
z Accessories like heater, blowers
z Thermocouple study, 10 probes/100cu.ft.
z Thermocouple study, loaded.

As per the USP 29, Chapter 1211 defines an acceptable cycle for depyrogenation as one
which gives a three log reduction in the endotoxin concentration at the end of the cycle.

Endotoxin Challenge of the Loaded Oven

z Endotoxin Challenge vials / Indicators containing 10000ng approx =100000EU/vial


or 1000ng approx = 10000EU/vial
z Minimum 1000EU/vail concentration
z Quantify more than 3 log reduction
z Minimum Lysate sensitivity Not Less Than 0.15EU/mL .Use LAL of sensitivity
0.125EU/mL,0.06EU/mL,0.03EU/mL.
VALIDATION PROTOCOL FOR DRY HEAT
DEPYROGENATION CYCLE

PREPARATION

1. Remove seals.
2. Aseptically remove stoppers and discard.
3. Cover endotoxin vials with a double layer of aluminum foil.

HEAT EXPOSURE OF ENDOTOXIN INDICATORS

1. All endotoxin indicators shall be labeled by a marking pen as to oven location.


2. Position one endotoxin indicator adjacent to each thermocouple.
3. Two vials per depyrogenation cycle shall be retained as a positive control. These controls
should be placed outside the dry heat oven.
4. Expose indicators to heat cycle.
5. After oven run is complete, carefully remove indicator vials.

ENDOTOXIN RECOVERY ASSAY

1. Initiate recovery by rehydrating the endotoxin indicator vials including positive controls
with 1mL of LRW. Immediately vortex each container for 5 minutes.

2. Controls:

a. Positive Endotoxin Indicator Controls


Make 10-fold and 2-fold serial dilutions to get the concentrations (8λ,4λ,2λ,λ,λ/2,λ/4)
This is the basis for determining the endotoxin content. Actual recovery concentration is
to be taken for calculation.

b. Standard Curve
Make a valid 4-point standard curve of the CSE using concentrations 2λ,λ,λ/2,λ/4 where
λ is the sensitivity of the Lysate or use a Lysate whose sensitivity is verified.

c. Negative Control
The LRW used above is the negative control.
CALCULATIONS

1. Recovered EU/mL from heat = Reciprocal of last dilution x λ


treated Sample of sample that was positive

2. Recovered EU/mL from Positive = Reciprocal of last dilution x λ


Endotoxin Indicator Controls of PC that was positive
(PC)

3. Log {Recovered EU/mL from PC} - Log {Recovered EU/mL from Sample}

= X - Log Reduction

INTERPRETATION OF RESULTS

1. The depyrogenation cycle was successful if there was a > 3-log reduction in endotoxin in
the heat exposed vials.
FLOW CHART FOR VALIDATION OF DRY HEAT STERILIZERS
OVENS / TUNNELS

Start with a 10000ng approx =100,000 EU Endotoxin Indicator vial.



Reconstitute the vial in 2mL of LRW, Vortex for 20 minutes.

Aliquot 0.1 mL into ampoules or vials used for your injectables. The vials / ampoules must
be the same ones used typically for that particular oven / tunnel.

Dry the vials / ampoules in a Laminar Flow hood overnight. Each vial / ampoule will
now contain approx 5,000 EU.

Keep at least 1 vial / amp as Positive Control (do not pass through oven / tunnel).
Mark as PPC-1, 2, etc.

Expose the rest (8 or 9 amps / vials to appropriate number) to the oven / tunnel
as per it’s typical depyrogenation cycle.

After the cycle, mark all the vials as NPC - 1,2,3 etc.

Reconstitute all PPC and NPC vials / amps in 1mL of LRW each. Vortex each
vial / amp for at least 5 minutes. Remember to be consistent.

For PPC vial / amp For NPC vials / amps
(approx 5,000 EU/mL) (Unknown - Assuming a 3 log
reduction to 5 EU/mL)

↓ 1:50 dilution ↓ 1:10 dilution


100 EU/mL 0.5 EU/mL
↓ 1:100 dilution ↓ 1:4 dilution
1 EU/mL 0.125 EU/mL
(do 2 - fold dilutions to run a standard dilution series)
8λ,4λ,2λ ,λ,λ / 2,λ / 4

Test all final dilutions in DUPLICATE with a LAL reagent of 0.125EU/mL sensitivity.

Results :

For a valid Depyrogenation cycle, a greater than 3-log reduction of Endotoxin is to be


achieved.
FLOW CHART FOR VALIDATION OF DRY HEAT STERILIZERS
OVENS / TUNNELS

Start with a 10000ng approx =100,000 EU Endotoxin Indicator vial.



Reconstitute the vial in 2 mL or 1mL of LRW, Vortex for 20 minutes.

Aliquot 0.2 mL or 0.1mL (depending on volume of reconstitution of the Endotoxin Indicator
vial that is 2mL or 1mL) into ampoules or vials used for your injectables. The vials /
ampoules must be the same ones used typically for that particular oven / tunnel.

Dry the vials / ampoules in a Laminar Flow hood overnight. Each vial / ampoule will
now contain approx 10,000 EU.

Keep at least 1 vial / amp as Positive Control (do not pass through oven / tunnel).
Mark as PPC-1, 2, etc.

Expose the rest (8 or 9 amps / vials to appropriate number) to the oven / tunnel
as per it’s typical depyrogenation cycle.

After the cycle, mark all the vials as NPC - 1,2,3 etc.

Reconstitute all PPC and NPC vials / amps in 1mL of LRW each. Vortex each
vial / amp for at least 5 minutes. Remember to be consistent.

For PPC vial / amp For NPC vials / amps
(approx 10,000 EU/mL) (Unknown - Assuming a 3 log
reduction to 10 EU/mL)
↓ 1:10 dilution ↓ 1:10 dilution
1000 EU/mL 1 EU/mL
↓ 1:10 dilution ↓ 1:8 dilution
100 EU/mL 0.125 EU/mL
↓ 1:10 dilution
10 EU/mL
↓ 1:10 dilution
1 EU/mL
(do 2 - fold dilutions to run a standard dilution series)
8λ,4λ,2λ ,λ,λ / 2,λ / 4

Test all final dilutions in DUPLICATE with a LAL reagent of 0.125 EU/mL sensitivity.

Results:

For a valid Depyrogenation cycle, a greater than 3-log reduction of Endotoxin is to be


achieved.
Locations of Vials in DHS

1 3
2
4

TOP 5

6 8
7
9
FRONT
Locations of Vials in Tunnel

3 7

6 8
2 4

1 5 9

Direction of conveyer belt


Examples of Product Validation

Preliminary Screening Test

Product : Gentamycin Inj.

Potency : 40 mg/mL

Endotoxin Limit : 0.71EU/mg

Lysate Sensitivity (λ): 0.125EU/mL

MVD : 1: 227

Test Dilution: 1:25, 1:50, 1:100, 1:200

Test Concentrations: 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, 0.2 mg/mL

Test Dilutions / Concentrations NPC PPC


1:25 − − + +
1:50 − − + +
1:100 − − + +
1:200 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion: No inhibition observed. Test dilution of 1:100 to be chosen for validating


the product.
LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION
AS AN END-PRODUCT ENDOTOXINS TEST

Product : Gentamycin Inj

Preparation : Product Concentration : 40 mg/mL

Endotoxin Limit : NMT 0.71 EU/mg. MVD: 1:227

Test Concentration / Dilution: 0.4 mg/mL; 1:100

Sample Preparation : 1:50

Results:

Test Endotoxin Concentration NEG NEG Test Log of Geometric


Material EU/mL. Control Product Endpt. Endpt Mean
Control EU/mL
2λ λ 0.5λ 0.25λ
CSE + + − − − 0.125 -0.903
Water + + − − − 0.125 -0.903
Control + + − − 0.125 -0.903 0.125
EU/mL
+ + − − 0.125 -0.903
Positive + + + − − 0.0625 -1.2041
Product + + + − − 0.0625 -1.2041 0.0625
Control + + + − − 0.0625 -1.2041 EU/mL
+ + + − − 0.0625 -1.2041
+ = Firm Gel − = No Gel or less than firm Gel

Interpretation: Test results are VALID

Comments : The end points of CSE in Water = 0.125 EU/mL. The end point of CSE in
Product = 0.0625 EU/mL which is within ± one two fold dilution of the LAL labeled
sensitivity λ. λ = 0.125 EU/mL.
Preliminary Screening Test

Product : Dextrose for inj. (5% W/V).

Endotoxin Limit : 0.5 EU/mL

Lysate Sensitivity (λ) : 0.06EU/mL

MVD : 1:8

Test Dilution : 1:2, 1:4, 1:8

Test Dilutions / Concentrations NPC PPC


1:2 − − + +
1:4 − − + +
1:8 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion: No inhibition observed. Test dilution of 1:4 to be chosen for validating the
product.
LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION
AS AN END-PRODUCT ENDOTOXINS TEST

Product : Dextrose for inj. (5% W/V).

Preparation : Endotoxin Limit : NMT 0.5EU/mL. MVD: 1:8

Test Dilution: 1:4 Sample Preparation: 1:2

Results:
Test Endotoxin NEG NEG Test Log of Geometric
Material Concentration EU/mL. Control Product Endpt. Endpt. Mean
Control EU/mL
2λ λ ½λ ¼λ
CSE + + − − − 0.0625 -1.2041
Water + + − − − 0.0625 -1.2041
Control + + − − 0.0625 -1.2041 0.0625 EU/mL
+ + − − 0.0625 -1.2041
Positive + + − − − 0.0625 -1.2041
Product + + − − − 0.0625 -1.2041 0.0625 EU/mL
Control + + − − − 0.0625 -1.2041
+ + − − − 0.0625 -1.2041
+ = Firm Gel − = No Gel or less than firm Gel

Interpretation: Test results are VALID.

Comments : The end points of CSE in Water = 0.0625 EU/mL. The end point of CSE in
Product = 0.0625 EU/mL which is within ± one two fold dilution of the LAL labeled
sensitivity λ. λ = 0.06 EU/mL.
Preliminary Screening Test

Product : Normal Saline

Endotoxin Limit : 0.5 EU/mL

Lysate Sensitivity (λ) : 0.06EU/mL

MVD : 1:8

Test Dilution : 1:2, 1:4, 1:8

Test Dilutions / Concentrations NPC PPC


1:2 − − + +
1:4 − − + +
1:8 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion: No inhibition observed. Test dilution of 1:4 to be chosen for validating the
product.
LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION
AS AN END-PRODUCT ENDOTOXINS TEST

Product : Normal Saline

Preparation :

Endotoxin Limit : NMT 0.5EU/mL. MVD : 1:8

Test Dilution : 1:4 Sample Preparation : 1:2

Results :
Test Endotoxin Concentration NEG NEG Test Log of Geometric
EU/mL. Control Product Endpt. Endpt. Mean
Control EU/mL
Material 2λ λ 0.5λ 0.25λ
CSE + + − − − 0.0625 -1.2041
Water + + − − − 0.0625 -1.2041
Control + + − − 0.0625 -1.2041 0.0625
EU/mL
+ + − − 0.0625 -1.2041
Positive + + + − − 0.03125 -1.5051
Product + + + − − 0.03125 -1.5051 0.03125
Control + + + − − 0.03125 -1.5051 EU/mL
+ + + − − 0.03125 -1.5051

+ = Firm Gel − = No Gel or less than firm Gel

Interpretation: Test results are VALID

Comments : The end points of CSE in Water = 0.0625 EU/mL. The end point of CSE in
Product = 0.03125 EU/mL which is within ± one two fold dilution of the LAL labeled
sensitivity λ. λ = 0.0625 EU/mL.
Preliminary Screening Test

Product : Benzathine Penicillin.

Potency : 400,000 units/mL

Endotoxin Limit : 0.01 EU/100 units

Lysate Sensity (λ) : 0.125EU/mL

MVD : 1:320

Test Dilution: 1:40, 1:80, 1:160, 1:320

Test Concentrations: 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, 0.2 mg/mL

Test Dilutions / Concentrations NPC PPC


1:25 − − + +
1:50 − − + +
1:100 − − + +
1:200 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion : No inhibition observed. Test dilution of 1:100 to be chosen for


validating the product.
LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION
AS AN END-PRODUCT ENDOTOXINS TEST

Product : Gentamycin Inj

Preparation : Product Concentration: 40mg/mL

Endotoxin Limit : NMT 0.71 EU/mg. MVD: 1:227

Test Concentration / Dilution: 0.4 mg/mL; 1:100 Sample Preparation: 1:50

Results :
Test Endotoxin NEG NEG Test Log of Geometric
Material Concentration EU/mL. Control Product Endpt. Endpt. Mean
Control EU/mL
2λ λ ½λ ¼λ
CSE + + − − − 0.125 -0.903
Water + + − − − 0.125 -0.903
Control + + − − 0.125 -0.903 0.125
EU/mL
+ + − − 0.125 -0.903
Positive + + + − − 0.0625 -1.2041
Product + + + − − 0.0625 -1.2041 0.0625
Control + + + − − 0.0625 -1.2041 EU/mL
+ + + − − 0.0625 -1.2041
+ = Firm Gel − = No Gel or less than firm Gel

Interpretation : Test results are : VALID INVALID

Comments : The end points of CSE in Water = 0.125 EU/mL. The end point of CSE in
Product = 0.06 EU/mL which is within ± one two fold dilution of the LAL labeled sensitivity
λ. λ = 0.125 EU/mL