EUROPEAN PHARMACOPOEIA 6.

0 Diphtheria antitoxin

01/2008:0085 Prepare mixtures of the solution of the reference preparation

and the test toxin such that each contains 2.0 ml of the
solution of the reference preparation, one of a graded series
BOTULINUM ANTITOXIN of volumes of the test toxin and sufficient of a suitable liquid
to bring the total volume to 5.0 ml. Allow the mixtures to
Immunoserum botulinicum stand at room temperature, protected from light, for 60 min.
Using four mice for each mixture, inject a dose of 1.0 ml
DEFINITION intraperitoneally into each mouse. Observe the mice for 96 h.
Botulinum antitoxin is a preparation containing antitoxic The test dose of toxin is the quantity in 1.0 ml of the mixture
globulins that have the power of specifically neutralising the made with the smallest amount of toxin capable of causing,
toxins formed by Clostridium botulinum type A, type B or despite partial neutralisation by the reference preparation,
type E, or any mixture of these types. the death of all four mice injected with the mixture within
the observation period.
PRODUCTION Determination of potency of the antitoxin. Prepare
It is obtained by fractionation from the serum of horses, solutions of each reference preparation in a suitable liquid
or other mammals, that have been immunised against such that each contains 0.25 IU of antitoxin per millilitre.
Cl. botulinum type A, type B and type E toxins. Prepare solutions of each test toxin in a suitable liquid such
that each contains 2.5 test doses per millilitre.
IDENTIFICATION Using each toxin solution and the corresponding reference
It specifically neutralises the types of Cl. botulinum toxins preparation in turn, determine the potency of the antitoxin.
stated on the label, rendering them harmless to susceptible Prepare mixtures of the solution of the test toxin and the
animals. antitoxin to be examined such that each contains 2.0 ml
of the solution of the test toxin, one of a graded series
POTENCY of volumes of the antitoxin to be examined, and sufficient of
Not less than 500 IU of antitoxin per millilitre for each a suitable liquid to bring the total volume to 5.0 ml. Also
of types A and B and not less than 50 IU of antitoxin per prepare mixtures of the solution of the test toxin and the
millilitre for type E. solution of the reference preparation such that each contains
2.0 ml of the solution of the test toxin, one of a graded
The potency of botulinum antitoxin is determined by series of volumes of the solution of the reference preparation
comparing the dose necessary to protect mice against the centred on that volume (2.0 ml) that contains 0.5 IU, and
lethal effects of a fixed dose of botulinum toxin with the sufficient of a suitable liquid to bring the total volume to
quantity of the standard preparation of botulinum antitoxin 5.0 ml. Allow the mixtures to stand at room temperature,
necessary to give the same protection. For this comparison a protected from light, for 60 min. Using four mice for each
reference preparation of each type of botulinum antitoxin, mixture, inject a dose of 1.0 ml intraperitoneally into each
calibrated in International Units, and suitable preparations mouse. Observe the mice for 96 h.
of botulinum toxins, for use as test toxins, are required. The The mixture that contains the largest volume of antitoxin
potency of each test toxin is determined in relation to the that fails to protect the mice from death contains 0.5 IU. This
specific reference preparation ; the potency of the botulinum quantity is used to calculate the potency of the antitoxin in
antitoxin to be examined is determined in relation to the International Units per millilitre.
potency of the test toxins by the same method.
The test is not valid unless all the mice injected with
International Units of the antitoxin are the specific mixtures containing 2.0 ml or less of the solution of the
neutralising activity for botulinum toxin type A, type B and reference preparation die and all those injected with mixtures
type E contained in stated amounts of the International containing more survive.
Standards which consist of dried immune horse sera of types
A, B and E. The equivalence in International Units of the LABELLING
International Standard is stated from time to time by the The label states the types of Cl. botulinum toxin neutralised
World Health Organisation. by the preparation.
Selection of animals. Use mice having body masses such
that the difference between the lightest and the heaviest
does not exceed 5 g. 01/2008:0086
Preparation of test toxins. CAUTION : Botulinum toxin is
extremely toxic : exceptional care must be taken in any DIPHTHERIA ANTITOXIN
procedure in which it is employed. Prepare type A, B and E
toxins from sterile filtrates of approximately 7-day cultures Immunoserum diphthericum
in liquid medium of Cl. botulinum types A, B and E. To the
filtrates, add 2 volumes of glycerol, concentrate, if necessary, DEFINITION
by dialysis against glycerol and store at or slightly below Diphtheria antitoxin is a preparation containing antitoxic
0 °C. globulins that have the power of specifically neutralising the
toxin formed by Corynebacterium diphtheriae.
Selection of test toxins. Select toxins of each type for use
as test toxins by determining for mice the L+/10 dose and PRODUCTION
the LD50, the observation period being 96 h. The test toxins It is obtained by fractionation from the serum of horses,
contain at least 1000 LD50 in an L+/10 dose. or other mammals, that have been immunised against
Determination of test doses of the toxins (L+/10 dose). diphtheria toxin.
Prepare solutions of the reference preparations in a suitable
liquid such that each contains 0.25 IU of antitoxin per IDENTIFICATION
millilitre. Using each solution in turn, determine the It specifically neutralises the toxin formed by C. diphtheriae,
test dose of the corresponding test toxin. rendering it harmless to susceptible animals.

General Notices (1) apply to all monographs and other texts 965
Gas-gangrene antitoxin, mixed
ASSAY
Not less than 1000 IU of antitoxin per millilitre for antitoxin
obtained from horse serum. Not less than 500 IU of antitoxin
per millilitre for antitoxin obtained from the serum of other
mammals.
The potency of diphtheria antitoxin is determined by
comparing the dose necessary to protect guinea-pigs or
rabbits against the erythrogenic effects of a fixed dose
of diphtheria toxin with the quantity of the standard
preparation of diphtheria antitoxin necessary to give the
same protection. For this comparison a reference preparation
of diphtheria antitoxin, calibrated in International Units,
and a suitable preparation of diphtheria toxin, for use as
a test toxin, are required. The potency of the test toxin
is determined in relation to the reference preparation ;
the potency of the diphtheria antitoxin to be examined is
determined in relation to the potency of the test toxin by
the same method.
The International Unit of antitoxin is the specific neutralising
activity for diphtheria toxin contained in a stated amount of
the International Standard, which consists of a quantity of
dried immune horse serum. The equivalence in International
Units of the International Standard is stated by the World
Health Organisation.
Preparation of test toxin. Prepare diphtheria toxin from
cultures of C. diphtheriae in a liquid medium. Filter the
culture to obtain a sterile toxic filtrate and store at 4 °C.
Selection of test toxin. Select a toxin for use as a test toxin
by determining for guinea-pigs or rabbits the lr/100 dose
and the minimal reacting dose, the observation period being
48 h. The test toxin has at least 200 minimal reacting doses
in the lr/100 dose.
Minimal reacting dose. This is the smallest quantity of toxin
which, when injected intracutaneously into guinea-pigs or
rabbits, causes a small, characteristic reaction at the site of
injection within 48 h.
The test toxin is allowed to stand for some months before
being used for the assay of antitoxin. During this time its
toxicity declines and the lr/100 dose may be increased.
Determine the minimal reacting dose and the lr/100 dose
at frequent intervals. When experiment shows that the
lr/100 dose is constant, the test toxin is ready for use and
may be used for a long period. Store the test toxin in the
dark at 0 °C to 5 °C. Maintain its sterility by the addition of
toluene or other antimicrobial preservative that does not
cause a rapid decline in specific toxicity.
Determination of test dose of toxin (lr/100 dose). Prepare
a solution of the reference preparation in a suitable liquid
such that it contains 0.1 IU of antitoxin per millilitre.
Prepare mixtures of the solution of the reference preparation
and of the test toxin such that each contains 1.0 ml of the
solution of the reference preparation, one of a graded series
of volumes of the test toxin and sufficient of a suitable liquid
to bring the total volume to 2.0 ml. Allow the mixtures to
stand at room temperature, protected from light, for 15 min
to 60 min. Using two animals for each mixture, inject a dose
of 0.2 ml intracutaneously into the shaven or depilated
flanks of each animal. Observe the animals for 48 h.
The test dose of toxin is the quantity in 0.2 ml of the mixture
made with the smallest amount of toxin capable of causing,
despite partial neutralisation by the reference preparation,
a small but characteristic erythematous lesion at the site of
injection.
Determination of potency of the antitoxin. Prepare a
solution of the reference preparation in a suitable liquid
such that it contains 0.125 IU of antitoxin per millilitre.
966 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 6.0
Prepare a solution of the test toxin in a suitable liquid such
that it contains 12.5 test doses per millilitre.
Prepare mixtures of the solution of the test toxin and of the
antitoxin to be examined such that each contains 0.8 ml
of the solution of the test toxin, one of a graded series
of volumes of the antitoxin to be examined and sufficient of
a suitable liquid to bring the total volume to 2.0 ml. Also
prepare mixtures of the solution of the test toxin and the
solution of the reference preparation such that each contains
0.8 ml of the solution of the test toxin, one of a graded
series of volumes of the solution of the reference preparation
centred on that volume (0.8 ml) that contains 0.1 IU and
sufficient of a suitable liquid to bring the total volume to
2.0 ml. Allow the mixtures to stand at room temperature,
protected from light, for 15 min to 60 min. Using two animals
for each mixture, inject a dose of 0.2 ml intracutaneously
into the shaven or depilated flanks of each animal. Observe
the animals for 48 h.
The mixture that contains the largest volume of antitoxin
that fails to protect the guinea-pigs from the erythematous
effects of the toxin contains 0.1 IU. This quantity is used to
calculate the potency of the antitoxin in International Units
per millilitre.
The test is not valid unless all the sites injected with mixtures
containing 0.8 ml or less of the solution of the reference
preparation show erythematous lesions and at all those
injected with mixtures containing more there are no lesions.
01/2008:0090
GAS-GANGRENE ANTITOXIN, MIXED
Immunoserum gangraenicum mixtum
DEFINITION
Mixed gas-gangrene antitoxin is prepared by mixing
gas-gangrene antitoxin (novyi), gas-gangrene antitoxin
(perfringens) and gas-gangrene antitoxin (septicum) in
appropriate quantities.
IDENTIFICATION
It specifically neutralises the alpha toxins formed by
Clostridium novyi (former nomenclature : Clostridium
oedematiens), Clostridium perfringens and Clostridium
septicum, rendering them harmless to susceptible animals.
ASSAY
Gas-gangrene antitoxin (novyi), not less than 1000 IU of
antitoxin per millilitre ; gas-gangrene antitoxin (perfringens),
not less than 1000 IU of antitoxin per millilitre ; gas-gangrene
antitoxin (septicum) not less than 500 IU of antitoxin per
millilitre.
Carry out the assay for each component, as prescribed in
the monographs on Gas-gangrene antitoxin (novyi) (0087),
Gas-gangrene antitoxin (perfringens) (0088) and
Gas-gangrene antitoxin (septicum) (0089).
01/2008:0087
GAS-GANGRENE ANTITOXIN (NOVYI)
Immunoserum gangraenicum
(Clostridium novyi)
DEFINITION
Gas-gangrene antitoxin (novyi) is a preparation containing
antitoxic globulins that have the power of neutralising
the alpha toxin formed by Clostridium novyi (Former