[Title 21 CFR K]
[Code of Federal Regulations (annual edition) - April 1, 1996 Edition]
[Title 21 - FOOD AND DRUGS]
[Chapter I - FOOD AND DRUG ADMINISTRATION,]
[Subchapter F - BIOLOGICS]
[Part 660 - ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS]
[Subpart K - Limulus Amebocyte Lysate]
[From the U.S. Government Publishing Office]
21
FOOD AND DRUGS
7
1996-04-01
1996-04-01
false
Limulus Amebocyte Lysate
K
Subpart K
FOOD AND DRUGS
FOOD AND DRUG ADMINISTRATION,
BIOLOGICS
ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS
Subpart K--Limulus Amebocyte Lysate
Sec. 660.100 Limulus Amebocyte Lysate.
The proper name of this product shall be Limulus Amebocyte Lysate.
The product is defined as an extract that is derived from the blood of
Limulus polyphemus and is capable of detecting bacterial endotoxins.
[45 FR 32299, May 16, 1980]
Sec. 660.101 U.S. Standard/Reference Preparations.
The following U.S. Standard/Reference preparations shall be obtained
from the Center for Biologics Evaluation and Research, Food and Drug
Administration, 8800 Rockville Pike, Bethesda, MD 20892, for use as
prescribed in this subpart:
(a) A U.S. Standard Endotoxin for determining the sensitivity of
Limulus Amebocyte Lysate.
(b) A U.S. Reference Limulus Amebocyte Lysate for establishing the
potency of Limulus Amebocyte Lysate.
[45 FR 32299, May 16, 1980, as amended at 49 FR 23834, June 8, 1984; 51
FR 15611, Apr. 25, 1986; 55 FR 11013, Mar. 26, 1990]
Sec. 660.102 Potency test.
A sample of each final filling of each lot of Limulus Amebocyte
Lysate and the U.S. Reference Lysate shall be tested in parallel with
the U.S. Standard Endotoxin. If the product is freeze-dried after
filling, the test shall be conducted on samples from each filling in
each drying chamber run. The procedure for rehydrating and mixing the
lysate for the potency test shall be that specified in the
manufacturer's package insert. A minimum of 8 vials and a maximum of 28
vials from each filling or, if freeze-dried, from each drying chamber
run representing all parts of the chamber load, shall be tested in
parallel with an equal number of tests from 1 or more vials of the U.S.
Reference Lysate. The test shall be performed as follows:
(a) Dilution of U.S. Standard Endotoxin. A single series of
consecutive two-fold dilutions, beginning with a concentration of the
U.S. Standard Endotoxin at least four-fold above the endpoint, shall be
prepared with a range selected to bracket the endpoint for both the U.S.
Reference Lysate and test lysate filling in each test performed.
(b) Test procedure. (1) Transfer 0.1 milliliter of each
concentration of U.S. Standard Endotoxin, as prepared in paragraph (a)
of this section, into each of two test tubes having an inside diameter
not greater than 10 millimeters, unless the use of another size test
[[Page 164]]
tube has been approved by the Director, Center for Biologics Evaluation
and Research.
(2) Add 0.1 milliliter of the U.S. Reference Lysate to one of the
tubes containing the lowest concentration of U.S. Standard Endotoxin.
Add 0.1 milliliter of test lysate to the second tube containing the
lowest concentration of U.S. Standard Endotoxin.
(3) Repeat the procedure in paragraphs (b)(1) and (2) of this
section for each dilution of the U.S. Standard Endotoxin and for each
vial of lysate to be tested from each filling of the test lot,
progressing from the lowest endotoxin concentration to the highest.
(4) Immediately following addition of the lysate to each tube, mix
the contents gently and place in a 37 deg. C water bath for 1 hour.
(c) Validity of the test. (1) Record the reaction in each tube as
either positive or negative. A positive reaction is demonstrated by a
firm gel that remains intact, at least momentarily, when the tube is
inverted 180 degrees. For Limulus Amebocyte Lysate that does not require
gelation as an indicator of reactions, the endpoint shall be determined
by the method specified in the labeling for the product.
(2) For each parallel test obtain the ratio of endpoints of
reference and test lysates. Calculate the standard deviations (S.D.) of
log ratios.
(3) The test is valid if the S.D. is less than or equal to the value
for the 99 percent fiducial upper limit of the S.D. of the sample size
tested. The S.D. table is shown in paragraph (d) of this section.
(4) If the S.D. is greater than the tabulated value, the test may be
expanded up to the maximum of 28 parallel tests and a new S.D. for log
ratios may be calculated.
(5) The tests are invalid due to excessive variability if the S.D.
is greater than the value in the S.D. table corresponding to the sample
size tested.
(6) If the S.D. is within the limits, the geometric mean (G.M.) of
the ratios shall be calculated.
(7) The endpoints of U.S. Reference and test lysates, ratios of
endpoints, S.D. of log ratios, and G.M. of ratios shall be calculated
and reported on the protocol submitted to the Director, Center for
Biologics Evaluation and Research.
(d) S.D. table. Ninety-nine percent fiducial upper limit on S.D. of
log2 (ratio):
------------------------------------------------------------------------
Upper
Sample size limit
------------------------------------------------------------------------
4........................................................... \1\ 1.02
8........................................................... 0.86
12.......................................................... 0.79
16.......................................................... 0.75
20.......................................................... 0.73
24.......................................................... 0.71
28.......................................................... 0.69
------------------------------------------------------------------------
\1\ Limits can be converted to log10 by multiplying each value by 0.3.
[45 FR 32299, May 16, 1980, as amended at 49 FR 23834, June 8, 1984; 52
FR 39637, Oct. 23, 1987; 55 FR 11013, Mar. 26, 1990]
Sec. 660.103 General requirements.
(a) Handling the horseshoe crabs. The horseshoe crabs (Limulus
polyphemus), from which blood is collected for production of the lysate,
shall be handled in a manner so as to minimize injury to each crab. The
horseshoe crabs shall be returned alive to their natural environment
after a single collection of their blood.
(b) Processing. The processing methods shall be those which have
been shown to yield consistently a potent and detection-specific final
product free of properties that would adversely affect the accuracy of
the test results when the Limulus Amebocyte Lysate is used by the
methods recommended by the manufacturer in the package insert.
(c) Final containers. Final containers at the time of filling shall
be sterile, nonpyrogenic, colorless, and transparent.
(d) Date of manufacture. The date of manufacture of each filling of
each lot shall be the date the manufacturer initiated the last valid
potency test for such filling. The results from this test shall be
reported on the protocol submitted to the Director, Center for Biologics
Evaluation and Research.
(e) Sterility test. A sterility test shall be performed on the bulk
lot and on each filling as prescribed in Sec. 610.12 of this chapter.
(f) Test for quality. A test for lysate quality shall be performed
as follows:
[[Page 165]]
(1) Samples from each of eight final containers from each filling
or, if freeze-dried, from each filling in each drying chamber run
representing all parts of the chamber load, shall be used.
(2) The volume of lysate required for a single test from each of the
final containers and a volume of distilled water equal to the volume of
sample used for a single test are combined into each of an appropriate
number of test tube and incubated for 24 hours in a 37 deg. C water
bath.
(3) The test passes if none of the samples yield a positive test.
(g) Test for residual moisture. (1) If the weight of the contents of
each final container is 3 milligrams or more, the test for residual
moisture shall be performed as prescribed in Sec. 610.13(a) of this
chapter.
(2)(i) If the weight of the contents of each final container is less
than 3 milligrams, the product is exempt from the test for residual
moisture. However, the manufacturer of such exempt product shall perform
the potency test described in Sec. 660.102 on at least 4 vials at 4-
month intervals on representative samples from each filling throughout
the dating period.
(ii) Upon the completion of each potency test, the results of all
tests performed shall be submitted to the Director, Center for Biologics
Evaluation and Research.
(h) Ancillary reagents and materials. All ancillary reagents and
materials accompanying the product that are used in the performance of a
test, as described by the manufacturer's recommended test procedures,
shall not affect adversely the performance of the Limulus Amebocyte
Lysate within the prescribed dating period.
[45 FR 32299, May 16, 1980, as amended at 49 FR 23834, June 8, 1984; 52
FR 39637, Oct. 23, 1987; 55 FR 11013, Mar. 26, 1990]
Sec. 660.104 Labeling.
In addition to the applicable labeling provisions of this chapter,
the following information is required:
(a) Final container labels. The final container label shall include
the following additional information:
(1) The sensitivity (geometric mean of the end points of the lot)
expressed as units/milliliter or nanograms/milliliter of the U.S.
Standard Endotoxin, determined by the potency test procedure in
Sec. 660.102.
(2) For final containers intended for multiple tests, a designated
area adequate for the user to identify the time that the product is
reconstituted.
(3) For final containers intended for multiple tests, a statement
identifying the period within which the product may be used after
reconstitution.
(4) For final containers intended for multiple tests, a statement
specifying storage conditions after reconstitution.
(b) Package label. The package label shall include the following
additional information:
(1) A reference to the package insert for the test method(s) to be
employed when using Limulus Amebocyte Lysate.
(2) A statement that the product shall not be rehydrated until
immediately prior to use.
(3) For products in final containers intended for multiple tests, a
statement identifying the period within which the product may be used
after reconstitution.
(4) For products in final containers intended for multiple tests, a
statement specifying storage conditions after reconstitution.
(c) Package insert. The package insert shall include the following
additional information:
(1) A statement that if the container of diluent used to rehydrate
the lysate has been entered previously or was not supplied by the
manufacturer of the lysate, the diluent must be tested, without addition
of test material.
(2) A warning statement that the tubes of material on test should
not be removed from incubation or disturbed prior to the time specified
for reading the test.
(3) A statement that the product shall not be rehydrated until
immediately prior to use.
(4) For products in final containers intended for multiple tests, a
statement identifying the period within which the product may be used
after reconstitution.
[[Page 166]]
(5) For products in final containers intended for multiple tests, a
statement specifying storage conditions after reconstitution.
[45 FR 32299, May 16, 1980]
Sec. 660.105 Samples and protocols; official release.
(a) For each final filling of each lot of Limulus Amebocyte Lysate,
or if freeze dried, from each drying chamber run representing all parts
of the chamber load, the following material shall be submitted to the
Director, Center for Biologics Evaluation and Research, Food and Drug
Administration, 8800 Rockville Pike, Bethesda, MD 20892:
(1) Samples. Not fewer than the number of vials of lysate used for
the potency test in Sec. 660.102, two of which shall be complete market
packages, packaged for distribution and including all ancillary reagents
and materials.
(2) Protocols. A protocol consisting of a complete summary of the
history of manufacture of each filling, the dates of testing, and the
results of all required tests.
(b) Official release. Limulus Amebocyte Lysate shall not be
distributed by the manufacturer until written notification of official
release of each filling is received from the Director, Center for
Biologics Evaluation and Research.
[45 FR 32299, May 16, 1980, as amended at 49 FR 23834, June 8, 1984; 51
FR 15611, Apr. 25, 1986; 52 FR 39637, Oct. 23, 1987; 55 FR 11013, Mar.
26, 1990]