[Federal Register Volume 62, Number 225 (Friday, November 21, 1997)]
[Notices]
[Pages 62472-62475]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-30706]
[[Page 62471]]
_______________________________________________________________________
Part V
Department of Health and Human Services
_______________________________________________________________________
Food and Drug Administration
_______________________________________________________________________
International Conference on Harmonisation; Guidance on Genotoxicity: A
Standard Battery for Genotoxicity Testing of Pharmaceuticals;
Availability; Notice
Federal Register / Vol. 62, No. 225 / Friday, November 21, 1997 /
Notices
[[Page 62472]]
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration
[Docket No. 97D-0112]
International Conference on Harmonisation; Guidance on
Genotoxicity: A Standard Battery for Genotoxicity Testing of
Pharmaceuticals; Availability
AGENCY: Food and Drug Administration, HHS.
ACTION: Notice.
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SUMMARY: The Food and Drug Administration (FDA) is publishing a
guidance entitled ``S2B Genotoxicity: A Standard Battery for
Genotoxicity Testing of Pharmaceuticals.'' The guidance was prepared
under the auspices of the International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human
Use (ICH). The guidance identifies a standard set of genotoxicity tests
that should be conducted for pharmaceutical registration and recommends
the extent of confirmatory experimentation in in vitro genotoxicity
tests in the standard battery. The guidance complements the ICH
guidance ``Guidance on Specific Aspects of Regulatory Genotoxicity
Tests for Pharmaceuticals'' (S2A).
DATES: Effective November 21, 1997. Submit written comments at any
time.
ADDRESSES: Submit written comments on the guidance to the Dockets
Management Branch (HFA-305), Food and Drug Administration, 12420
Parklawn Dr., rm. 1-23, Rockville, MD 20857. Copies of the guidance are
available from the Drug Information Branch (HFD-210), Center for Drug
Evaluation and Research, Food and Drug Administration, 5600 Fishers
Lane, Rockville, MD 20857, 301-827-4573.
FOR FURTHER INFORMATION CONTACT:
Regarding the guidance: Robert E. Osterberg, Center for Drug
Evaluation and Research (HFD-520), Food and Drug Administration, 9201
Corporate Blvd., Rockville, MD 20850, 301-827-2123.
Regarding ICH: Janet J. Showalter, Office of Health Affairs (HFY-
20), Food and Drug Administration, 5600 Fishers Lane, Rockville, MD
20857, 301-827-0864.
SUPPLEMENTARY INFORMATION: In recent years many important initiatives
have been undertaken by regulatory authorities and industry
associations to promote international harmonization of regulatory
requirements. FDA has participated in many meetings designed to enhance
harmonization and is committed to seeking scientifically based
harmonized technical procedures for pharmaceutical development. One of
the goals of harmonization is to identify and then reduce differences
in technical requirements for drug development among regulatory
agencies.
ICH was organized to provide an opportunity for tripartite
harmonization initiatives to be developed with input from both
regulatory and industry representatives. FDA also seeks input from
consumer representatives and others. ICH is concerned with
harmonization of technical requirements for the registration of
pharmaceutical products among three regions: The European Union, Japan,
and the United States. The six ICH sponsors are the European
Commission, the European Federation of Pharmaceutical Industries
Associations, the Japanese Ministry of Health and Welfare, the Japanese
Pharmaceutical Manufacturers Association, the Centers for Drug
Evaluation and Research and Biologics Evaluation and Research, FDA, and
the Pharmaceutical Research and Manufacturers of America. The ICH
Secretariat, which coordinates the preparation of documentation, is
provided by the International Federation of Pharmaceutical
Manufacturers Associations (IFPMA).
The ICH Steering Committee includes representatives from each of
the ICH sponsors and the IFPMA, as well as observers from the World
Health Organization, the Canadian Health Protection Branch, and the
European Free Trade Area.
In the Federal Register of April 3, 1997 (62 FR 16026), FDA
published a draft tripartite guideline entitled ``Genotoxicity: A
Standard Battery for Genotoxicity Testing of Pharmaceuticals'' (S2B).
The notice gave interested persons an opportunity to submit comments by
June 2, 1997.
After consideration of the comments received and revisions to the
guidance, a final draft of the guidance was submitted to the ICH
Steering Committee and endorsed by the three participating regulatory
agencies on July 16, 1997.
In accordance with FDA's Good Guidance Practices (62 FR 8961,
February 27, 1997), this document has been designated a guidance,
rather than a guideline.
Genotoxicity tests are in vitro and in vivo tests designed to
detect compounds that induce genetic damage directly or indirectly by
various mechanisms. Compounds that are positive in tests that detect
such damage have the potential to be human carcinogens and/or mutagens,
i.e., may induce cancer and/or heritable defects. The guidance
addresses two areas of genotoxicity testing for pharmaceuticals: (1)
Identification of a standard set of tests that should be conducted for
registration and (2) the extent of confirmatory experimentation in in
vitro genotoxicity tests in the standard battery. The guidance is
intended to be used together with the ICH S2A guidance entitled
``Guidance on Specific Aspects of Regulatory Genotoxicity Tests for
Pharmaceuticals'' (61 FR 18198, April 24, 1996) as ICH guidance
principles for testing pharmaceuticals for potential genotoxicity.
This guidance represents the agency's current thinking on a
recommended standard battery for genotoxicity testing of a
pharmaceutical. It does not create or confer any rights for or on any
person and does not operate to bind FDA or the public. An alternative
approach may be used if such approach satisfies the requirements of the
applicable statute, regulations, or both.
As with all of FDA's guidances, the public is encouraged to submit
written comments with new data or other new information pertinent to
this guidance. The comments in the docket will be periodically
reviewed, and, where appropriate, the guidance will be amended. The
public will be notified of any such amendments through a notice in the
Federal Register.
Interested persons may, at any time, submit written comments on the
guidance to the Dockets Management Branch (address above). Two copies
of any comments are to be submitted, except that individuals may submit
one copy. Comments are to be identified with the docket number found in
brackets in the heading of this document. The guidance and received
comments may be seen in the office above between 9 a.m. and 4 p.m.,
Monday through Friday. An electronic version of this guidance is
available on the Internet (http://www.fda.gov/cder/guidance.htm).
The text of the guidance follows:
S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of
Pharmaceuticals\1\
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\1\ This guidance represents the agency's current thinking on a
recommended standard battery for genotoxicity testing of a
pharmaceutical. It does not create or confer any rights for or on
any person and does not operate to bind FDA or the public. An
alternative approach may be used if such approach satisfies the
requirements of the applicable statute, regulations, or both.
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1. Introduction
Two fundamental areas in which harmonization of genotoxicity
testing for
[[Page 62473]]
pharmaceuticals is considered necessary are the scope of this
guidance: (I) Identification of a standard set of tests that should
be conducted for registration. (II) The extent of confirmatory
experimentation in in vitro genotoxicity tests in the standard
battery. Further issues that were considered necessary for
harmonization can be found in the ICH guidance S2A ``Guidance on
Specific Aspects of Regulatory Genotoxicity Tests for
Pharmaceuticals.'' The two ICH guidances on genotoxicity complement
each other and therefore should be used together as ICH guidance
principles for testing of a pharmaceutical for potential
genotoxicity.
2. General Purpose of Genotoxicity Testing
Genotoxicity tests can be defined as in vitro and in vivo tests
designed to detect compounds that induce genetic damage directly or
indirectly by various mechanisms. These tests should enable a hazard
identification with respect to damage to DNA and its fixation.
Fixation of damage to DNA in the form of gene mutations, larger
scale chromosomal damage, recombination and numerical chromosome
changes is generally considered to be essential for heritable
effects and in the multistep process of malignancy, a complex
process in which genetic changes may play only a part. Compounds
which are positive in tests that detect such kinds of damage have
the potential to be human carcinogens and/or mutagens, i.e., may
induce cancer and/or heritable defects. Because the relationship
between exposure to particular chemicals and carcinogenesis is
established for man, while a similar relationship has been difficult
to prove for heritable diseases, genotoxicity tests have been used
mainly for the prediction of carcinogenicity. Nevertheless, because
germ line mutations are clearly associated with human disease, the
suspicion that a compound may induce heritable effects is considered
to be just as serious as the suspicion that a compound may induce
cancer. In addition, the outcome of such tests may be valuable for
the interpretation of carcinogenicity studies.
3. The Standard Test Battery for Genotoxicity
Registration of pharmaceuticals requires a comprehensive
assessment of their genotoxic potential. It is clear that no single
test is capable of detecting all relevant genotoxic agents.
Therefore, the usual approach should be to carry out a battery of in
vitro and in vivo tests for genotoxicity. Such tests are
complementary rather than representing different levels of
hierarchy.
The general features of a standard test battery can be outlined
as follows:
(i) It is appropriate to assess genotoxicity in a bacterial
reverse mutation test. This test has been shown to detect relevant
genetic changes and the majority of genotoxic rodent carcinogens.
(ii) DNA damage considered to be relevant for mammalian cells
and not adequately measured in bacteria should be evaluated in
mammalian cells. Several mammalian cell systems are in use: Systems
that detect gross chromosomal damage (in vitro tests for structural
and numerical chromosomal aberrations), systems that detect
primarily gene mutations (see Note 1), and a system that detects
gene mutations and clastogenic effects (mouse lymphoma tk assay)
(see Note 2). The information given in Notes 3 and 4 demonstrates
that with appropriate test protocols (see section 5 of this
document) the various in vitro tests for chromosomal damage and the
mouse lymphoma tk assay yield results with a high level of
congruence for compounds that are regarded as genotoxic but yield
negative results in the bacterial reverse mutation assay. Therefore,
these systems are currently considered interchangeable when used
together with other genotoxicity tests in a standard battery for
genotoxicity testing of pharmaceuticals, if these test protocols are
used.
(iii) An in vivo test for genetic damage should usually be a
part of the test battery to provide a test model in which additional
relevant factors (absorption, distribution, metabolism, excretion)
that may influence the genotoxic activity of a compound are
included. As a result, in vivo tests permit the detection of some
additional genotoxic agents (see Note 5). An in vivo test for
chromosomal damage in rodent hematopoietic cells fulfills this need.
This in vivo test for chromosomal damage in rodents could be either
an analysis of chromosomal aberrations in bone marrow cells or an
analysis of micronuclei in bone marrow or peripheral blood
erythrocytes.
The following standard test battery is recommended based upon
the considerations mentioned above:
(i) A test for gene mutation in bacteria.
(ii) An in vitro test with cytogenetic evaluation of chromosomal
damage with mammalian cells or an in vitro mouse lymphoma tk assay.
(iii) An in vivo test for chromosomal damage using rodent
hematopoietic cells.
For compounds giving negative results, the completion of this 3-test
battery, performed and evaluated in accordance with current
recommendations, will usually provide a sufficient level of safety
to demonstrate the absence of genotoxic activity (see Note 6).
Compounds giving positive results in the standard test battery may,
depending on their therapeutic use, need to be tested more
extensively (see ICH S2A ``Guidance on Specific Aspects of
Regulatory Genotoxicity Tests for Pharmaceuticals'').
The suggested standard set of tests does not imply that other
genotoxicity tests are generally considered inadequate or
inappropriate (e.g., tests for measurement of DNA adducts, DNA
strand breaks, DNA repair or recombination). Such tests serve as
options in addition to the standard battery for further
investigation of genotoxicity test results obtained in the standard
battery. Furthermore, molecular techniques to study mechanisms of
genotoxicity in the standard battery systems may be useful for risk
assessment. Only under extreme conditions in which one or more tests
comprising the standard battery cannot be employed for technical
reasons, alternative validated tests can serve as substitutes. For
this to occur, sufficient scientific justification should be
provided to support the argument that a given standard battery test
is not appropriate.
The standard battery does not include an independent test
designed specifically to test for aneuploidy. However, information
on this type of damage may be derived from the tests for chromosomal
damage in vitro and in vivo. Elements of the standard protocols that
provide such information are elevations in the mitotic index,
polyploidy induction and micronucleus evaluation. There is also
limited experimental evidence that aneuploidy inducers can be
detected in the mouse lymphoma tk assay (see Note 4). In such cases,
further testing may be needed.
4. Modifications of the 3-Test Battery
The following sections give situations where the standard 3-test
battery may need modification.
4.1 Limitations to the Use of Bacterial Test Organisms
There are circumstances where the performance of the bacterial
reverse mutation test does not provide appropriate or sufficient
information for the assessment of genotoxicity. This may be the case
for compounds that are excessively toxic to bacteria (e.g., some
antibiotics) and compounds thought or known to interfere with the
mammalian cell replication system (e.g., topoisomerase inhibitors,
nucleoside analogues, or inhibitors of DNA metabolism). For these
cases, usually two in vitro mammalian cell tests should be performed
using two different cell types and of two different endpoints (gene
mutation (see Note 1) and chromosomal damage). Nevertheless, it is
still important to perform the bacterial reverse mutation test (see
Note 7); either a full test or a limited (range-finding) test (see
section 5 of this document) may be appropriate.
4.2 Compounds Bearing Structural Alerts for Genotoxic Activity
Structurally alerting compounds (see Note 8) are usually
detectable in the standard 3-test battery. However, compounds
bearing structural alerts that have given negative results in the
standard 3-test battery may necessitate limited additional testing.
The choice of additional test(s) or protocol modification(s) depends
on the chemical nature, the known reactivity, and metabolism data on
the structurally alerting compound under question (see Note 9 and
ICH S2A ``Guidance on Specific Aspects of Regulatory Genotoxicity
Tests for Pharmaceuticals'').
4.3 Limitations to the Use of Standard In Vivo Tests
There are compounds for which standard in vivo tests do not
provide additional useful information. These include compounds for
which data from studies on toxicokinetics or pharmacokinetics
indicate that they are not systemically absorbed and therefore are
not available for the target tissues in standard in vivo
genotoxicity tests. Examples of such compounds are some radioimaging
agents, aluminum-based antacids, and some dermally applied
pharmaceuticals. In cases where a modification of the route of
administration does not provide sufficient target tissue exposure,
it may be appropriate to base the evaluation only on in vitro
testing.
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4.4 Additional Genotoxicity Testing in Relation to the
Carcinogenicity Bioassay
4.4.1 Evidence for Tumor Response
Additional genotoxicity testing in appropriate models may be
conducted for compounds that were negative in the standard 3-test
battery but which have shown effects in carcinogenicity bioassay(s)
with no clear evidence for a nongenotoxic mechanism. To help
understand the mechanism of action, additional testing can include
modified conditions for metabolic activation in in vitro tests or
can include in vivo tests measuring genetic damage in target organs
of tumor induction (e.g., liver UDS test, 32P-postlabeling, mutation
induction in transgenes, molecular characterization of genetic
changes in tumor-related genes).
4.4.2 Structurally Unique Chemical Classes
On rare occasions, a completely novel compound in a unique
structural chemical class will be introduced as a pharmaceutical.
When such a compound will not be tested in chronic rodent
carcinogenicity bioassays, further genotoxicity evaluation may be
invoked.
5. Standard Procedures for In Vitro Tests
Reproducibility of experimental results is an essential
component of research involving novel methods or unexpected
findings; however, the routine testing of chemicals with standard,
widely used genotoxicity tests need not always be completely
replicated. These tests are sufficiently well characterized and have
sufficient internal controls that repetition can usually be avoided
if protocols with built-in confirmatory elements, such as those
outlined below, are used.
For both bacterial and mammalian cell gene mutation tests, the
results of a range-finding test can be used to guide the selection
of concentrations to be used in the definitive mutagenicity test. By
these means, a range-finding test may supply sufficient data to
provide reassurance that the reported result is the correct one. In
bacterial mutagenicity tests, preliminary range-finding tests
performed on all bacterial strains, with and without metabolic
activation, with appropriate positive and negative controls, and
with quantification of mutants, may be considered a sufficient
replication of a subsequent complete test. Similarly, a range-
finding test may also be a satisfactory substitute for a complete
repeat of a test in gene mutation tests with mammalian cells other
than the mouse lymphoma tk assay (see below) if the range-finding
test is performed with and without metabolic activation, with
appropriate positive and negative controls, and with quantification
of mutants (see Note 10).
For the cytogenetic evaluation of chromosomal damage in vitro,
the test protocol includes the conduct of tests with and without
metabolic activation, with appropriate positive and negative
controls, where the exposure to the test articles is 3 to 6 hours
and a sampling time of approximately 1.5 normal cell cycles from the
beginning of the treatment. A continuous treatment without metabolic
activation up to the sampling time of approximately 1.5 normal cell
cycles is needed in case of a negative result for the short
treatment period without metabolic activation. Certain chemicals may
be more readily detected by longer treatment or delayed sampling
times, e.g., some nucleoside analogues or some nitrosamines.
Negative results in the presence of a metabolic activation system
may need confirmation on a case-by-case basis (see Note 11). In any
case, information on the ploidy status should be obtained by
recording the incidence of polyploid cells as a percentage of the
number of metaphase cells. An elevated mitotic index or an increased
incidence of polyploid cells may give an indication of the potential
of a compound to induce aneuploidy. In such cases, further testing
may be needed.
For the mouse lymphoma tk assay, the test protocol includes the
conduct of tests with and without metabolic activation, with
appropriate positive and negative controls, where the exposure to
the test articles is 3 to 4 hours. A continuous treatment without
metabolic activation for approximately 24 hours is needed in case of
a negative result for the short treatment without metabolic
activation (see Note 4). Negative results in the presence of a
metabolic activation system may need confirmation on a case by case
basis (see Note 11). In any case, an acceptable mouse lymphoma tk
assay includes (i) the incorporation of positive controls, which
induces mainly small colonies and (ii) colony sizing for positive
controls, solvent controls, and at least one positive test compound
dose (should any exist), including the culture that gave the
greatest mutant frequency.
Following such testing, further confirmatory testing in the case
of clearly negative or positive test results is not usually needed.
Ideally, it should be possible to declare test results as
clearly negative or clearly positive. However, test results
sometimes do not fit the predetermined criteria for a positive or
negative call and therefore are declared ``equivocal.'' The
application of statistical methods aids in data interpretation,
however, adequate biological interpretation is of critical
importance. Nonetheless, further testing is usually indicated for
equivocal results.
6. Notes
(1) Test approaches currently accepted for the assessment of
mammalian cell gene mutation involve the tk locus using mouse
lymphoma L5178Y cells or human lymphoblastoid TK6 cells, the hprt
locus using CHO cells, V79 cells, or L5178Y cells, or the gpt locus
using AS52 cells.
(2) The molecular dissection of mutants induced at the tk locus
shows a broad range of genetic events including point mutations,
deletions, translocations, recombinations, etc. Small colony mutants
have been shown to predominantly lack the tkb allele as a
consequence of structural or numerical alterations or
recombinational events. There is some evidence that other loci, such
as hprt or gpt are also sensitive to large deletion events. However,
due to the X-chromosomal origin of the hprt gene which is probably
flanked by essential genes, large scale deletion events or numerical
alterations often do not give rise to mutant colonies, thus limiting
the sensitivity of this genetic locus relative to the tk locus for
the detection of a wide range of genetic changes.
(3) With respect to the cytogenetic evaluation of chromosomal
damage, it is not uncommon for the systems currently in use, i.e.,
several systems with permanent mammalian cells in culture and human
lymphocytes either isolated or in whole blood, to give different
results for the same test compound. However, there is evidence that
some of the differences observed have been due to protocol
differences. This may be minimized by using the procedures described
in section 5 of this document.
For the great majority of presumptive genotoxic compounds that
were negative in a bacterial reverse mutation assay, the data on
chromosomal damage in vitro and mouse lymphoma tk results are in
agreement. Several reliable studies indicate that the mouse lymphoma
tk assay is able to detect compounds that induce structural and
numerical chromosomal damage. For safety testing of pharmaceuticals,
the mouse lymphoma tk assay is considered an acceptable alternative
to the direct analysis of chromosomal damage in vitro. Although
colony sizing is an essential element of the mouse lymphoma tk assay
test protocol, it gives only limited information on the type of
damage induced in mutant colonies. Further mechanistic
investigations may be used to assess the nature of cytogenetic
changes induced by clastogens and aneuploidy inducers in the mouse
lymphoma tk assay. Such information could be provided by studies to
demonstrate the loss of the tk gene or the loss of the chromosome
carrying the tk gene.
(4) The detection of a number of different nucleoside analogues
and base analogues is enhanced for the mouse lymphoma tk assay when
the treatment protocol for both agar and microtitre methods includes
a 24-hour treatment regimen in the absence of an exogenous metabolic
activation system. Similarly, the detection of aneuploidy inducers
is enhanced if a 24-hour treatment regimen is used with the
microtitre method. Currently, there is no evidence to support this
conclusion for the soft agar method. The specificity of the test
protocol, i.e., to obtain correct test results for presumptive
nongenotoxic compounds, does not change significantly using a 24-
hour treatment in the microtitre method. For the soft agar method,
there appears to be a reduction in specificity under the same
treatment regimen. Based on this information, the microtitre method
is recommended for use in the standard battery.
(5) There are a small but significant number of genotoxic
carcinogens that are reliably detected by the bone marrow tests for
chromosomal damage that have yielded negative/weak/conflicting
results in the pairs of in vitro tests outlined in the standard
battery options, e.g., bacterial reverse mutation plus one of a
selection of possible tests with cytogenetic evaluation of
chromosomal damage or bacterial mutation plus the mouse lymphoma tk
assay. Carcinogens such as procarbazine, hydroquinone, urethane and
benzene fall into this category.
(6) The continuing evolution of short-term tests and test
methodologies will afford new,
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more sensitive, more practical, more expeditious, and more
economical techniques for detection of genotoxic compounds. Some of
these may ultimately replace the genotoxicity tests used for
regulatory purposes. Among the more promising tests, the in vitro
micronucleus test appears to offer potential for screening purposes.
(7) Some antibacterial agents, albeit highly toxic to the tester
strains, are detected as genotoxic at very low, sublethal
concentrations in the bacterial reverse mutation test (e.g.,
nitrofuran antibiotics).
(8) Certain structurally alerting molecular entities are
recognized as being causally related to the carcinogenic and/or
mutagenic potential of chemicals. Examples of structural alerts
include alkylating electrophilic centers, unstable epoxides,
aromatic amines, azo-structures, N-nitroso-groups, aromatic nitro-
groups.
(9) For some classes of compounds with specific structural
alerts, it is established that specific protocol modifications/
additional tests are necessary for optimum detection of genotoxicity
(e.g., molecules containing an azo-group, glycosides, compounds such
as nitroimidazoles requiring nitroreduction for activation,
compounds such as phenacetin requiring another rodent S9 for
metabolic activation). The additional testing needed when the chosen
3-test battery yields negative results for a structurally alerting
test compound could consist of such modifications.
(10) The dose range-finding study should: (i) Give information
on the shape of the toxicity dose-response curve if the test
compound exhibits toxicity, (ii) include highly toxic
concentrations, and (iii) include quantification of mutants in the
cytotoxic range. If a compound is not toxic, then mutants should
nevertheless be quantified.
(11) A repetition of a test using the identical source and
concentration of the metabolic activation system is usually not
necessary. A modification of the metabolic activation system may be
indicated for certain chemical classes where knowledge is available
on specific requirements of metabolism. This would usually invoke
the use of an external metabolizing system which is known to be
competent for the metabolism/activation of the class of compound
under test.
7. Glossary
Cytogenetic evaluation: Chromosome structure analysis in mitosis
or meiosis by light microscopy.
DNA adduct: (Covalent) binding of chemicals to DNA.
DNA repair: Reconstitution of damaged DNA sequence.
DNA strand breaks: Single or double strand scissions in the DNA.
Numerical chromosome changes: Chromosome numbers different from
the original haploid or diploid set of chromosomes; for cell lines,
chromosome numbers different from the modal chromosome set.
Recombination: Breakage and balanced or unbalanced rejoining of
DNA.
Transgene: An exogenous or foreign gene inserted into the host
genome, into either somatic cells or germ line cells.
Dated: November 15, 1997.
William K. Hubbard,
Associate Commissioner for Policy Coordination.
[FR Doc. 97-30706 Filed 11-20-97; 8:45 am]
BILLING CODE 4160-01-F