[Federal Register Volume 59, Number 98 (Monday, May 23, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-12492]
[[Page Unknown]]
[Federal Register: May 23, 1994]
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Part II
Department of Health and Human Services
_______________________________________________________________________
Food and Drug Administration
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21 CFR Parts 173 and 573
Secondary Direct Food Additives Permitted in Food for Human
Consumption; Food Additives Permitted in Feed and Drinking Water of
Animals; Aminoglycoside 3'-Phosphotransferase II; Final Rule
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration
21 CFR Parts 173 and 573
[Docket No. 93F-0232]
Secondary Direct Food Additives Permitted in Food for Human
Consumption; Food Additives Permitted in Feed and Drinking Water of
Animals; Aminoglycoside 3'-Phosphotransferase II
AGENCY: Food and Drug Administration, HHS.
ACTION: Final rule.
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SUMMARY: The Food and Drug Administration (FDA) is amending the food
additive regulations to provide for the safe use of aminoglycoside 3'-
phosphotransferase II (APH(3')II) as a processing aid in the
development of new varieties of tomato, oilseed rape, and cotton.
APH(3')II is a protein encoded by the kanamycin resistance (kanr)
gene. This action is in response to a petition filed by Calgene, Inc.
DATES: Effective May 23, 1994; written objections and requests for a
hearing by June 22, 1994.
ADDRESSES: Submit written objections to the Dockets Management Branch
(HFA-305), Food and Drug Administration, rm. 1-23, 12420 Parklawn Dr.,
Rockville, MD 20857.
FOR FURTHER INFORMATION CONTACT: Nega Beru, Center for Food Safety and
Applied Nutrition (HFS-206), Food and Drug Administration, 200 C St.,
SW., Washington, DC 20204, 202-254-9523.
SUPPLEMENTARY INFORMATION:
Table of Contents
I. Introduction
A. Regulatory History
B. Scope of the Regulation
C. Determination of Safety
II. Use of the kanr Gene as a Selectable Marker in Transgenic
Plants
A. Background
B. Need for a Selectable Marker
C. Identity of the Additive
D. Use and Intended Technical Effects
III. Safety Evaluation
A. APH(3')II
1. Direct effects of ingestion
2. Effects on the therapeutic efficacy of orally administered
antibiotics
a. APH(3')II in human foods
b. APH(3')II in animal feed
B. The kanr Gene
1. Potential transfer of the kanr gene to intestinal
microorganisms and cells lining the intestinal lumen
a. Relevant source of kanr gene available for possible
transformation
b. Effect of digestion on the availability of the kanr gene
for transformation
c. Calculation of worst-case transformation frequencies
2. Potential transfer of the kanr gene to soil
microorganisms
3. Food Advisory Committee discussions regarding potential
horizontal transfer of the kanr gene
4. Agency conclusions
IV. Response to Comments
A. Regulatory Issues
B. Food Safety
1. Glycosylation
2. In vitro digestibility studies
3. Copy number of the kanr gene and expression level of
APH(3')II
4. The potential for side effects from consumption of
genetically engineered foods
5. Relevance of clinical studies
C. Possible Effect on Clinical Efficacy of Orally Administered
Kanamycin or Neomycin.
D. Fate of the kanr Gene in the Environment
1. Potential transfer of the kanr gene from crops to
microorganisms
2. Potential transfer of the kanr gene to other crops and
to wild relatives
E. Possible Effects of Consumption of Animal Feeds Containing
APH(3')II on Animals and Their Gut Microflora
F. Labeling of Foods Containing the kanr Gene and APH(3')II
V. Conclusions
VI. Inspection of Documents
VII. Environmental Impact
VIII. Objections
IX. References
I. Introduction
A. Regulatory History
In accordance with 21 CFR 10.85, Calgene, Inc., submitted to FDA on
November 26, 1990, a request for advisory opinion regarding whether the
kanr gene, a selectable marker, may be used in the production of
genetically engineered tomato, cotton, and oilseed rape plants intended
for human food and animal feed uses (kanr Gene: Safety and use in
the production of genetically engineered plants, Docket Number 90A-
0416). In the Federal Register of May 1, 1991 (56 FR 20004), FDA
announced that the request had been received and solicited comments
from interested persons. The data submitted to the agency with the
request for advisory opinion and the comments received were made
available to the public at the Dockets Management Branch.
Subsequent to the submission of the request for advisory opinion,
FDA published its ``Statement of Policy: Foods Derived From New Plant
Varieties'' (the 1992 policy statement) in the Federal Register of May
29, 1992 (57 FR 22984). This policy statement clarified FDA's
interpretation of the Federal Food, Drug, and Cosmetic Act (the act)
with respect to human foods and animal feeds derived from new plant
varieties, including plants developed by new methods of genetic
modification such as recombinant deoxyribonucleic acid (DNA)
techniques.
In the 1992 policy statement, FDA stated that the postmarket
authority under section 402(a)(1) of the act (21 U.S.C. 342(a)(1))
would continue to be the primary legal tool for ensuring the safety of
whole foods derived from genetically modified plants. FDA also noted
that under the statutory definition of ``food additive'' in section
201(s) of the act (21 U.S.C. 321(s)), the transferred genetic material
and the intended expression products could be subject to regulation as
food additives, if such material or expression products were not
generally recognized as safe (GRAS) (57 FR 22984 at 22990). FDA further
stated that the agency would use its food additive authority to the
extent necessary to ensure public health protection (such as when an
intended expression product in a food differs significantly in
structure, function, or composition from substances found currently in
food) (57 FR 22984 at 22990).
The 1992 policy statement specifically discussed selectable markers
that provide antibiotic resistance in product selection and
development. With such markers, both the antibiotic resistance gene and
the gene product, unless removed, are expected to be present in foods
derived from such plants. FDA stated:
Selectable marker genes that produce enzymes that inactivate
clinically useful antibiotics theoretically may reduce the
therapeutic efficacy of the antibiotic when taken orally if the
enzyme in the food inactivates the antibiotic. FDA believes that it
will be important to evaluate such concerns with respect to
commercial use of antibiotic resistance marker genes in food,
especially those that will be widely used.
(See 57 FR 22984 at 22988.)
Subsequently, in January 1993, Calgene requested that FDA convert
its request for advisory opinion to a food additive petition under
section 409 of the act. FDA then announced in the Federal Register of
July 16, 1993 (58 FR 38429), that a food additive petition (FAP 3A4364)
had been filed by Calgene, Inc., 1920 Fifth St., Davis, CA 95616,
proposing that the food additive regulations be amended to provide for
the safe use of APH(3')II as a processing aid in the development of new
varieties of tomato, oilseed rape, and cotton.
After completing its review of the data submitted by Calgene, FDA
convened a public meeting of its Food Advisory Committee on April 6
through 8, 1994, to undertake a scientific discussion of the agency's
approach to evaluating the safety of whole foods produced by new
biotechnologies; a genetically modified tomato developed by Calgene
containing the kanr gene served as an example and focus of the
discussion. The membership of the standing committee was supplemented
with temporary members and consultants to the committee, representing
scientific disciplines appropriate to the evaluation of foods derived
from new plant varieties developed using recombinant DNA techniques.
At the meeting, Calgene presented a summary of the data they
considered adequate to show safety of the tomato, and FDA presented its
evaluation of the data. The committee was asked to comment on the
approach used by FDA to evaluate whole foods and specifically, on the
approach used for the Calgene tomato (Ref. 1). During committee
discussion of the Calgene and FDA presentations, the committee members
generally expressed the view that the approach used by FDA to evaluate
the safety of the tomato, including the safety of the kanr gene,
was appropriate and that all relevant scientific questions had been
adequately addressed.
In regard to the use of the kanr gene, Calgene and the agency
presented, and the committee discussed, such issues as the potential
allergenicity of APH(3')II and the potential for ingested APH(3')II to
inactivate orally administered antibiotics. Most of the discussion
concerning the kanr gene focused on the potential transfer of the
gene to microorganisms in the gastrointestinal (GI) tract or in the
environment. In evaluating Calgene's food additive petition for the use
of the kanr gene product, APH(3')II, in the development of new
varieties of tomato, oilseed rape, and cotton, FDA has considered the
committee's discussions and recommendations on this subject, which are
summarized in section III.B.3. of this document.
B. Scope of the Regulation
Having completed its evaluation and having considered the
deliberations of the Food Advisory Committee, the agency is amending
the food additive regulations to permit the use of APH(3')II in the
development of genetically modified tomatoes, oilseed rape, and cotton
intended for food use. Only the translation product of the kanr
gene, APH(3')II, and not the gene itself, is being regulated as a food
additive. As the 1992 policy statement indicated, FDA does not
anticipate that transferred genetic material (deoxyribonucleic acid
(DNA)) would itself be regulated as a food additive (57 FR 22984 at
22990). DNA is present in the cells of all living organisms, including
every plant and animal used for food by humans or animals, and is
efficiently digested (Ref. 2). In this respect, the DNA that makes up
the kanr gene does not differ from any other DNA and does not
itself pose a safety concern as a component of food.
This final rule is being promulgated after consideration of the
issues relating to the safety of the use of APH(3')II in the selection
of transgenic plants. In addition, as noted above, because of the
property of the kanr gene to confer antibiotic resistance, the
agency has considered the possibility that the gene might be
transferred to other organisms (discussed in section III.B. of this
document).
Potential safety issues specific to particular food products that
contain the kanr gene are not addressed by the agency in this
document because such issues are beyond the scope of this rulemaking.
For example, issues associated with other co-transferred DNA sequences,
including other genes intended to impart specific traits, and issues
related to potential genetic instability are not addressed because such
issues will vary with specific products.
Developers of new plant varieties are responsible for addressing
potential safety issues associated with specific food products
resulting from the transfer of genetic materials and for ensuring the
safety of the food products that they market. The policy statement
contains a ``Guidance to Industry'' section (57 FR 22984 at 22991) that
outlines an approach for the safety evaluation of foods derived from
transgenic plants and suggests that the agency be consulted, as needed,
to resolve critical issues.
As noted, issues related to genetic instability are not addressed
because such issues are not unique to the kanr gene but apply to
any transferred genetic material irrespective of the transfer
techniques used. Genetic instability could arise as a result of
insertion of multiple copies of a given construct, especially if
insertion occurs at multiple loci. Recombinations of the transferred
DNA could cause deletions, duplications, or rearrangements within the
plant genome (Ref. 3). Hence, in the 1992 policy statement, the agency
noted that the genetic stability of a new plant variety is an important
safety consideration and further stated that, ``Factors that favor
stability include a minimum number of copies of the introduced genetic
material, and insertion at a single site.'' (57 FR 22984 at 23004).
In developing new plant varieties, developers are therefore
responsible for following good manufacturing and good agricultural
practices to ensure that they have developed a genetically stable
transgenic plant. As a practical matter, this would ordinarily include
using such techniques as segregation and Southern blot analysis to
ensure that new plant varieties chosen for development have the new
genetic material inserted into a single locus and that the number of
copies of inserted DNA at a given site is limited to the minimum
sufficient to achieve the intended effect.
C. Determination of Safety
Under section 409(c)(3)(A) of the act, a food additive cannot be
approved for a particular use unless a fair evaluation of the data
available to FDA establishes that the additive is safe for that use.
The concept of safety embodied in the Food Additives Amendment of 1958
is explained in the legislative history of the provision: ``Safety
requires proof of a reasonable certainty that no harm will result from
the proposed use of an additive. It does not--and cannot--require proof
beyond any possible doubt that no harm will result under any
conceivable circumstance.'' (H. Rept. 2284, 85th Cong., 2d sess.
(1958)). FDA has incorporated this concept of safety into its food
additive regulations. Under 21 CFR 170.3(i), a food additive is
``safe'' if ``there is a reasonable certainty in the minds of competent
scientists that the substance is not harmful under the intended
conditions of use.''
The agency has reviewed the data and studies submitted in the
request for advisory opinion, material that was submitted subsequent to
the conversion of the request for advisory opinion to a food additive
petition, the deliberations of the Food Advisory Committee that took
place at the April 1994 meeting, as well as other information in its
files. In addition, the agency has considered the comments that were
received in response to the Federal Register notice announcing receipt
of the request for advisory opinion. The comments are addressed in
section IV. of this document. As discussed below, FDA has concluded,
based upon its review, that the use of aminoglycoside 3'-
phosphotransferase II is safe for use as a processing aid in the
development of new varieties of tomato, oilseed rape, and cotton
intended for food use.
II. Use of the kanr Gene As a Selectable Marker in Transgenic
Plants
A. Background
Developers have for many years used plant breeding techniques to
introduce desirable genetic traits into new varieties that can be used
in agriculture. Traditionally, breeders have relied on selection of
mutants and on hybridization between different varieties of the same
species to achieve this goal. More recently, recombinant DNA techniques
(commonly referred to as ``genetic engineering'' techniques) have come
into use to generate new plant varieties with desirable
characteristics. Recombinant DNA techniques involve the isolation, and
subsequent introduction into a host plant, of discrete DNA segments
containing the gene(s) of interest. This introduction of exogenous DNA
into a cell, resulting in its acquisition of a new phenotype, is
commonly referred to as ``transformation,'' and transformed plants that
contain genetic material derived from sources other than the host plant
itself are called transgenic.
The desired gene(s) may be introduced into a host plant by one of
several methods, including: (1) Direct DNA uptake by the plant cells
mediated by chemical or electrical treatments; (2) microinjection of
DNA directly into plant cells; (3) biolistics, or firing tiny particles
coated with the DNA of interest into plant cells; and (4) the use of a
bacterium, such as the soil bacterium Agrobacterium tumefaciens, as a
vehicle to carry the DNA into plant cells. (For a discussion of these
processes, see Ref. 4).
B. Need for a Selectable Marker
Transformation of plant cells by introducing exogenous DNA is an
inefficient process and, in general, only a small proportion of cells
will successfully take up, integrate, and express the new genetic
material (Ref. 5). Further, the few cells that do so are not readily
distinguishable from the vast majority of cells that do not. Therefore,
developers of transgenic plants need a means to distinguish cells that
are successfully transformed from those that are not. Selectable
markers, such as the kanr gene, perform this function.
The kanr gene is linked to the gene (or genes) of interest and
then this genetic material is inserted into plant cells. Because plant
cells are sensitive to the antibiotic kanamycin, incorporation of the
kanr gene into cells and subsequent expression of APH(3')II
provides a convenient method for selecting successfully transformed
cells. Kanr works as a marker because only successfully
transformed cells (which contain both the kanr and the desired
genetic material) survive when grown in a kanamycin-containing medium.
These cells are subsequently regenerated into transgenic plants.
C. Identity of the Additive
APH(3')II1 (CAS Reg. No. 58943-39-8) is encoded by the
kanr gene, which was originally isolated as a component of
transposon Tn52 from the bacterium Escherichia coli (Refs. 6 and
7). APH(3')II is an enzyme with an apparent molecular weight of 25,000
that catalyzes the transfer of a phosphate group from adenosine 5'-
triphosphate (ATP) to a hydroxyl group of aminoglycoside antibiotics
(see below), thereby inactivating the antibiotics.
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\1\Other names for this enzyme include neomycin
phosphotransferase II (NPT II), neomycin phosphotransferase, and
kanamycin phosphotransferase II.
\2\A transposon is a segment of DNA that is mobile and has the
capacity to move from one site in the genome to another. Transposons
vary in size and frequently contain, as does Tn5, antibiotic
resistance genes in addition to genes coding for functions concerned
with movement of the transposon.
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APH(3')II inactivates the aminoglycoside antibiotics neomycin,
kanamycin, paromomycin, ribostamycin, gentamicins A and B, as well as
butirosins (Refs. 8 and 9). Of the antibiotics that are inactivated by
APH(3')II, only neomycin and kanamycin are currently approved for use
in humans or animals in the United States (Refs. 10 and 11).3
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\3\Gentamicin, which is used therapeutically, is composed of a
complex mixture of the antibiotic substances produced by
Micromonospora purpurea that contain primarily gentamicin C1
(25 to 50 percent), gentamicin C1a (10 to 35 percent), and
gentamicins C2a and C2 (25 to 55 percent) (Ref. 10).
Gentamicins A and B are at most minor components of the commercial
drug. Thus, APH(3')II does not confer resistance to gentamicin that
is used therapeutically (Ref. 12).
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The APH(3')II evaluated in this document is the enzyme whose
synthesis is directed by the kanr gene derived from transposon
Tn5. This enzyme is not to be confused with enzymes that may be
similarly named (e.g., a type I aminoglycoside phosphotransferase
encoded by a gene isolated from transposon Tn601) or other bacterial
enzymes (including acetyltransferases, nucleotidyltransferases, and
phosphotransferases) that inactivate kanamycin and neomycin (Refs. 8
and 12).
D. Use and Intended Technical Effects
Aminoglycoside antibiotics exert their effect on bacteria by
binding to bacterial ribosomes and inhibiting protein synthesis.
Phosphorylation of the antibiotics by APH(3')II interferes with this
binding and thus prevents the antibiotics from inhibiting protein
synthesis (Ref. 13). In this way, cells that contain the kanr gene
and that express APH(3')II are rendered resistant to the action of the
antibiotics. In plant cells, the antibiotics exert their effect on
mitochondria and chloroplasts where protein synthesis takes place on
ribosomes that resemble bacterial ribosomes (Ref. 14).
The proposed use of the kanr gene and gene product APH(3')II
is as a processing aid in the development of new varieties of tomato,
cotton, and oilseed rape intended for food use. As discussed above,
because transformation of plant cells is an inefficient process, the
presence of APH(3')II and the consequent ability of the plant cells to
grow in the presence of antibiotics is used to distinguish between
transformed and nontransformed cells. Therefore, the intended technical
effect of APH(3')II is to permit, in the early phases of development of
genetically modified plants, the selection of transformants carrying
the kanr gene along with the genetic material of interest.
However, APH(3')II has no intended technical effect in the final plant
or final crop product.
III. Safety Evaluation
A. APH(3')II
Safety issues associated with APH(3')II can be divided into two
areas: (1) Those associated with the direct effects of ingestion of the
protein, including the possibility of allergenicity; and (2) those
associated with the biological activity of APH(3')II (i.e., the effect
of the enzyme on the therapeutic efficacy of orally administered
antibiotics).
1. Direct Effects of Ingestion
Calgene provided evidence that APH(3')II is rapidly inactivated by
stomach acid, is degraded by digestive enzymes, and is not modified by
glycosylation (i.e., does not contain sugar molecules attached to the
protein) when produced in the transgenic plants under consideration. In
addition, Calgene noted that enzymes such as APH(3')II are heat labile.
Thus, Calgene concluded that APH(3')II does not possess any of the
characteristics associated with allergenic proteins such as proteolytic
stability, glycosylation, or heat stability (Ref. 15). In April 1992,
Calgene also conducted protein and DNA sequence comparisons using
sequences in four separate databases (GenBank, EMBL, PIR 29, and Swiss-
Prot) and established that APH(3')II does not have significant homology
to any proteins listed as food allergens or toxins in these databases.
FDA agrees with Calgene that the characteristics of APH(3')II do
not raise a safety concern. First, each whole food, on average,
contains several thousands of different proteins (Ref. 16). As a class,
proteins are rarely toxic (Ref. 17) and APH(3')II is not known to be
toxic. Second, APH(3')II is a phosphorylating enzyme, and all plants
and animals that are part of the food supply contain such
phosphorylating enzymes without adverse consequences. Third, APH(3')II
has been shown to be rapidly degraded under simulated gastric
conditions (Refs. 18 through 21). Finally, the estimated dietary
exposure to APH(3')II is very low (480 g APH(3')II per person
per day,4 or 0.16 part per million in the diet, based on a 100-
percent market share for tomatoes containing APH(3')II (Ref. 18)).
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\4\Because oils produced from transgenic cottonseed and rapeseed
would not contribute APH(3')II to the human diet (see also section 2
below), the exposure estimate was derived exclusively for tomatoes.
The agency made several conservative assumptions in arriving at the
probable per capita exposure to APH(3')II of 480 g/person/
day. For example, FDA assumed that all tomatoes contain APH(3')II at
a level of 0.1 percent of total protein although, of the two lines
intended for commercialization by Calgene, one contains less than
0.01 percent and the other less than 0.002 percent of APH(3')II (as
a percentage of total protein). Second, FDA included APH(3')II in
processed products in its estimate although high temperature
treatment used in the production of processed products would be
expected to result in loss of enzymatic activity of APH(3')II. In
summary, the exposure estimate represents a theoretical maximum
rather than a realistic estimate of exposure to APH(3')II.
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Based upon the available evidence, the agency believes that this
protein does not possess any properties that would distinguish it
toxicologically from other phosphorylating enzymes in the food supply.
Further, because of the low exposure levels and normal digestibility of
APH(3')II, the agency concludes that no limits other than good
manufacturing practice are needed to ensure the safety of the
petitioned use of APH(3')II (Ref. 20).5
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\5\A recently published study (Ref. 22) also showed that
APH(3')II is rapidly degraded under simulated mammalian digestive
conditions. In addition, in an acute mouse feeding study, the
investigations showed that feeding highly exaggerated doses of
purified APH(3')II caused no deleterious effects.
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2. Effects on the Therapeutic Efficacy of Orally Administered
Antibiotics
a. APH(3')II in human foods. i. Relevant source of APH(3')II.
Calgene considered whether APH(3')II could affect the therapeutic
efficacy of orally administered aminoglycoside antibiotics. In doing
so, Calgene stated that only APH(3')II from fresh tomatoes is relevant
because it is the only form that is enzymatically active. Processed
tomato products (such as processed whole tomatoes, chili, juice, pulp,
paste, catsup, and soup) are subjected to temperatures in the range of
82 to 100 deg.C; these temperatures would be expected to inactivate
the APH(3')II enzyme. For edible oils extracted from cottonseed and
rapeseed, high temperature treatment, solvent extraction, and
subsequent purification steps generally included in the processing of
such oils would also be expected to inactivate APH(3')II.
FDA agrees that high temperature treatment denatures proteins and
inactivates enzymes and therefore, processed products that contain
tomatoes with the kanr gene are unlikely to contain any
enzymatically active APH(3')II. In addition, purified oils essentially
do not contain protein; therefore, oils derived from transgenic
cottonseed and rapeseed modified using the kanr gene would not be
expected to contain active or inactive APH(3')II (Refs. 18 and 23).
Thus, FDA agrees that fresh tomatoes from plants developed using the
kanr gene are the only source of active APH(3')II.
ii. Effect of APH(3')II in fresh tomatoes on the therapeutic
efficacy of orally administered antibiotics. Calgene performed several
experiments intended to address whether APH(3')II consumed as a
component of fresh tomatoes could render orally-administered kanamycin
ineffective. These experiments were performed under simulated gastric
and intestinal conditions (i.e., appropriate pH, reagent
concentrations, temperature, and reaction times) chosen to reflect
conditions expected in vivo. In some studies both tomato extract and
nonfat milk were added to determine whether the presence of additional
food-source proteins in the simulated gastric and intestinal fluids
might slow the proteolytic degradation of APH(3')II by competition.
After evaluating the loss of immunologically detectable APH(3')II,
Calgene concluded that, under normal gastric and intestinal conditions,
APH(3')II would be effectively degraded before the enzyme could
inactivate kanamycin or neomycin and therefore, APH(3')II would not
interfere with orally administered kanamycin or neomycin therapy. The
results of Calgene's experiments were the same whether done in the
presence or the absence of tomato extract and nonfat milk.
In addition, Calgene presented the results of in vitro degradation
studies performed under simulated abnormal gastric conditions, such as
may exist in patients treated with drugs that reduce stomach acidity.
Calgene stated that these studies demonstrated that APH(3')II is not
degraded in neutralized (pH 7.0) simulated gastric fluid and thus,
APH(3')II may remain active in such abnormal gastric conditions.
However, Calgene pointed out that, even under those conditions,
APH(3')II would not be expected to inactivate orally administered
kanamycin or neomycin because the concentration of ATP, which the
enzyme requires to inactivate kanamycin and neomycin, would be
limiting. In support of this contention, Calgene presented data from
the published literature on ATP levels in fresh fruits and vegetables.
Calgene then estimated ATP intake and calculated the fraction of
neomycin that would be phosphorylated assuming that all of the
available ATP reacted with the antibiotic. Under the worst-case
situation (high intake of ATP-containing food, low dose of antibiotic)
Calgene's calculations showed that only a small fraction (no more than
1.5 percent) of the antibiotic would be inactivated. Moreover, Calgene
presented data that showed that no significant inactivation of
kanamycin was observed during in vitro studies conducted with tomato
extract containing APH(3')II and kanamycin over a 4-hour incubation
period.
iii. Agency conclusions. The agency has evaluated the data and
other information presented by Calgene (Refs. 18 through 21 and 24).
FDA agrees that Calgene's in vitro digestion studies show that, as is
the case for dietary protein in general, the biological activity of
APH(3')II is destroyed during gastric and intestinal phases of
digestion. Further, the agency has determined that any active APH(3')II
that might remain would not significantly inactivate kanamycin or
neomycin in the gut because the small amount of ATP in fruits and
vegetables would limit the amount of antibiotic that could be
phosphorylated. ATP is an extremely labile molecule that is susceptible
to inactivation both by heat (e.g., cooking) and by enzymes, such as
alkaline phosphatases (Ref. 25), that are found in the intestine.
Because the ATP in meat, poultry, fish, and cooked vegetables would be
broken down by cooking, the primary source of ATP in the
gastrointestinal (GI) tract of patients would be uncooked fruits and
vegetables. However, the amount of ATP in a variety of fruits and
vegetables would provide enough ATP to inactivate only a small
percentage of kanamycin or neomycin, even if one makes the conservative
assumption that all of the ATP in these fruits and vegetables would
survive the alkaline phosphatases in the intestines and would be
available for catalytic phosphorylation of kanamycin or neomycin.
In addition, the agency has considered the patient population
likely to be exposed to aminoglycoside antibiotics. Oral
aminoglycosides are most commonly administered to either pre-operative
patients (prior to bowel surgery) or patients with hepatic
encephalopathy. Neither patient population would be expected to be
ingesting tomatoes or any other fresh fruits and vegetables; therefore
there is little or no risk of inactivating the oral antibiotic in these
patients (Refs. 24 and 26). For these reasons, FDA concludes that the
presence of APH(3')II in food will not compromise the therapeutic use
of orally administered kanamycin or neomycin.
b. APH(3')II in animal feed. Calgene also considered the potential
inactivation of neomycin that is used in animal feeds manufactured
using cottonseed meal and rapeseed meal obtained from transgenic
plants. The transgenic tomato was not considered because only small
amounts of tomato and tomato byproducts are used in the animal feed
industry. Further, neomycin is primarily used to treat calves and swine
whereas tomato byproducts, to the extent that they are used in animal
feed, are primarily used as ingredients in cattle diets (Ref. 27).
Calgene analyzed neomycin levels both in nontransgenic medicated
cottonseed and rapeseed meals and in transgenic medicated cottonseed
and rapeseed meals over a storage period of 56 days (considered a
worst-case situation) and concluded that there was no significant
inactivation of neomycin.
FDA reviewed the data submitted by Calgene and concludes that there
was no significant difference with respect to neomycin stability
between medicated cottonseed and rapeseed meals prepared from
transgenic cottonseed and rapeseed containing APH(3')II, and
appropriate controls (Ref. 28). Therefore, the agency concludes that
transgenic strains of cottonseed and rapeseed containing APH(3')II have
no apparent untoward effect regarding the stability of neomycin and
that the therapeutic efficacy of neomycin in animal feed will not be
affected. The agency also considers this conclusion applicable to other
aminoglycoside antibiotics, e.g., gentamicin, when orally administered.
B. The Kanr Gene
The agency also evaluated issues relevant specifically to the
safety of the use of the kanr gene in tomato, oilseed rape, and
cotton. In particular, FDA evaluated the potential for horizontal
transfer of the gene and subsequent expansion of the population of
antibiotic-resistant pathogens. The agency evaluated whether efficacy
of oral antibiotic treatment of humans or animals could be compromised
by consumption of food containing the kanr gene either because of
the development of resistant intestinal microflora in humans and
animals or because the cells lining the intestinal lumen might become
transformed. In addition, the agency considered the possible transfer
of the kanr gene from transgenic plants to soil microorganisms and
expansion of the antibiotic-resistant bacterial population.
1. Potential Transfer of the kanr Gene to Intestinal
Microorganisms and Cells Lining the Intestinal Lumen
Calgene presented theoretical and experimental evidence to
demonstrate that the potential for compromise of antibiotic therapy by
horizontal transfer of the kanr gene to gut microorganisms or
intestinal epithelial cells is not of significant concern. Calgene
considered the sources of the kanr gene, the role digestion plays
in degrading DNA, and possible DNA transfer mechanisms.
a. Relevant source of the kanr gene available for
transformation. Calgene considered potential transfer of the kanr
gene only from fresh tomatoes because processing is expected to
inactivate the kanr gene in processed tomato products and in food
products derived from cotton and oilseed rape. The kanr gene is
not expected to survive procedures used to process tomatoes because
heating processes, such as those used in commercial processing, can
directly degrade DNA or can damage DNA by releasing cellular DNA-
degrading enzymes.
The kanr gene is also not expected to survive the process of
oil production from cottonseed and rapeseed. Mechanical grinding or
flaking of oilseeds during the production of oils and meals from
oilseeds is expected to liberate degradative enzymes normally present
within the cell that would degrade the kanr gene. In addition, oil
processing also includes high temperatures and solvent extractions,
both of which would be expected to inactivate the kanr gene.
Moreover, because DNA is hydrophilic, it is unlikely to fractionate
into oil, which is hydrophobic, during the extraction of oil from
cottonseed and rapeseed. Therefore, intact DNA, including the kanr
gene, is not expected to survive the production of oils and animal
feeds from cottonseed and rapeseed.
b. Effect of digestion on the availability of the kanr gene
for possible transformation. Calgene demonstrated that most if not all
of the DNA comprising the kanr gene ingested by humans will be
degraded in the stomach and upper small intestine before it reaches the
lower small intestine, cecum, and colon, and would be unavailable for
potential transformation of gut microorganisms. Calgene estimated that
99.9 percent of fresh tomato DNA would be digested to fragments smaller
than 1,000 base pairs. This estimate was based on in vitro studies that
found that only 0.1 percent of DNA could be detected as fragments of
1,000 base pairs or longer after exposure to stomach-simulating fluids
for 10 minutes and to intestinal-simulating fluids for another 10
minutes. Thus most of the DNA remaining after digestion would be
smaller than the kanr gene which is about 1,000 base pairs long.
Regarding animal feed, food-producing animals consume primarily
processed forms of cottonseed and rapeseed, in which, as discussed
above, the kanr gene is not expected to remain intact. In
addition, researchers have shown that nucleic acids introduced into the
rumens of calves, or incubated with calf, sheep, or cow rumen contents
in vitro, were rapidly and completely degraded to nucleotides and
nucleosides (Ref. 29).
c. Calculation of worst-case transformation frequencies. In its
submission, Calgene addressed the potential for horizontal transfer of
the kanr gene. Natural transformation, i.e., the uptake and
incorporation into the genome of free DNA, is known to occur in some
bacterial species. This is the only possible mechanism by which
intestinal microflora could take up free DNA (Ref. 30). However, none
of the species known to be present in the GI tract has been found
capable of acquiring exogenous DNA by natural transformation.
Nonetheless, to consider the worst-case scenario, Calgene assumed that
all microbes in the intestine would be able to take up and incorporate
exogenous DNA at a frequency found for certain species of the genus
Streptococcus. Calgene noted that although the firm developed its
transformation model for certain Streptococcus species, they are not
aware of any information indicating that Streptococcus species found in
the GI tract can be naturally transformed.
To undergo natural transformation, the recipient bacterium must be
transformation-competent, i.e., ready to take up DNA. As noted, none of
the bacterial species that occur in the GI tract is known to be capable
of becoming transformation-competent. In addition, the genome of a
recipient bacterium should contain DNA homologous to the incoming DNA
(Refs. 31 and 32). Because the genomes of intestinal Streptococci or
other intestinal bacteria are not expected to exhibit homology to the
DNA constructs containing the kanr gene6, Calgene assumed
that the kanr gene could only undergo ``illegitimate''
recombination, a process that does not require significant DNA
homology. Calgene noted that illegitimate recombination occurs in
microorganisms at a much lower rate than homologous recombination.
---------------------------------------------------------------------------
\\6One population that does contain DNA segments homologous with
part of the kanr construct is E. coli, because the kanr
construct contains part of an E. coli gene. Although E. coli
constitutes one of the predominant species of aerobic GI tract
bacteria, E. coli is not transformation-competent under conditions
that prevail in the GI tract (Ref. 33). Thus, transformation of E.
coli due to homologous recombination is not an issue.
---------------------------------------------------------------------------
Under the foregoing worst-case assumptions, Calgene estimated that
if a person consumes fresh tomatoes at the 90th percentile level (i.e.,
eats more tomatoes than 89 percent of the individuals in the
population), the transformation frequency of the intestinal
microorganisms with the kanr gene will be approximately
3 x 10-15 transformants per day. This transformation frequency is
more than 5 orders of magnitude less than the frequency of mutation to
kanamycin resistance per bacterial replication, i.e., 10-9 (Ref.
12). Thus, Calgene showed that for every 300,000 bacteria that mutate
to kanamycin resistance per replication (generally a matter of hours),
there would be, at most, under worst-case conditions, one kanamycin-
resistant bacterium per day added to that number due to transformation.
Calgene stated that the potential for food-producing animals to
experience decreased efficacy of antibiotic therapy as a result of
pathogenic intestinal microflora incorporating and expressing the
kanr gene would be similar to that described for humans, i.e.,
equally improbable. In reaching this conclusion, Calgene relied on the
finding that DNA is rapidly and completely digested in the gut of food
animals (Ref. 29) and on the contention that the worst-case
transformation scenario described above for human gut microorganisms
also applies to microorganisms found in the gut of food-producing
animals.
With respect to epithelial cells lining the intestinal lumen,
Calgene provided information that no transformation of human epithelial
cells has been demonstrated in vivo (Ref. 2). In addition, even if
transformed, intestinal epithelial cells are terminally differentiated
(i.e., do not divide) and have a relatively short life span (Ref. 34),
and thus would continually be shed and replaced by nontransformed
cells.
2. Potential Transfer of the kanr Gene to Soil Microorganisms
Calgene also considered the possibility that the kanr gene
might be transferred to soil microorganisms, thereby increasing the
level of antibiotic-resistant organisms in the environment. Calgene
pointed out that the only plausible mechanism by which gene transfer
could occur between plants and bacteria is through natural
transformation. Taking this mechanism into consideration and using
worst-case assumptions similar to those discussed above for intestinal
microorganisms, Calgene calculated that, at worst, kanamycin-resistant
transformants resulting from plant DNA left in the fields would
represent not more than one in 10 million of the existing kanamycin-
resistant soil population.
3. Food Advisory Committee Discussions Regarding Potential Horizontal
Transfer of the Kanr Gene
As part of its discussion of the scientific issues related to the
evaluation of Calgene's genetically engineered tomato, the Food
Advisory Committee discussed the possibility that the kanr gene
might be transferred to microorganisms in the GI tract and in the
environment (Ref. 1).
The committee members concluded that transfer of the kanr gene
consumed as a component of tomatoes to microorganisms in the GI tract
was highly unlikely based on published data in the scientific
literature. Similarly, the committee members judged that the potential
for transfer of the kanr gene from plants to microorganisms in the
environment is highly unlikely based on the members' knowledge of
mechanisms of gene transfer. In addition, members of the committee
pointed out that the rate at which such transfer could take place, if
at all, was of so small a magnitude that, coupled with the high
prevalence of kanamycin resistant organisms already present in the
environment, it would not cause a significant environmental impact.
Some members of the committee, while convinced by the information
presented at the meeting that the transfer of the kanr gene from
tomato plants to microorganisms in the soil was improbable, expressed
concern regarding the use of the kanr gene in other crops that may
be grown on a wide scale. In addition, some committee members were
concerned that a determination of safety with regard to the use of
kanr gene in Calgene's tomato might signal to producers that it is
now permissible to use the kanr gene in other crops. In light of
such concerns, these committee members advised that use of the
kanr gene in other crops should be evaluated on a case-by-case
basis.
4. Agency Conclusions
The agency has considered the recommendations of the members of the
Food Advisory Committee. The agency agrees that the potential transfer
of the kanr gene, as well as other antibiotic resistance marker
genes, from crops to microorganisms should be evaluated on a case-by-
case basis. As noted, Calgene petitioned for the use of the kanr
gene product, APH(3')II, in the development of genetically engineered
cotton and oilseed rape in addition to tomato. As discussed below, the
agency has evaluated data and information concerning horizontal
transfer of the kanr gene from its use in all three crops. This is
consistent with the committee's advice that safety of the use of the
kanr gene be evaluated on a case-by-case basis. In addition,
Calgene's petition seeks to amend the food additive regulations to
permit the use of APH(3')II only in tomato, cotton, and oilseed rape;
approval of Calgene's petition would not mean that developers could use
the kanr gene in crops other than those identified in the
petition.
FDA has also evaluated the information submitted by Calgene and has
determined that the probability of transfer of the kanr gene to
gut microflora is remote and that even under worst-case conditions, the
number of microorganisms that would be converted to kanamycin
resistance is negligible when compared to the reported prevalence of
gut microflora that are already resistant to kanamycin (Ref. 35). This
conclusion applies to both humans and animals. The agency has
determined that exposure to foods that contain the kanr gene will
not compromise the efficacy of antibiotic treatment because the
likelihood of increasing the number of antibiotic resistant
microorganisms is extremely low. Further, the agency has determined
that there is no evidence that free DNA containing the kanr gene,
even if present, can transform cells lining the GI tract (Ref. 2).
FDA has also evaluated the information submitted by Calgene
concerning soil microorganisms and agrees with Calgene that there would
be no increase in kanamycin-resistant soil microorganisms because it is
highly unlikely that the kanr gene could move from the plant
genome into soil microorganisms via horizontal gene transfer. Further,
the agency has determined that, even if such transfer could occur, the
rate at which it could occur is such that it would not result in a
detectable increase over the existing background population of
kanamycin-resistant bacteria (Ref. 36). Based on the foregoing, FDA has
concluded that the use of the kanr gene does not pose safety
concerns in terms of increase in the population of antibiotic-resistant
pathogens due to the potential for horizontal transfer of the gene.
IV. Response to Comments
FDA received 47 comments on Calgene's request for an advisory
opinion on the use of the kanr gene in the development of new
varieties of tomato, oilseed rape, and cotton plants. Comments were
received from members of academia, industry and industry-related
organizations, State and Federal agencies, environmental groups and
other nonprofit organizations, and individual consumers. Additionally,
several comments on the agency's 1992 policy statement addressed the
use of the kanr gene.
Most of the comments supported the use of the kanr gene in
crop development, stating that there were no health or environmental
issues precluding its use. Several comments expressed opinions on a
wide range of issues including regulatory approaches for genetically
engineered foods, concerns relating to human and animal food safety,
and to the environmental effects of the kanr gene, and whether
foods containing the kanr gene and APH(3')II should be specially
labeled.
A. Regulatory Issues
Some comments stated that it was not appropriate for FDA to
evaluate the safety of the kanr gene and APH(3')II under an
advisory opinion and that the kanr gene and APH(3')II should be
treated as food additives by FDA. FDA has discussed above the basis for
its decision not to regulate the DNA that makes up the kanr gene
itself as a food additive. Further, in light of Calgene's conversion of
its request for advisory opinion on the use of the kanr gene to a
food additive petition, the comment concerning the regulation of
APH(3')II as a food additive no longer requires a response.
B. Food Safety
Several comments stated that the presence in food of APH(3')II
raised no food safety concerns whatsoever. Others questioned whether
Calgene had supplied adequate data to ensure the safety of the
kanr gene and gene product, APH(3')II, when present in food. The
substantive questions raised are discussed in sections IV.B.1 through 5
of this document.
1. Glycosylation
Two comments stated that APH(3')II might be glycosylated (i.e.,
might contain sugar molecules attached to the protein via the amino
acid asparagine (N-linked) or via the amino acids serine, threonine, or
hydroxyproline (O-linked)) when produced in tomatoes or other plants
and, therefore, might become a food allergen. One of the comments
asserted that for this reason, Calgene should be required to test
whether APH(3')II is glycosylated. The comments, however, did not
provide any information showing that glycosylated APH(3')II is likely
to be, or is, allergenic.
At this time, FDA is unaware of any practical method to predict or
assess the potential for new proteins in food to induce allergenicity.
Although many food allergens that have been characterized at a
structural level are glycosylated (Ref. 37), the agency is not aware of
any information on structural or other properties of glycosylated
proteins that would be predictive of their allergenicity. As noted, the
comments did not provide such information. Moreover, glycosylated
proteins are widespread in food. For these reasons, glycosylation is
not a useful positive predictor of a potential allergenic effect.
Accordingly, FDA did not request that Calgene determine whether
APH(3')II is glycosylated.
Nevertheless, in a submission dated October 24, 1991, entitled
``Response to Public Comments,'' Calgene addressed whether APH(3')II is
likely to be glycosylated and concluded that it is not. Calgene noted
that APH(3')II lacks the amino terminal sequence of amino acids
(commonly referred to as a ``signal peptide'') that is necessary to
direct the protein into the cellular compartments where glycosylation
occurs. Calgene also asserted that the unchanged molecular weight of
APH(3')II in plants (relative to the molecular weight of bacterial
APH(3')II, which is not glycosylated) supports the conclusion that
APH(3')II is not glycosylated in plants. Finally, Calgene stated that
the amino acid sequence (asparagine-X-serine/threonine) that is
required to direct N-linked glycosylation to specific asparagine
moieties is not present in APH(3')II. (Calgene noted that a
corresponding argument for the lack of the appropriate amino acid
sequence to direct O-linked glycosylation cannot be made because the
sequences that direct O-linked glycosylation have not been defined.)
FDA has considered the information and arguments submitted in the
comments and Calgene's response and has concluded that the available
evidence indicates that APH(3')II is not glycosylated in plants.
However, even if glycosylation had been demonstrated, FDA emphasizes
that glycosylation alone does not necessarily establish that APH(3')II
is likely to produce an allergenic response because the positive
predictive value of glycosylation with respect to the potential for
inducing allergenicity has not been demonstrated.
2. In Vitro Digestibility Studies
In its original submission, Calgene presented the results of in
vitro digestibility studies that demonstrated that APH(3')II enzymatic
activity is rapidly decreased in simulated gastric fluid and in
simulated intestinal fluid.
One comment asserted that Calgene should provide a more thorough
study of degradation of APH(3')II in the digestive tract because the
conditions of the in vitro digestibility study submitted by Calgene did
not fully mimic the complex environments of the human gut. The comment
further asserted that it was not clear whether the digestibility data
also apply to neonates and to people with coeliac disorders or ulcers
who can absorb peptides and intact proteins through their intestines.
The comment noted that the applicability of the data to neonates would
be of special importance should kanr be used in soybeans because
soy protein is a major component of some infant formulas. Importantly,
however, the comment presented no information to provide a basis for
concluding that the absorption of APH(3')II occurs, or that if it does,
such absorption presents a health concern greater than that posed by
the absorption of any other protein in the diet.
As discussed above, FDA has evaluated the studies presented by
Calgene to demonstrate the normal digestibility of the enzyme and
concurs with Calgene's conclusion that APH(3')II is rapidly degraded
under normal conditions in the GI tract. Therefore, FDA believes that
the intestinal transfer of intact or large fragments of APH(3')II is
not likely to occur in individuals with normal GI tracts.
In regard to the possibility of increased intestinal absorption of
proteins in neonates and individuals with special conditions (e.g.,
ulcers), FDA has concluded that there is no reason to expect that
absorption of the intact or partially digested APH(3')II protein would
present a safety problem different from absorption of any other protein
in the diet. As discussed above, proteins, as a class, are rarely
toxic. Furthermore, APH(3')II is a phosphorylating enzyme and does not
contain any properties that would distinguish it toxicologically from
any other phosphorylating enzymes that historically have been part of
the food supply without adverse consequences. Finally, because Calgene
did not petition FDA for the use of APH(3')II in soybeans, it is not
necessary to address the comment concerning the applicability of
Calgene's digestibility data to neonates fed soybean-derived formulas.
3. Copy Number of the kanr Gene and Expression Level of APH(3')II
In its submission of November 26, 1990, Calgene stated that it did
not intend to commercialize lines that contained more than 10 copies of
the kanr gene. In addition, Calgene also declared that, in
tomatoes, the APH(3')II level would be no more than 0.1 percent of the
total protein of the tomato and that processing procedures would
destroy APH(3')II in processed tomatoes and edible oils extracted from
cottonseed and rapeseed.
One comment asserted that Calgene inadequately described the
methods by which it would ensure that no lines with greater than 10
copies of the kanr gene would be marketed. The comment further
asserted that many of the analyses offered by Calgene to prove the
safety of the kanr gene depend on estimates of the number of genes
per cell and that, if the company cannot ensure this relatively low
level of gene incorporation, many of its safety arguments are
undermined. The comment, however, did not identify which of Calgene's
safety analyses depended on estimates of the numbers of genes per cell.
The comment may have been referring to Calgene's assumption that
each plant cell would contain 10 copies of the gene when it calculated
a worst-case frequency of transformation of microorganisms with the
kanr gene that would result from use of the gene in transgenic
plants. However, the agency notes that the outcome of those
calculations, i.e., Calgene's conclusion that the transformation
frequency of microorganisms with the kanr gene is insignificant,
would not change had Calgene assumed much higher gene copy numbers in
its calculations. Therefore, FDA's safety assessment does not depend on
precise estimates of gene copy number. Nor does the comment provide a
basis for concluding that it is necessary to have precise methods for
ensuring that no plants with more than 10 copies of the gene will be
marketed.
A second comment maintained that Calgene provided an inadequate
description of the quality control and assurance procedures the company
would use to ensure that APH(3')II would be kept to no more than 0.1
percent of total protein of the tomato, and that a number of the
company's safety analyses rely on the amount of APH(3')II in the food.
The comment, however, did not identify which of Calgene's safety
analyses relied on estimates of the concentration of APH(3')II in the
food.
FDA has determined that there is no need to set a tolerance for the
amount of APH(3')II that will be consumed because the agency knows of
no reason why this protein would have any properties that would
distinguish it toxicologically from any other phosphorylating enzymes
in the food supply. Also, as discussed above, APH(3')II will not affect
efficacy of orally administered antibiotics because APH(3')II is
rapidly digested under normal conditions in the GI tract, and even in
abnormal gastric conditions where APH(3')II may not be rapidly
digested, the amount of ATP available in food would allow only a small
proportion of kanamycin and neomycin to be inactivated. Therefore, the
agency concludes that there is no need to require quality control and
assurance procedures to ensure that the APH(3')II level will be no more
than 0.1 percent of the total protein in commercial tomato varieties.
A third comment argued that Calgene did not provide data to
establish that APH(3')II would not be present after tomato processing
and after extraction of edible oils.
The agency's exposure estimates included an assumption that
APH(3')II would be present in both processed tomatoes and fresh
tomatoes even though the high temperatures involved in processing
inactivate enzymes and therefore, processed tomato products are
unlikely to contain enzymatically active APH(3')II (Ref. 18). In
addition, well-established processing procedures used to extract edible
oils from oilseed crops do not extract significant amounts of protein
(Ref. 23). Therefore, exposure to APH(3')II obtained from rapeseed oil
and cottonseed oil would be negligible (Ref. 18). The comment did not
present any information to contradict FDA's analysis and conclusion on
this point.
4. The Potential for Side Effects From Consumption of Genetically
Engineered Foods
One comment asked whether there might be side effects from
consumption of genetically engineered foods, and if so, whether these
side effects would be short term or long term. Another comment noted
that food plants and humans exhibit complex and unpredictable behavior
and that therefore, the safety of a food substance should be based on
thoughtfully gathered empirical evidence.
The comments did not point to any specific side effects of
genetically engineered foods. FDA has evaluated the safety of APH(3')II
and has determined that it is safe for its proposed use. This safety
assessment is in fact based on empirical evidence, such as the
structure and function of APH(3')II, the low level at which APH(3')II
occurs in foods, the digestibility of APH(3')II, and the inability of
APH(3')II to interfere with clinically useful antibiotics under usual
conditions of use for the antibiotics.
5. Relevance of Clinical Studies
Several comments noted that a National Institutes of Health (NIH)
gene therapy trial in which cancer patients were infused with cells
containing the kanr gene, and which was cited by Calgene as strong
evidence for the safety of the kanr gene, provides little
information concerning the safety of the kanr gene and APH(3')II
in food. One comment also noted that the combination of data from the
in vitro studies and the gene therapy study was an inadequate basis for
a safety determination of the kanr gene and APH(3')II in food that
millions of people might eat.
In determining that APH(3')II is safe for its proposed food
additive use, FDA did not rely on the NIH gene therapy trial. However,
FDA does believe that the in vitro degradation data provide important
information that should be and was considered by the agency as part of
its overall safety assessment of the kanr gene and APH(3')II, as
discussed earlier in this document.
C. Possible Effect on Clinical Efficacy of Orally Administered
Kanamycin or Neomycin
Several comments questioned whether the presence of APH(3')II in
tomatoes or other foods might compromise the clinical efficacy of
orally administered kanamycin or neomycin. One comment noted that
Calgene claimed that at most only 76,800 people annually were
administered kanamycin or neomycin orally, and argued that those people
deserved not to be put at risk. The comment further requested that
Calgene be required to perform animal studies on the effects of
ingestion of APH(3')II on the efficacy of orally administered kanamycin
and neomycin. The comment asserted that if APH(3')II were shown to
compromise clinical efficacy of kanamycin or neomycin, food containing
APH(3')II should be appropriately labeled.
Other comments observed that ingested APH(3')II would not impair
the efficacy of orally administered kanamycin and neomycin, that these
antibiotics are rarely administered orally, and that the kanr gene
is therefore a good choice as a selectable marker gene.
FDA agrees with Calgene that kanamycin and neomycin are rarely
administered orally. The primary clinical role for orally administered
neomycin, and to a lesser extent kanamycin, is cleansing the bowel of
microbes prior to bowel surgery. This use is relatively minor because
of severe side effects (auditory nerve damage and kidney damage) that
may result from the antibiotic that is absorbed from the GI tract (Ref.
38).
As discussed above, for most individuals receiving oral kanamycin
or neomycin, APH(3')II will be inactivated by the acidic environment of
the stomach and degraded by the digestive enzymes present in the GI
tract. More important, even for patients receiving simultaneous
treatment to reduce stomach acidity, the amount of ATP available from
food would allow, at most, only a small fraction of kanamycin or
neomycin to be inactivated. The comment advocating animal studies did
not contradict directly or indirectly FDA's analysis concerning the
inactivation and degradation of APH(3')II or the information concerning
ATP levels. FDA has therefore determined that the presence of APH(3')II
in food will not compromise therapy with orally administered kanamycin
or neomycin. On this basis, FDA has concluded that neither animal
studies on the effects of ingestion of APH(3')II on the efficacy of the
antibiotics, nor special labeling of foods containing APH(3')II for
patients receiving orally administered kanamycin or neomycin, are
necessary.
D. Fate of the kanr Gene in the Environment
1. Potential Transfer of the kanr Gene From Crops to
Microorganisms
One comment posited a connection between ``the prophylactic use of
antibiotics [resulting] in antibiotic-resistant bacteria reaching the
human population'' with a health risk from the possible addition of up
to ``10 antibiotic genes [sic] in most of the cells of major crops.''
The comment agreed with Calgene's documentation that the widespread use
of antibiotics has led to an increase in antibiotic-resistant bacteria
in the environment, but went on to postulate that this was evidence
that introducing antibiotic-resistance genes into plants has human
health implications.
The comment further asserted that the ``scientific question is
whether the resistance genes in the crops can be transferred by any
mechanism [to] organisms that might be human pathogens,'' and that the
company should be required experimentally to ``determine the rates of
gene transfer to soil bacteria from plant debris, the persistence or
selection of organisms containing such genes in soil ecosystems, and
other important factors in the assessment of the likelihood of releases
compromising the use of antibiotics.'' The comment noted that Calgene
analyzed these issues ``in some detail,'' but with ``arm chair
calculations, most based on extrapolations from experiments done with
other organisms under other circumstances.''
A second comment noted that Calgene had supplied information that
three kinds of bacteria, with and without plasmids7 carrying
antibiotic resistance genes, had little effect on several measures of
soil ecosystems, but wrote that the ``relevance of experiments on
bacteria to releases of plants is marginal, at best.'' A third comment
asserted, without any supporting evidence, that ``genetic resistance to
antibiotics in these plants could be transferred by plasmids to
microorganisms in the soil and elsewhere in the food chain.''
---------------------------------------------------------------------------
\7\Plasmids are self-replicating units of DNA commonly found in
bacteria and are responsible for transfer of antibiotic resistance
between bacteria.
---------------------------------------------------------------------------
FDA agrees that increasing the number and prevalence of antibiotic-
resistant microbes may have serious human health implications if those
microbes are themselves pathogens of humans or domesticated animals, or
share the same microenvironment as such pathogens. FDA considers the
relevant scientific question to be whether there would be a meaningful
increase in antibiotic-resistant pathogenic microbes in the human
environment due to transfer of the kanr gene from plants to
microbes. This issue was also the subject of considerable discussion at
the April 1994 Food Advisory Committee meeting. As discussed in detail
above, FDA has determined, based on the body of evidence presented by
Calgene and based on the discussions of the Food Advisory Committee
(Ref. 1), that the transfer of the kanr gene from plants to
microbes will not occur at a detectable frequency and overall will
result in no significant increase in the numbers of antibiotic-
resistant microbes. Regarding whether Calgene should be required to
determine experimentally the rate of transfer, the agency notes that
Calgene's calculations represent worst-case scenarios, and the agency
believes it would not be useful to do experiments to attempt to measure
that which is too small to measure.
Regarding the relevance of experiments on bacterial releases to the
environment, FDA finds that information concerning the lack of an
environmental effect from the release of microbes with and without
antibiotic resistance genes is of limited direct relevance to the
environmental effects of plants with antibiotic resistance genes. The
agency did not rely on this information in reaching its determination
that there will be no significant increase in the antibiotic-resistant
microorganism population of the soil.
Finally the claim that the kanr gene could be transferred from
plants to bacteria by plasmids is without basis because there is no
evidence that plasmids exist in plants.
2. Potential Transfer of the kanr Gene to Other Crops and to Wild
Relatives
Comments were also received on the potential transfer of the
kanr gene to other crops and wild relatives. These comments
address environmental issues and do not bear on the safety of APH(3')II
for its proposed food additive use and are therefore addressed in
section VII. of this document.
E. Possible Effects of Consumption of Animal Feeds Containing APH(3')II
on Animals and Their Gut Microflora
One comment argued that empirical evidence should be gathered to
assess the potential effects of modified foods on animals and their gut
microflora.
The agency is aware of no information that APH(3')II would affect
animals or their gut microflora any differently than any other protein
in the diet, nor did the comment provide such information. The comment
may have been referring to the theoretical potential for APH(3')II in
animal feed to affect efficacy of neomycin administered to animals, and
the theoretical potential for the gut microflora to take up the
kanr gene and become resistant to neomycin. As discussed above,
the likelihood of transfer of the kanr gene to gut microflora of
food animals is extremely remote. Also, as discussed above, FDA has
evaluated the study presented by Calgene addressing the possibility of
inactivation of neomycin by APH(3')II in animal feed and has concluded
that the therapeutic efficacy of neomycin in animals would not be
affected by consumption of feed containing transgenic cottonseed and
rapeseed modified through the use of the kanr gene.
F. Labeling of Foods Containing the Kanr Gene and APH(3')II
One comment asserted that APH(3')II should be labeled as an
ingredient. The comment further stated that, if FDA exempted APH(3')II
from ingredient labeling requirements (based on its classification as a
processing aid that is present at insignificant levels in a finished
food and has no technical or functional effect in that food), FDA
should require special labeling if the ingestion of food containing
APH(3')II could compromise the clinical efficacy of orally administered
kanamycin or neomycin.
FDA's authority over food labeling is based on section 403 of the
act (21 U.S.C. 343). Section 403(i) of the act requires that, in the
case of foods fabricated from two or more ingredients, a food product
bear on the label the common or usual name of each ingredient, unless
compliance with the requirement for labeling is impracticable or
results in deception or unfair competition. FDA considers an
``ingredient'' to be a substance used to fabricate (i.e., manufacture
or produce) a food. FDA does not consider those substances that are
inherent components of food to be ingredients that must be disclosed in
the food's label.
A genetic substance introduced into a plant by breeding becomes an
inherent part of the plant as well as of all foods derived from the
plant. Consistent with FDA's general approach on ingredient labeling,
the agency has not treated as an ingredient a new constituent of a
plant introduced by breeding, regardless of the method used to develop
the new plant variety. The comment provides no basis for FDA to deviate
from its current practice in the case of APH(3')II.8 Accordingly,
FDA has determined that neither the kanr gene nor APH(3')II is an
ingredient that, under section 403(i) of the act, must be individually
identified in labels of foods containing them.
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\8\Furthermore, APH(3')II satisfies the definition of
``processing aid'' in Sec. 101.100(a)(3)(ii)(c) (21 CFR
101.100(a)(3)(ii)(c)) and will be regulated as such by this final
rule. As the comment acknowledges, FDA's labeling regulations exempt
processing aids like APH(3')II from the labeling requirements of
section 403(i)(2) of the act. Thus, even if APH(3')II were properly
considered an ingredient, its presence in a food would not be
required to be disclosed in the food's labeling.
---------------------------------------------------------------------------
FDA has also determined that the presence of APH(3')II is not a
material fact that must be disclosed in the labeling of foods that
contain the enzyme. Under section 403(a)(1) of the act (21 U.S.C.
343(a)(1)), a food is misbranded if its labeling is false or
misleading. Under section 201(n) of the act (21 U.S.C. 321(n)),
labeling is misleading if it fails to reveal all facts that are ``* * *
material with respect to consequences which may result from the use of
the article * * *.'' As discussed at length above, FDA has determined
that the ingestion of food containing APH(3')II will not compromise the
clinical efficacy of orally administered kanamycin or neomycin. Because
the consequences alleged in the comment--compromise of clinical
efficacy--will not occur, the presence of APH(3')II is not a material
fact requiring disclosure.
V. Conclusions
FDA has evaluated data in the petition and other relevant material
and concludes that the proposed use of APH(3')II as a processing aid in
the development of new varieties of tomato, oilseed rape, and cotton is
safe, and that 21 CFR parts 173 and 573 should be amended as set forth
below.
VI. Inspection of Documents
In accordance with Secs. 171.1(h) and 571.1(h) (21 CFR 171.1(h) and
571.1(h)), the petition and the documents that FDA considered and
relied upon in reaching its decision to approve the petition are
available for inspection at the Center for Food Safety and Applied
Nutrition by appointment with the information contact person listed
above. As provided in 21 CFR 171.1(h) and 571.1(h), the agency will
delete from the documents any materials that are not available for
public disclosure before making the documents available for inspection.
VII. Environmental Impact
Calgene's initial submission requesting an advisory opinion
regarding whether the kanr gene may be used in the production of
genetically engineered tomato, cotton, and oilseed rape plants included
an environmental assessment (EA). The agency received comments on this
EA. As noted earlier, the request for advisory opinion was later
converted to a food additive petition at Calgene's request at which
time Calgene submitted an updated EA. At the time the notice of filing
was published in the Federal Register, FDA announced that the
petitioner's EA was being made available to the public at the Dockets
Management Branch (address above) and expressly solicited comments on
the EA. No additional comments were received in response to this
request for comments. The comments received on the original EA are
discussed below.
One comment asserted that the kanr gene could spread from
tomato, cotton, and oilseed rape plants to other crops and related
weeds by pollen flow when the kanr gene-containing crops are grown
near nontransgenic crops, and in locations where the kanr-gene
containing crops have wild relatives. The comment noted that transfer
of the kanr gene would create a problem if it were to make wild
and weedy relatives more difficult to control.
The comment also criticized the Calgene submission for not
addressing whether it is ``wise to contribute foreign genes to the gene
pools of wild plants even where the plants do not become weeds or
manifest other obviously harmful traits'' and stated that Calgene's
submission ``too easily dismissed the problem of outcrossing from the
engineered oilseed rape.'' The comment noted that oilseed rape has wild
and weedy relatives with which it can breed, and that ``it is not
sufficient to rely on traditional commercial control practices to
control gene flow,'' but that the rate of gene flow must be
experimentally determined and then ``controlled by procedures that are
demonstrated, not assumed, to work.''
The agency has considered the potential for adverse environmental
effects from the commercial use of cotton, tomato, and oilseed rape
plants modified to contain the kanr gene. The agency notes that it
is possible for cotton and tomato plants to transfer the kanr gene
to neighboring plants of the same species via cross-pollination,
although commercially grown cotton and tomatoes are primarily self-
pollinating. Oilseed rape plants are also capable of pollinating
sexually compatible wild relatives, although not all crosses with wild
relatives prove fertile. Importantly, however, introduction of the
kanr gene will not confer a competitive advantage upon a plant
receiving it. That is, the gene will not enhance the plant's capacity
to compete with other plants for available resources. In particular,
there will be no selective pressure on plants containing the kanr
gene because kanamycin will not be present in the environment in
sufficient concentrations to create such pressure. First, there are no
specific therapeutic uses of kanamycin that would result in its
widespread application to agricultural crops. Also, kanamycin does not
accumulate in the environment from production by soil microbes or by
land application of animal wastes (Ref. 36). Accordingly, FDA has
concluded that transfer of the kanr gene to other crops or related
weeds will have no significant adverse environmental effects.
With regard to the comment about outcrossing from engineered
oilseed rape, the comment provided no information to show that the
transfer of the kanr gene to wild or weedy relatives of oilseed
rape will be any more frequent or have any greater significance than
the transfer of other genes from cultivated oilseed rape. FDA is aware
of no human health or environmental concern associated with such
transfer. Therefore, the agency does not agree that the cultivation of
kanr-containing oilseed rape should be subject to control
practices any different from those used traditionally.
The agency has carefully considered the potential environmental
effects of this action, including those described in the comments
discussed in this document. FDA has concluded that the action will not
have a significant impact on the human environment and that an
environmental impact statement is not required. The agency's finding of
no significant impact and the evidence supporting that finding,
contained in an environmental assessment, may be seen in the Dockets
Management Branch between 9 a.m. and 4 p.m., Monday through Friday.
VIII. Objections
Any person who will be adversely affected by this regulation may at
any time on or before June 22, 1994, file with the Dockets Management
Branch (address above) written objections thereto. Each objection shall
be separately numbered, and each numbered objection shall specify with
particularity the provisions of the regulation to which objection is
made and the grounds for the objection. Each numbered objection on
which a hearing is requested shall specifically so state. Failure to
request a hearing for any particular objection shall constitute a
waiver of the right to a hearing on that objection. Each numbered
objection for which a hearing is requested shall include a detailed
description and analysis of the specific factual information intended
to be presented in support of the objection in the event that a hearing
is held. Failure to include such a description and analysis for any
particular objection shall constitute a waiver of the right to a
hearing on the objection. Three copies of all documents shall be
submitted and shall be identified with the docket number found in
brackets in the heading of this document. Any objections received in
response to the regulation may be seen in the Dockets Management Branch
between 9 a.m. and 4 p.m., Monday through Friday.
IX. References
The following references have been placed on display in the Dockets
Management Branch (address above) and may be seen by interested persons
between 9 a.m. and 4 p.m., Monday through Friday.
1. Transcript of meeting of the Food Advisory Committee, FDA,
Herndon, VA, April 6 through 8, 1994.
2. Hoskins, L.C., ``Host and Microbial DNA in the Gut Lumen,''
The Journal of Infectious Diseases, 137:694-698, 1978.
3. Memorandum from T.A. Cebula, FDA, to N. Beru, FDA, November
2, 1993.
4. Potrykus, I., ``Gene Transfer to Plants: Assessment of
Published Approaches and Results,'' in ``Annual Review of Plant
Physiology and Plant Molecular Biology,'' Briggs, W.R., R.L. Jones,
and V. Walbot, 42:205-225, 1991.
5. Fraley, R.T. et al., ``Genetic Transformation in Higher
Plants,'' Critical Reviews in Plant Sciences, 4:1-46, 1985.
6. Beck, E. et al., ``Nucleotide Sequence and Exact Localization
of the Neomycin Phosphotransferase Gene From Transposon Tn5,''
Gene, 19:327-336, 1982.
7. Jorgensen, R.A. et al., ``A Restriction Enzyme Cleavage Map
of Tn5 and Location of a Region Encoding Neomycin Resistance,''
Molecular and General Genetics, 177:65-72, 1979.
8. Davies, J. et al., ``Plasmid-determined Resistance to
Antimicrobial Agents,'' Annual Review of Microbiology, 32:469-518,
1978.
9. Goldman, P.R. et al., ``Purification and Spectrophotometric
Assay of Neomycin Phosphotransferase II,'' Biochemical and
Biophysical Research Communications, 69:230-236, 1976.
10. U.S. Pharmacopeia (U.S.P.), The National Formulary (NF)
1990, U.S.P. XXII, NF XVII, U.S. Pharmacopeial Convention, Inc.,
Mack Printing Co., Easton, PA.
11. Prescott, J.F., and J.D. Baggot, ``Aminoglycosides and
Aminocyclitols,'' in Antimicrobial Therapy in Veterinary Medicine,
Blackwell Scientific Publications, Boston, MA, pp. 121-152, 1988.
12. Davies, J.E., ``Aminoglycoside-aminocyclitol Antibiotics and
Their Modifying Enzymes,'' in ``Antibiotics in Laboratory
Medicine,'' 2d ed., Lorian, V., editor, pp. 790-809, 1986.
13. Dickie, P. et al., ``Effect of Enzymatic Adenylation on
Dihydrostreptomycin Accumulation in Escherichia coli Carrying the R-
factor: Model Explaining Aminoglycoside Resistance by Inactivating
Mechanisms,'' Antimicrobial Agents and Chemotherapy, 14:569-580,
1978.
14. Nap, J.P et al., ``Biosafety of Kanamycin-resistant
Transgenic Plants,'' Transgenic Research, 1:239-249, 1992.
15. Taylor, S.L. et al.,''Food Allergens: Structure and
Immunologic Properties,'' Annals of Allergy, 59:93-99, 1987.
16. Darnel, J. et al., ``Molecular Cell Biology,'' 2d ed., p.
116, Scientific American Books, Inc.
17. Pariza, M.W. et al., ``Determining the Safety of Enzymes
Used in Food Processing,'' Journal of Food Protection, 46:453-468,
1988.
18. Memorandum from Z. Olempska-Beer, FDA, to N. Beru, FDA,
August 10, 1993.
19. Memorandum from Z. Olempska-Beer, FDA, to J. Maryanski, FDA,
July 14, 1992.
20. Memorandum from C.B. Johnson, FDA, to V. Zenger, FDA,
September 7, 1993.
21. Memorandum from C.B. Johnson, FDA, to J. Maryanski, FDA,
July 14, 1992.
22. Fuchs, R.L. et al., ``Safety Assessment of the Neomycin
Phosphotransferase II (NPTII) Protein,'' Biotechnology, 11:1543-
1547, 1993.
23. USDA Agricultural Handbook No. 8, Table I, Item 1401.
24. Memorandum from Z. Olempska-Beer, FDA, to N. Beru, FDA,
August 9, 1993.
25. Orten, J.M. and O.W. Neuhaus, Human Biochemistry, 10th ed.,
pp. 537-538, C.V. Mosby Co., St. Louis, MO, 1982.
26. Memorandum from A.T. Sheldon, FDA, to J. Maryanski, FDA,
March 30, 1993.
27. Memorandum from S.A. Giduck, FDA, to V. Zenger, FDA, July
21, 1992.
28. Memorandum from J.D. McCurdy, FDA, to V. Zenger, FDA,
October 13, 1993.
29. McAllan, A.B. et al., ``Degradation of Nucleic Acids in the
Rumen,'' British Journal of Nutrition, 29:467-474, 1973.
30. Stewart, G.J. et al., ``The Biology of Natural
Transformation,'' Annual Review of Microbiology, 40:211-235, 1986.
31. Taylor, D.E., ``Genetics of Campylobacter and
Helicobacter,'' Annual Review of Microbiology, 46:35-64, 1992.
32. Stewart, G.J., ``The Mechanism of Natural Transformation,''
in ``Gene Transfer in the Environment,'' Levy, S. B. and R. V.
Miller, editors, pp. 139-164, McGraw-Hill, New York, 1989.
33. Bergmans, H.E.N. et al., ``Transformation in Escherichia
coli: Stages in the Process,'' Journal of Bacteriology, 146:564-570,
1981.
34. Lapkin, M., ``Proliferation and Differentiation of Normal
and Diseased Gastrointestinal Cells,'' in ``Physiology of the
Gastrointestinal Tract,'' 2d ed., Johnson, L.R. et al., editors,
Raven Press, New York, NY, 1987.
35. Levy, S.B. et al., ``High Frequency of Antimicrobial
Resistance in Human Fecal Flora,'' Antimicrobial Agents and
Chemotherapy, 32: 1801-1806, 1988.
36. Memorandum from J. Glover-Glew, FDA, to N. Beru, FDA,
December 15, 1993.
37. Sweeney, M.J. et al., ``Immunology of Food Allergens,'' in
``Handbook of Food Allergens,'' pp. 13-35, Breneman, J.C., editor,
Marcel Dekker, Inc., New York, 1987.
38. Goodman, L.S. and Gilman, A., ``The Pharmacological Basis of
Therapeutics,'' 7th ed., pp. 1157-1160, MacMillan Publishing Co.,
New York, 1980.
List of Subjects
21 CFR Part 173
Food additives.
21 CFR Part 573
Animal feeds, Food additives.
Therefore, under the Federal Food, Drug, and Cosmetic Act and under
authority delegated to the Commissioner of Food and Drugs, 21 CFR parts
173 and 573 are amended as follows:
PART 173--SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR
HUMAN CONSUMPTION
1. The authority citation for 21 CFR part 173 continues to read as
follows:
Authority: Secs. 201, 402, 409 of the Federal Food, Drug, and
Cosmetic Act (21 U.S.C. 321, 342, 348).
2. New Sec. 173.170 is added to subpart B to read as follows:
Sec. 173.170 Aminoglycoside 3'-phosphotransferase II.
The food additive aminoglycoside 3'-phosphotransferase II may be
safely used in the development of genetically modified cotton, oilseed
rape, and tomatoes in accordance with the following prescribed
conditions:
(a) The food additive is the enzyme aminoglycoside 3'-
phosphotransferase II (CAS Reg. No. 58943-39-8) which catalyzes the
phosphorylation of certain aminoglycoside antibiotics, including
kanamycin, neomycin, and gentamicin.
(b) Aminoglycoside 3'-phosphotransferase II is encoded by the
kanr gene originally isolated from transposon Tn5 of the
bacterium Escherichia coli.
(c) The level of the additive does not exceed the amount reasonably
required for selection of plant cells carrying the kanr gene along
with the genetic material of interest.
PART 573--FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF
ANIMALS
3. The authority citation for 21 CFR part 573 continues to read as
follows:
Authority: Secs. 201, 402, 409 of the Federal Food, Drug, and
Cosmetic Act (21 U.S.C. 321, 342, 348).
4. New Sec. 573.130 is added to subpart B to read as follows:
Sec. 573.130 Aminoglycoside 3'-phosphotransferase II.
The food additive aminoglycoside 3'-phosphotransferase II may be
safely used in the development of genetically modified cotton, oilseed
rape, and tomatoes in accordance with the following prescribed
conditions:
(a) The food additive is the enzyme aminoglycoside 3'-
phosphotransferase II (CAS Reg. No. 58943-39-8) which catalyzes the
phosphorylation of certain aminoglycoside antibiotics, including
kanamycin, neomycin, and gentamicin.
(b) Aminoglycoside 3'-phosphotransferase II is encoded by the
kanr gene originally isolated from transposon Tn5 of the bacterium
Escherichia coli.
(c) The level of the additive does not exceed the amount reasonably
required for selection of plant cells carrying the kanr gene along
with the genetic material of interest.
Dated: May 17, 1994.
Fred R. Shank,
Director, Center for Food Safety and Applied Nutrition.
Linda A. Suydam,
Interim Deputy Commissioner for Operations.
David A. Kessler,
Commissioner of Food and Drugs.
[FR Doc. 94-12492 Filed 5-18-94; 12:39 pm]
BILLING CODE 4160-01-P
