Environmental Risk Assessment of Plant Incorporated Protectant (PIP)
Inert Ingredients

This environmental risk assessment of PIP inert ingredients is divided
into three sections which cover herbicide tolerance markers, antibiotic
resistance markers, and other markers. 

Herbicide Tolerance Markers

Phosphinothricin acetyltransferase (PAT)

SUMMARY

The Agency has conducted an environmental risk assessment of the
plant-incorporated protectant (PIP) inert ingredient phosphinothricin
acetyltransferase (PAT), and the genetic material necessary for its
production. Topics covered in this assessment include mode of action,
ecological effects, endangered species considerations, and gene flow
from a modified plant to wild or weedy relatives. Data cited in this
assessment were submitted to the Agency in support of Dekalb’s DBT 418
and Ciba Seed’s Event 176 Bt corn registrations, and Syngenta’s COT
102 Bt cotton registration. Ecological data and published information on
the biology of this protein indicate that this PIP inert ingredient is
not a known toxin and/or pathogen of plant or animal species. Due to the
low human health risks associated with this protein, the Agency has
granted an exemption from the requirement of a tolerance for this PIP
inert ingredient (40 CFR 180.1151; 62 FR 17719, Aug. 11, 1997).

MODE OF ACTION

The PAT enzyme is an acetyltransferase that catalyzes the acetylation
the active ingredients L-phosphinothricin (L-PPT) and
demethylphosphinothricin (DMPT), conferring tolerance to glufosinate
ammonium herbicides. L-PPT and DMPT inhibit glutamine synthetase,
resulting in the accumulation of toxic ammonium ions and a decrease in
the amount of glutamine, an essential amino acid used in many anabolic
processes. In the presence of acetyl-CoA, the PAT protein catalyses the
acetylation of the free amino group of L-PPT to N-acetyl-L-PPT, a
compound that does not inhibit glutamine synthetase (OECD 2002). The PAT
enzyme is highly specific for L-PPT and does not acetylate other L-amino
acids, such as glutamate (Thompson et al., 1987).

ENVIRONMENTAL Effects

The Agency assessed the toxicity of PAT protein to representative
non-target organisms that could be exposed to this PIP inert ingredient.
Toxicity evaluations included the following Tier I studies: Mammalian
(mouse), Avian (northern bobwhite quail), Freshwater Fish (channel
catfish), Aquatic Invertebrate [cladoceran (Daphnia magna)], and
Non-target Arthropod (honey bee larvae, lady beetle, green lacewing,
collembola, and earthworm). Tier IV field studies of non-target
abundance are also discussed below.

The wild mammal hazard assessment was performed on the basis of rodent
acute oral toxicity data prepared for human health risk assessment
purposes which utilized pure PAT protein. Submitted data indicate no
significant adverse effects among mice dosed with up to 2500 mg/kg body
weight PAT protein (DBT 418 MRID 439995-02). 

The remaining single species studies were maximum hazard dose
evaluations, where PAT protein toxicity was evaluated by feeding select
non-target organisms corn pollen, grain, or leaf tissue, which has been
shown to contain the PIP inert ingredient PAT and the active ingredient
Cry1Ab or VIP3A. Note: Studies submitted in support of the COT102
registration utilized corn plant material in place of cotton plant
material. 

An avian study, where northern bobwhite quail were dosed with
lyophilized DBT 418 corn leaf tissue, reported no adverse effects to the
test species at field exposure levels (MRID 439995-10). The freshwater
fish corn grain feeding study indicated that channel catfish diets may
contain up to 50% PAT-containing corn grain with no abnormalities or
adverse effects on fish growth (final report not submitted as of
9-5-03). In three Event 176 and four COT102 corn pollen dietary toxicity
studies, no adverse effects to Daphnia magna, a representative of
aquatic invertebrate species, honey bee larvae, lady beetle, or green
lacewing were seen at field level exposures (Event 176 MRIDs 433236-10,
433396-02, 434157-03; COT102 MRIDs 457921-01, 457665-09, and 458358-08).
Results of DBT 418 and COT102 corn leaf tissue feeding studies indicate
that PAT does not adversely affect reproduction of Collembola, a
representative plant tissue decomposer (DBT 418 MRID 439995-12; COT102
MRID 458358-10). Finally, no adverse effects were seen among earthworms
exposed for 14 days to soil containing lyophilized DBT 418 corn leaf
tissue (MRID 439995-13).

A semi-field study of honeybee colonies showed that the overall ability
of colonies to produce brood, manage stores of food and recruit new bees
was not affected by exposure to the VIP3A corn containing the PAT
protein (MRID 458358-13). Field studies that evaluated arthropod
abundance in plots of stacked VIP3A x Cry1Ab Bt corn were also
conducted. To date, results show that beneficial arthropods were
significantly more abundant in plots containing Bt plants than in plots
treated with conventional chemical insecticides (interim report MRID
458358-07).

The reviewed data showed no toxicity to non-target test species. As a
result of these findings and knowledge of the mode of action of PAT, the
Agency concludes that no unreasonable adverse effects on non-target
organisms are expected from exposure to PAT protein.

Endangered Species Considerations

Because this PIP inert ingredient is neither a known toxin and/or
pathogen of plant or animal species, EPA does not expect that exposure
to the PAT protein will result in a “may affect” finding for any
endangered or threatened species. 

Gene Flow Considerations

Gene flow may occur when the PAT protein is expressed in a plant that
can form viable hybrids in nature with wild or weedy relatives of that
species. With the assistance of the October 2004 SAP, EPA has identified
the following plants as not having wild or weedy relatives in the United
States, its possessions, or territories, with which they can produce
viable hybrids in nature: almond (Prunus communis), apricot (Prunus
armeniaca), asparagus (Asparagus officinale) avocado (Persea americana),
banana (Musa acuminata), barley (Hordeum vulgare), bean (Phaseolus
vulgaris), black-eyed pea (Vigna unguiculata), cacao (Theobroma cacao),
celery (Apium graveolens), chickpea (Cicer arietinum), citrus (Citrus
spp.), coffee (Coffea arabicua), corn (Zea maize), cucumber (Cucumis
sativus), eggplant (Solanum melongena), guava (Psidium guajava), kiwi
(Actinidia spp.), mango (Mangifera indica), nectarine (Prunus persica),
okra (Abelmoschus esculentus), olive (Olea europaea), papaya (Carica
papaya), parsley (Petroselinum crispum), pea (Pisum sativum), peach
(Prunus persica), peanut (Arachis hypogaea), pineapple (Ananas comosus),
pistachio (Pistacia vera), plum (Prunus domestica), potato (Solanum
tuberosum), soybean (Glycine max), spinach (Spinacia oleracea),
starfruit (Averrhoa carambola), taro (Colocasia esculenta), tomato
(Lycopersicon lycopersicum), or watermelon (Citrullus lanatus). Since
hybridization with wild or weedy relatives is not known to occur among
plants included on this list, EPA has concluded that, when introduced
into these species, PAT presents a low probability of risk to human
health and the environment.

CP4 enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS)

SUMMARY

The Agency has conducted an environmental risk assessment of the
plant-incorporated protectant (PIP) inert ingredient CP4
enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS), and the genetic
material necessary for its production. Topics covered in this assessment
include mode of action, ecological effects, endangered species
considerations, and gene flow from a modified crop to wild or weedy
relatives. Data cited in this assessment was submitted to the Agency in
support of Monsanto’s MON 810 Bt Corn registration. Ecological data
and published information on the biology of this protein indicate that
this PIP inert ingredient is not a known toxin and/or pathogen of plant
or animal species. Due to the low human health risks associated with
this protein, the Agency has granted an exemption from the requirement
of a tolerance for this PIP inert ingredient (40 CFR 180.1174; 61 FR
40340, Aug. 2, 1996).

MODE OF ACTION

When conventional plants are treated with glyphosate they cannot produce
the aromatic amino acids needed to grow. Glyphosate blocks biosynthesis
of aromatic amino acids by binding to 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS) in plants (Steinrucken and Amrhein, 1980). The CP4
EPSPS enzyme, isolated from the Agrobacterium tumefaciens strain CP4,
functions in the presence of glyphosate and thus confers tolerance to
glyphosate herbicides when expressed in plants. The EPSPS enzyme is
naturally present in plants, bacteria, and fungi (Levin and Sprinson,
1964).

ENVIRONMENTAL Effects

The Agency assessed the toxicity of CP4 EPSPS protein to representative
non-target organisms that could be exposed to the PIP inert ingredient.
Toxicity evaluations included the following Tier I studies: Mammalian
(mouse), Avian (juvenile northern bobwhite quail), and Freshwater Fish
(channel catfish). 

The wild mammal hazard assessment was performed on the basis of rodent
acute oral toxicity data prepared for human health risk assessment
purposes which utilized pure CP4 EPSPS protein. Submitted data indicate
no significant adverse effects among mice dosed with up to 572 mg/kg
body weight for CP4 EPSPS protein (MRID 436433-03). 

The avian study was a maximum hazard dose evaluation, where CP4 EPSPS
protein toxicity was evaluated by feeding northern bobwhite quail MON
801 corn grain. Results of this feeding study showed no mortality among
northern bobwhite quail fed diets containing MON 801 corn grain (MRID
435332-05).

Results of a freshwater fish corn grain feeding study reported that no
adverse effects on fish growth and survival were seen among channel
catfish fed MON 801 corn (MRID No. 438879-01).

The reviewed data showed no toxicity to non-target mammalian and avian
test species. As a result of these findings, knowledge of the mode of
action of CP4 EPSPS, and the presence of a similar enzyme, EPSPS, in all
plants, the Agency concludes that no unreasonable adverse effects on
non-target organisms are expected from exposure to the CP4 EPSPS
protein.

Endangered Species Considerations

Because this PIP inert ingredient is neither a known toxin and/or
pathogen of plant or animal species, EPA does not expect that exposure
to the CP4 EPSPS protein will result in a “may affect” finding for
any endangered or threatened species.

Gene Flow CONSIDERATIONS

Gene flow may occur when the CP4 EPSPS protein is expressed in a plant
that can form viable hybrids in nature with wild or weedy relatives of
that species. With the assistance of the October 2004 SAP, EPA has
identified the following plants as not having wild or weedy relatives in
the United States, its possessions, or territories, with which they can
produce viable hybrids in nature: almond (Prunus communis), apricot
(Prunus armeniaca), asparagus (Asparagus officinale) avocado (Persea
americana), banana (Musa acuminata), barley (Hordeum vulgare), bean
(Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), cacao
(Theobroma cacao), celery (Apium graveolens), chickpea (Cicer
arietinum), citrus (Citrus spp.), coffee (Coffea arabicua), corn (Zea
maize), cucumber (Cucumis sativus), eggplant (Solanum melongena), guava
(Psidium guajava), kiwi (Actinidia spp.), mango (Mangifera indica),
nectarine (Prunus persica), okra (Abelmoschus esculentus), olive (Olea
europaea), papaya (Carica papaya), parsley (Petroselinum crispum), pea
(Pisum sativum), peach (Prunus persica), peanut (Arachis hypogaea),
pineapple (Ananas comosus), pistachio (Pistacia vera), plum (Prunus
domestica), potato (Solanum tuberosum), soybean (Glycine max), spinach
(Spinacia oleracea), starfruit (Averrhoa carambola), taro (Colocasia
esculenta), tomato (Lycopersicon lycopersicum), or watermelon (Citrullus
lanatus). Since hybridization with wild or weedy relatives is not known
to occur among plants included on this list, EPA has concluded that,
when introduced into these species, CP4 EPSPS presents a low probability
of risk to human health and the environment.

Glyphosate oxidoreductase (GOX)

SUMMARY

The Agency has conducted an environmental risk assessment of the
plant-incorporated protectant (PIP) inert ingredient glyphosate
oxidoreductase (GOX), and the genetic material necessary for its
production. Topics covered in this assessment include mode of action,
ecological effects, endangered species considerations, and gene flow
from a modified crop to wild or weedy relatives. Data cited in this
assessment was submitted to the Agency in support of Monsanto’s MON
810 Bt Corn registration. Ecological data and published information on
the biology of this protein indicate that this PIP inert ingredient is
not a known toxin and/or pathogen of plant or animal species. Due to the
low human health risks associated with this protein, the Agency has
granted an exemption from the requirement of a tolerance for this PIP
inert ingredient (40 CFR 180.1190; 62 FR 52509, Oct. 8, 1997).

MODE OF ACTION

GOX catalyzes the conversion of glyphosate to aminomethylphosphonic acid
(AMPA) and glyoxylate; by increasing the speed of AMPA degradation, GOX
confers glyphosate tolerance to plants. The GOX proteins that EPA
evaluated for registration of MON 810 Bt Corn and for the GOX tolerance
exemption were derived from a gene originally isolated from
Achromobacter sp. strain LBAA. Two modified GOX proteins are specified
in the tolerance exemption. They are designated GOX and GOXv247. The GOX
protein has the same amino acid sequence as the native protein, with an
additional four amino acid sequence on the N-terminus (remnants of an
added signal sequence). In GOXv247, in addition to the four amino acids
at the N-terminus from the signal sequence, the gene sequence was
altered resulting in changes to three amino acids in the protein
compared to the native protein. These protein variants are similar to
the native GOX protein in terms of molecular weight, immunoreactivity,
amino acid sequence, and enzymatic activity.

ENVIRONMENTAL Effects

The Agency assessed the toxicity of GOX protein to mice, a
representative non-target mammalian species that could be exposed to the
PIP inert ingredient in the animal’s natural environment. The Tier 1
hazard assessment was performed on the basis of rodent acute oral
toxicity data prepared for human health risk assessment purposes which
utilized pure GOX protein. Submitted data indicate no significant
adverse effects among mice dosed with up to 91.3 and 104 mg/kg body
weight for GOX and GOXv247 proteins, respectively (MRID 439037-07 and
439037-08).  

The reviewed data showed no toxicity to the non-target mammalian test
species. As a result of this finding, and knowledge of the mode of
action of the enzyme, the Agency concludes that no unreasonable adverse
effects on non-target organisms are expected from exposure to GOX
protein.

Endangered Species Considerations

Because this PIP inert ingredient is neither a known toxin and/or
pathogen of plant or animal species, EPA does not expect that exposure
to the GOX protein will result in a “may affect” finding for any
endangered or threatened species.

GENE FLOW CONSIDERATIONS

Gene flow may occur when the GOX protein is expressed in a plant that
can form viable hybrids in nature with wild or weedy relatives of that
species. With the assistance of the October 2004 SAP, EPA has identified
the following plants as not having wild or weedy relatives in the United
States, its possessions, or territories, with which they can produce
viable hybrids in nature: almond (Prunus communis), apricot (Prunus
armeniaca), asparagus (Asparagus officinale) avocado (Persea americana),
banana (Musa acuminata), barley (Hordeum vulgare), bean (Phaseolus
vulgaris), black-eyed pea (Vigna unguiculata), cacao (Theobroma cacao),
celery (Apium graveolens), chickpea (Cicer arietinum), citrus (Citrus
spp.), coffee (Coffea arabicua), corn (Zea maize), cucumber (Cucumis
sativus), eggplant (Solanum melongena), guava (Psidium guajava), kiwi
(Actinidia spp.), mango (Mangifera indica), nectarine (Prunus persica),
okra (Abelmoschus esculentus), olive (Olea europaea), papaya (Carica
papaya), parsley (Petroselinum crispum), pea (Pisum sativum), peach
(Prunus persica), peanut (Arachis hypogaea), pineapple (Ananas comosus),
pistachio (Pistacia vera), plum (Prunus domestica), potato (Solanum
tuberosum), soybean (Glycine max), spinach (Spinacia oleracea),
starfruit (Averrhoa carambola), taro (Colocasia esculenta), tomato
(Lycopersicon lycopersicum), or watermelon (Citrullus lanatus). Since
hybridization with wild or weedy relatives is not known to occur among
plants included on this list, EPA has concluded that, when introduced
into these species, GOX presents a low probability of risk to human
health and the environment.

Antibiotic Resistance Marker

Neomycin phosphotransferase II (NPTII)

SUMMARY

The Agency has conducted an environmental risk assessment of the
plant-incorporated protectant (PIP) inert ingredient neomycin
phosphotransferase II (NPTII), and the genetic material required for its
production. Topics covered in this assessment include mode of action,
ecological effects, endangered species considerations, and gene flow
from a modified crop to wild or weedy relatives. Data cited in this
assessment was submitted to the Agency in support of Monsanto’s
NewLeaf Potato and YieldGard Plus Corn registrations and is discussed in
more detail in the Bacillus thuringiensis Plant-Incorporated Protectant
and MON863 Biopesticide Registration Action Documents. Ecological data
and published information on the biology of this protein indicate that
this PIP inert ingredient is not a known toxin and/or pathogen of plant
or animal species. Due to the low human health risks associated with
this protein, the Agency has granted an exemption from the requirement
of a tolerance for this PIP inert ingredient (40 CFR 180.1134; 59 FR
49125, Sep. 28, 1994).

MODE OF ACTION

The NPTII enzyme inactivates, by phosphorylation, a range of
aminoglycoside antibiotics, including neomycin and kanamycin.  NPTII
occurs naturally in bacteria, is not known to be toxic, and degrades
rapidly under simulated gastric conditions.  Microbially-produced and
plant-produced NPTII enzymes have similar molecular weights and terminal
amino acids, indicating that glycosylation and post-transcription
modifications do not occur with plant-expressed NPTII.

ENVIRONMENTAL Effects

The Agency assessed the toxicity of NPTII protein to representative
non-target organisms that could be exposed to the PIP inert ingredient.
Toxicity evaluations included the following Tier I studies: Mammalian
(mouse), Avian (juvenile northern bobwhite quail), Freshwater Fish
(channel catfish), Aquatic Invertebrate [cladoceran (Daphnia magna)],
and Non-target Arthropod (collembola, lady beetle, and monarch
butterfly). A Tier IV field study of non-target abundance is also
discussed below.

The wild mammal hazard assessment was performed on the basis of rodent
acute oral toxicity data prepared for human health risk assessment
purposes which utilized pure NPTII protein. Submitted data indicate no
significant adverse effects among mice dosed with up to 5000 mg/kg body
weight NPTII protein (MRID 430547-01). 

The remaining single species studies were maximum hazard dose
evaluations, where NPTII protein toxicity was evaluated by feeding
select non-target organisms NewLeaf or YieldGard Plus plant material,
which has been shown to contain the PIP inert ingredient NPTII and the
active ingredient Cry3A (NewLeaf) or Cry3Bb1 (YieldGard Plus). The avian
studies, where northern bobwhite quail were fed potato tuber tissue for
the NewLeaf and corn grain for the YieldGard Plus evaluations, reported
no adverse effects to the test species at field exposure levels (NewLeaf
MRIDs 429322-14, 429322-15; MON863 MRID 449043-15). The freshwater fish
corn grain feeding study indicated that channel catfish diets may
contain up to 35% NPTII protein-containing corn grain meal with no
adverse effects on fish growth, feed conversion efficiency, survival,
behavior, or body composition (MON863 MRID 449043-19). In a corn pollen
feeding study, no adverse effects to Daphnia magna, a representative of
aquatic invertebrate species, were seen at field level exposure (MON863
MRID 449043-18). In separate corn pollen dietary toxicity studies, no
adverse effects to adult and larval lady beetles were seen at field
level exposure (diet composed of 50% pollen) (MON863 MRIDs 455382-04,
453613-02). A dietary toxicity study with the monarch butterfly also
showed no adverse effects to larvae when exposed to field levels of corn
pollen (MON863 MRID 455382-05). Finally, results of a corn leaf tissue
feeding study indicated that the NPTII protein does not adversely affect
reproduction of Collembola, a representative plant tissue decomposer
(MON863 MRID 449043-17).

A field study that evaluated arthropod abundance in YieldGard Plus
fields was also conducted. Results showed that beneficial arthropods
(i.e. lady beetles, damsel bugs, flower flies, soldier beetles, big eyed
bugs, spiders, minute pirate bugs, green lacewings, brown lacewings,
stink bugs, and ground beetles) were significantly more abundant in
plots containing Bt plants than in plots treated with conventional
chemical insecticides (MRID 456530-03).

The reviewed data showed no toxicity to non-target test species. As a
result of these findings, and knowledge of the mode of action of the
enzyme, the Agency concluded that no unreasonable adverse effects on
non-target organisms are expected from exposure to NPTII protein.

Endangered Species Considerations

Because this PIP inert ingredient is neither a known toxin and/or
pathogen of plant or animal species, EPA does not expect that exposure
to the NPTII protein will result in a “may affect” finding for any
endangered or threatened species.

Gene Flow CONSIDERATIONS

EPA anticipates that gene flow may occur if NPTII is expressed in plants
that have wild or weedy relatives in the United States, its possessions,
or territories. However, no unreasonable adverse effects to the
environment are expected from movement of this inert into wild plant
populations, because the trait is unlikely to confer selective advantage
to recipient plants. 

EPA considers the frequency of horizontal gene transfer (HGT) from
NPTII-expressing plants to microbes to be very low. Studies conducted
under a range of test conditions could not demonstrate HGT from plants
to microbes, nor is there a clear mechanism for such transfer (Nap et
al., 1992; Redenbaugh et al. 1994; Schlüter et al., 1995; SAP Report,
2001). Nonetheless, if HGT was to occur it would not have a significant
affect on existing populations of neomycin and kanamycin resistant
bacteria, because resistance to these groups of antibiotics is
widespread in naturally occurring microbes in humans and the
environment. 

Due to the low probability that gene flow from NPTII-containing plants
would confer a selective advantage on wild or weedy relatives of that
species and the low frequency of HGT from NPTII-expressing plants to
microbes, EPA has concluded that this inert marker poses low risk to
human health and the environment when used in any plant as part of a
PIP.

OTHER MARKERS

Beta-D-glucuronidase (GUS) from Escherichia coli

SUMMARY

The Agency has conducted an environmental risk assessment of the
Escherichia coli-derived plant-incorporated protectant (PIP)
beta-D-glucuronidase (GUS), and the genetic material necessary for its
production. Topics covered in this assessment include mode of action,
ecological effects, endangered species considerations, and gene flow
wild or weedy relatives. Data cited in this assessment were submitted to
the Agency in support of Monsanto’s Bollgard II Bt cotton registration
and are discussed in the Bollgard II BRAD. Ecological data and published
information on the biology of this protein indicate that this PIP inert
ingredient is not a known toxin and/or pathogen of plant or animal
species. Due to the low human health risks associated with this protein,
the Agency has granted an exemption from the requirement of a tolerance
for this PIP inert ingredient (40 CFR 180.1216; 66 FR 42961, Aug. 16,
2001).

MODE OF ACTION

E.coli (-glucuronidase (GUS), an enzyme that catalyzes the hydrolysis of
glucuronides, is introduced into plants to serve as a visual marker of
transformation (  HYPERLINK
"http://www.sciencedirect.com/science?_ob=ArticleURL&_aset=V-WA-A-W-ZC-M
sSAYWW-UUA-U-AABBVZEBBC-AABAUCUABC-VVVEDZEDD-ZC-U&_rdoc=4&_fmt=full&_udi
=B6T30-4DTP5DS-2&_coverDate=02%2F01%2F2005&_cdi=4932&_orig=search&_st=13
&_sort=d&view=c&_acct=C000001678&_version=1&_urlVersion=0&_userid=14684&
md5=f12d80d9007d0ed5200a40da0cd22384" \l "bib6#bib6"  Guivarc'h et al.,
1996 ). GUS from E.coli has a pH optimum near 7.0 and maintains
enzymatic activity for approximately 2 hours at 55º C, but is rapidly
inactivated at 60º C. This enzyme is ubiquitous in the digestive system
of humans and other vertebrates. Other types of GUS enzyme are present
in the liver, spleen, kidneys, salivary glands, breast milk and a
variety of other tissues in humans, other vertebrates and a number of
invertebrates, as well as in the fruit, seed coat, and endosperm of
various plants.

ENVIRONMENTAL Effects

The Agency assessed the toxicity of GUS protein to representative
non-target organisms that could be exposed to the PIP inert ingredient.
Toxicity evaluations included the following Tier I studies: Mammalian
(mouse), Avian (juvenile northern bobwhite quail), Freshwater Fish
(channel catfish), and Non-target Arthropod (collembola). 

The wild mammal hazard assessment was performed on the basis of rodent
acute oral toxicity data prepared for human health risk assessment
purposes which utilized pure GUS protein. Submitted data indicate no
significant adverse effects among mice dosed with up to 100.0 mg/kg body
weight GUS protein (MRID 449888-00). 

The remaining single species studies were maximum hazard dose
evaluations, where GUS protein toxicity was evaluated by feeding select
non-target organisms Bollgard II plant tissue, which has been shown to
contain the PIP inert ingredient GUS and the active ingredients Cry1Ac
and Cry2Ab in cotton leaf and seed material. The avian cottonseed meal
feeding study reported no adverse effects to northern bobwhite quail at
field exposure levels (MRID 450863-16). The freshwater fish cottonseed
meal feeding study indicated that channel catfish diets may contain up
to 20% GUS protein-containing cotton seed meal with no adverse effects
on fish growth, feed conversion efficiency, survival, behavior, or body
composition (MRIDs 450863-18 and 453371-03). Finally, results of a
cotton tissue feeding study indicated that the GUS protein is non-toxic
to Collembola, a representative plant tissue decomposer, and did not
adversely affect the rate of Collembola reproduction (MRID 450863-14). 

The reviewed data showed no toxicity to non-target test species. As a
result of these findings, and the natural occurrence of this protein in
a variety of species, and knowledge of the mode of action of the enzyme,
the Agency concluded that no unreasonable adverse effects on non-target
organisms are expected from exposure to GUS protein.

Endangered Species Considerations

Because this PIP inert ingredient is neither a known toxin and/or
pathogen of plant or animal species, EPA does not expect that exposure
to the GUS protein will result in a “may affect” finding for any
endangered or threatened species.

Gene Flow CONSIDERATIONS

EPA anticipates that gene flow may occur if GUS is expressed in plants
that have wild or weedy relatives in the United States, its possessions,
or territories. However, due to the low probability that gene flow from
a GUS-containing plant would confer a selective advantage on wild or
weedy relatives of that species, EPA has concluded that this inert
marker poses low risk to human health and the environment when used in
any plant as part of a PIP.

Phosphomannose isomerase (PMI)

SUMMARY

The Agency has conducted an environmental risk assessment of the
Escherichia coli-derived plant-incorporated protectant (PIP)
phosphomannose isomerase (PMI), and the genetic material required for
its production. Topics covered in this assessment include mode of
action, ecological effects, endangered species considerations, and gene
flow from a modified crop to wild or weedy relatives. Data cited in this
assessment were submitted to the Agency in support of Syngenta’s
MIR604 Bt corn registration. Ecological data and published information
on the biology of this protein indicate that this PIP inert ingredient
is not a known toxin and/or pathogen of plant or animal species. Due to
the low human health risks associated with this protein, the Agency has
granted an exemption from the requirement of a tolerance for this PIP
inert ingredient (40 CFR 180.1252; 69 FR 26770, May 14, 2004).

MODE OF ACTION

The PMI protein is a ubiquitous enzyme involved in carbohydrate
metabolism. Plant cells take up mannose and convert it to
mannose-6-phosphate, an inhibitor of glycolysis. PMI activity converts
mannose-6-phosphate to fructose-6-phosphate, an intermediate of
glycolysis, which positively supports growth of transformed cells (Todd
and Tague, 2001). PMI, or a highly homologous enzymatic protein, is
expressed in many species including enteric bacteria, fungi, insects,
some species of nematodes, and mammals including monkeys, mice and man.

ENVIRONMENTAL Effects

The Agency assessed the toxicity of PMI protein to representative
non-target organisms that could be exposed to the PIP inert ingredient.
Toxicity evaluations included the following Tier I studies: Mammalian
(mouse) and Freshwater Fish (rainbow trout).

The wild mammal hazard assessment was performed on the basis of rodent
acute oral toxicity data prepared for human health risk assessment
purposes which utilized pure PMI protein. Submitted data indicate no
significant adverse effects among mice dosed with up to 5,050 mg/kg of
dosing solution or 3,080 mg/kg of PMI protein (MRID 459344-07).

The freshwater fish study was a maximum hazard dose evaluation, where
PMI protein toxicity was evaluated by feeding juvenile rainbow trout
Event MIR604 corn grain, which has been shown to contain the PIP inert
ingredient PMI and the active ingredient Cry1Ab. Results of the feeding
study indicate that rainbow trout diets may contain up to 50% PMI
protein-containing corn seed meal with no adverse effects on fish weight
or length (MRID 461556-17). 

The reviewed data showed no toxicity to non-target mammalian and
freshwater fish test species used in the evaluations. As a result of
these findings, and the natural occurrence of this protein in a variety
of microbe, insect, plant and mammalian species, the Agency has
concludes that no unreasonable adverse effects on non-target organisms
are expected from exposure to PMI protein.

Endangered Species Considerations

Because this PIP inert ingredient is neither a known toxin and/or
pathogen of plant or animal species, EPA does not expect that exposure
to the PMI protein will result in a “may affect” finding for any
endangered or threatened species.

Gene Flow CONSIDERATIONS

EPA anticipates that gene flow may occur if PMI is expressed in plants
that have wild or weedy relatives in the United States, its possessions,
or territories. However, due to the low probability that gene flow from
a GUS-containing plant would confer a selective advantage on wild or
weedy relatives of that species, EPA has concluded that this inert
marker poses low risk to human health and the environment when used in
any plant as part of a PIP.

References

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Levin, J.G. and Sprinson, D.B. (1964). The enzymatic formation and
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Nap, J.P., Bijvoet, J. and Strikema, W.J. (1992). Biosafety of
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OECD (2002). Module II: Herbicide biochemistry, herbicide metabolism,
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Redenbaugh, K., Hiatt, W., Martineau, B., Lindemann, J. and Emlay, D.
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SAP Report No. 2000-17 (2001). Bt Plant-Pesticides Risk and Benefit
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Schlüter, K., Futterer, J. and Potrykus, I. (1995). “Horizontal”
gene transfer from a transgenic potato line to a bacterial pathogen
(Erwinia chryanthemi) occurs - if at all - at an extremely low
frequency. Bio/Technology 13:1094-1098.

Steinruken, H.C., Amrhein, N. (1980). The herbicide glyphosate is a
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Thompson, C.J., N.R.Movva, R. Tizard, R. Crameri, J.E. Davies, M.
Lauwereys, J. Botterman. (1987). Characterization of the
herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J.
6:2519-23.

Todd, R. and B.W. Tague (2001). Phosphomannose Isomerase: A versatile
selectable marker for Arabidopsis thaliana germ-line transformation.
Plant Molecular Biology Reporter 19: 307-319. pp 758-5016.

UNPUBLISHED STUDIES (identified by MRID Numbers)

429322-14	Cambell, S.; Beavers, J.; Jaber, M. (1993) A Dietary Toxicity
Study with Russet Burbank Potatoes in the Northern Bobwhite: Lab Project
Number: 139-356: WL-93-11: 93-081E. Unpublished study prepared by
Wildlife International Ltd. 87 p.

429322-15	Campbell, S.; Beavers, J.; Jaber, M. (1993) A Dietary Toxicity
Study with Russet Burbank Potatoes in the Northern Bobwhite: Lab Project
Number: 139-357: WL-93-12. Unpublished study prepared by Wildlife
International Ltd. 50 p.

430547-01	Naylor, M. (1992) Acute Oral Toxicity Study of (inert
ingredient) in Albino Mice: Lab Project Number: 91177: ML-91-409: EHL
91177. Unpublished study prepared by Monsanto Co. Environmental Health
Lab. 161 p.

433236-10	Collins, M. (1994) Bt Maize Pollen (PHO176-0194): Acute
Toxicity to Daphnids (Daphnia magna) under Static-Renewal Conditions:
Lab Project Number: 94/3/5217: 1781/0394/6419/110. Unpublished study
prepared by Springborn Laboratories, Inc. 59 p.

433396-02	Obrycki, J. (1994) Feeding, Development, and Survival of the
Lady Beetle Predator Coleomegilla maculata (Coleoptera:coccinellidae) on
Transgenic "Bt" Maize (Corn) Pollen: Final Report: Lab Project Number:
PHO176/0194 ISU. Unpublished study prepared by Iowa State University,
Department of Entomology. 62 p.

434157-03	Maggi, V. (1994) Evaluation of the Dietary Effect(s) of
Transgenic Bt Maize (Corn) Pollen (Sample PH0176-0294) on Honeybee
Development: Lab Project Number: CAR 176-94. Unpublished study prepared
by California Agricultural Research, Inc. 57 p.

435332-05	Campbell, S.; Beavers, J. (1994) A Dietary Toxicity Study with
MON 80187 Meal in the Northern Bobwhite: Lab Project Number: WL 94-150:
139-387. Unpublished study prepared by Wildlife International Ltd. 32 p.

436433-03	Naylor, M. (1993) Acute Oral Toxicity Study of (inert
ingredient) in Albino Mice: Lab Project Number: 92223. Unpublished study
prepared by Monsanto Co. 179 p.

438879-01	Jackson, L.; Robinson, E.; Nida, D. et al. (1995) Evaluation
of the European Corn Borer Resistant Corn Line MON 801 as a Feed
Ingredient for Catfish: Lab Project Number: 94-01-39-16: 14066:
95-459-720. Unpublished study prepared by Mississippi State University
Delta Research and Extension Center. 39 p.

439037-07	Nickson, T.; Bailey, M.; Harrison, L. et al. (1994)
Preparation and Confirmation of Doses for Acute Oral Toxicity Study in
Mice with (Inert Ingredient): Lab Project Number: 93-02-30-03: 12967:
93-447-703. Unpublished study prepared by Monsanto Co. 42 p.

439037-08	Naylor, M. (1994) Acute Oral Toxicity Study of (Inert
Ingredient) in Albino Mice: Lab Project Number: ML-93-206: 93079.
Unpublished study prepared by Monsanto Environmental Health Lab. 204 p.

439995-02	Merriman, T. (1996) An Acute Oral Toxicity Study in Mice with
(inert ingredient): Lab Project Number: DGC-95-A18: 3406.2: 3406.1.
Unpublished study prepared by DEKALB Genetics Corp. and Springborn
Laboratories (SLI). 50 p.

439995-10	Palmer, S.; Beavers, J. (1996) Lyophilized DBT418 Leaf Tissue:
A Dietary Toxicity Study with the Northern Bobwhite: Lab Project Number:
DGC-95-A13: 438-101: 438/102495/QLC-TRAN/CHP106. Unpublished study
prepared by DEKALB Genetics Corp. and Wildlife Int'l. Ltd. 51 p.

439995-12	Collins, M. (1996) Transgenic Maize Leaf Tissue and
Microbially Produced CryIA(c) Protein: Chronic Toxicity To Collembola
(Folsomia candida), Under Static Conditions: Lab Project Number:
DGC-95-A16: 13601.1195.6101.123: 96-3-6408. Unpublished study prepared
by DEKALB Genetics Corp. and Springborn Laboratories, Inc. (SLI). 141 p.

439995-13	Garvey, N. (1996) Lyophilized DBT418 Maize Leaf Tissue and
Microbially Produced CryIA(c) Protein: Acute (14-Day) Toxicity To
Earthworms (Eisenia foetida): Lab Project Number: DGC-95-A15:
13601.1095.6100.630: 96-2-6377. Unpublished study prepared by DEKALB
Genetics Corp. and Springborn Laboratories, Inc. (SLI). 81 p.

449043-15	Gallagher, S.; Grimes, J.; Beavers, J. (1999) Bacillus
thuringiensis Protein 11231 in Corn Grain: A Dietary Toxicity Study with
the Northern Bobwhite: Lab Project Number: WL-99-014: 16161: 139-44.
Unpublished study prepared by Monsanto Company and Wildlife
International Ltd. 43 p.

449043-17	Teixeira, D. (1999) Assessment of Chronic Toxicity of Corn
Tissue Containing the Bacillus thuringiensis Protein 11098 to Collembola
(Folsomia candida): Lab Project Number: SB-98-296: 15988: 252.6149.
Unpublished study prepared by Monsanto Company and Springborn Labs. 55
p.

449043-18	Drottar, K.; Krueger, H. (1999) Bacillus thuringiensis Protein
11098 in Corn Pollen: A 48-Hour Static-Renewal Acute Toxicity with the
Cladoceran (Daphnia magna): Lab Project Number: WL-98-295: 16163:
139A-236. Unpublished study prepared by Monsanto Company and Wildlife
International Ltd. 31 p.

449043-19	Meng, L.; Robinson, E. (1999) Evaluation of Insect Protected
Corn Lines MON 853 and MON 859 as a Feed Ingredient for Catfish: Lab
Project Number: XX-98-297: 16164. Unpublished study prepared by Monsanto
Company and Mississippi State University. 32 p.

449888-00	Monsanto Company (1999) Submission of Toxicity Data in Support
of the Petition for Tolerance of (Inert Ingredient) as a Plant
Pesticide. Transmittal of 1 Study.

450863-14	Teixiera, D. (2000) Assessment of Chronic Toxicity of Cotton
Tissue Containing Insect Protection Protein 2 to Collembola (Folsomia
candida): Amended Final Report: Lab Project Number: 252.6152: SB-99-063:
MSL 16174. Unpublished study prepared by Springborn Laboratories, Inc.
49 p.

450863-16	Gallagher, S.; Grimes, J.; Beavers, J. (2000) Insect
Protection Protein 2 In Cottonseed Meal: A Dietary Toxicity Study with
the Northern Bobwhite: Lab Project Number: 139-449: WL-99-065: MSL
16178. Unpublished study prepared by Wildlife International, Ltd. 40 p.

450863-18	Li, M.; Robinson, E. (2000) Evaluation of Cottonseed Meal
Derived from Insect Protected Cotton Lines 15813 and 15985 as a Feed
Ingredient for Catfish: Final Report: Lab Project Number: XX-99-056: MSL
16179: 99-858E. Unpublished study prepared by Thad Cochran National
Warmwater Aquaculture Center. 22 p.

453371-03	Li, M.; Robinson, E. (2001) Evaluation of Cottonseed Meal
Derived from Insect Protected Cotton Lines 15813 and 15985 as a Feed
Ingredient for Catfish: Lab Project Number: XX-99-056: MSL 16179.
Unpublished study prepared by Thad Cochran National Warmwater
Aquaculture Center 23 p.

453613-02	Bryan, R.; Porch, J.; Krueger, H. (2001) Dietary Effects of
Transgenic BT Corn Pollen Expressing a Variant of Cry3Bb1 Protein on
Adults of the Ladybird Beetle, Hippodamia convergens: Final Report: Lab
Project Number: WL-2000-158: MSL-17171: 139-453. Unpublished study
prepared by Wildlife International, Ltd. 36 p.

455382-04	Duan, J.; McKee, M.; Nickson, T. (2001) Dietary Effects of
Transgenic Bacillus thuringiensis (Bt) Corn Pollen Expressing a Variant
of Cry3Bb1 Protein on Larvae of the Ladybird Beetle, Coleomegilla
maculata: Lab Project Number: MSL-16907: 00-01-39-26. Unpublished study
prepared by Monsanto Company. 39 p.

455382-05	Sears, M.; Mattila, H. (2001) Determination of the Toxicity of
Corn Pollen Expressing a Cry3Bb1 Variant Protein to First Instar Monarch
Butterfly Larvae (Danaus plexippus) via Laboratory Bioassay: Lab Project
Number: MSL-17235: 01-01-39-26. Unpublished study prepared by University
of Guelph. 33 p.

456530-03	Head, G. (2002) Research on the Effects of Corn Rootworm
Protected Transgenic Corn Events on Nontarget Organisms: Preliminary
Results: Lab Project Number: 00-CR-032E-7. Unpublished study prepared by
Monsanto Company. 70 p.

457665-09	Teixeira, D. (2002) Assessment of the Chronic Toxicity of
VIP3A and VIP3A/Cry1Ab Maize Pollen to Pink-Spotted Lady Beetle
(Coleomegilla maculata): Lab Project Number: 1781.6623: PACHA:
PACHA-00-05AN. Unpublished study prepared by Springborn Smithers
Laboratories. 79 p. {OPPTS 885.4340}

457921-01	Putt, A. (2002) VIP3A Maize (Corn) Pollen--Acute Toxicity to
Daphnids (Daphnia magna) Under Static-Renewal Conditions: Lab Project
Number: 1781.6616: SSB-003-00. Unpublished study prepared by Springborn
Smithers Laboratories. 42 p.

458358-07	Valchos, D.; Habig, C. (2002) Environmental Safety Assessment
of Bacillus thurnigiensis VIP3A Protein and VIP3A Cotton Event COT102 to
Non-Target Organisms: Unpublished study prepared by Syngenta Seeds, Inc.
and Exponent. 37 p.

458358-08	Teixeira, D. (2002) VIP3A Inbred Maize (Corn) Pollen: Toxicity
to Green Lacewing (Chrysoperla carnea): Lab Project Number: 1781.6624.
Unpublished study prepared by Springborn Smithers Laboratories. 69 p.
{OPPTS 885.4340}

458358-10	Privalle, L. (2002) Impact of VIP3A and Cry1AB Transgenic
Maize (Corn) Leaf Tissue (Samples LLPACHA-0100, LLBT11-0100, and
LLPACHABT11-0100) On 28-Day Survival and Reproduction of Collembola
(Folsomia candida): Lab Project Number: PACHA-00-03: SSB-006-01: PACHA.
Unpublished study prepared by Syngenta Seeds, Inc. 21 p.

458358-13	Dively, G. (2002) Impact of Transgenic Lepidopeteran-Resistant
VIP3A Field Corn (Maize) on Honey Bee Colonies in a Semi-Field Setting.
Unpublished study prepared by Wye Research and Education Center,
University of Maryland. 34 p.

459344-07	Kuhn, J. (1999) Acute Oral Toxicity Study in Mice: Final
Report: (Inert Ingredient): Final Report: Lab Project Number: 4708-98:
PMI: PMI-98-01. Unpublished study prepared by Stillmeadow, Inc. 24 p.
{OPPTS 870.1100}

461556-17	Hutchings, M.; Caunter, J. (2003) A 28-Day Laboratory Study to
Evaluate the Effects of Modified Cry3A Maize Fish Feed (FFMIR604-0103)
on the Growth of Juvenile Rainbow Trout (Oncorhynchus mykiss). Project
Number: 03/0294/A, 2033039. Unpublished study prepared by Astrazeneca Uk
Ltd. 19 p.

 Tier I testing consists of maximum dose single species hazard
assessments that are used to evaluate the potential for toxicity,
infectivity, and pathogenicity of a pesticidal agent to nontarget
organisms.  

 Tier IV testing refers to field testing under simulated or actual field
conditions. These studies are designed on a case-by-case basis to
evaluate specific problems that cannot be resolved by lower tier
testing.

		

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