Request for Temporary Exemption from the Requirements of a Tolerance

AF36 on Corn

EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY NOTICE OF
FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER 
(7/1/2006)

EPA has received a pesticide petition  from Interregional Research
Project Number 4 (IR-4), Rutgers University, 500 College Road East,
Suite 201W, Princeton, NJ 08540 on behalf of the Arizona Cotton Research
and Protection Council, 3721 East Wier Avenue Phoenix, Arizona
85040-2933  proposing pursuant to section 408(d) of the Federal  Food,
Drug, and Cosmetic Act (FFDCA),  21 U.S.C. 346a(d), to amend 40 CFR part
180.1206 to establish a temporary tolerance exemption for the microbial
pesticide Aspergillus flavus AF36 in or on Corn.

Pursuant to section 408(d)(2)(A)(i) of the FFDCA, as amended,
Interregional Research Project Number 4 (IR-4), Rutgers University, 500
College Road East, Suite 201W, Princeton, NJ 08540 on behalf of the
Arizona Cotton Research and Protection Council has submitted the
following summary of information, data, and arguments in support of the
pesticide petition.  This summary was prepared by Interregional Research
Project Number 4 (IR-4) Rutgers University, 500 College Road East, Suite
201W, Princeton, NJ 08540 on behalf of the Arizona Cotton Research and
Protection Council 3721 East Wier Avenue Phoenix, Arizona 85040-2933 and
EPA has not fully evaluated the merits of the pesticide petition.  The
summary may have been edited by EPA if the terminology used was unclear,
the summary contained extraneous material, or the summary
unintentionally made the reader conclude that the findings reflected
EPA’s position and not the position of the petitioner. 

I.   Interregional Research Project Number 4 (IR-4), Rutgers University,
500 College Road East, Suite 201W, Princeton, NJ 08540 on behalf of the
Arizona Cotton Research and Protection Council 3721 East Wier Avenue
Phoenix, Arizona 85040-2933]  Petition Summary

    

A.  Product name and Proposed Use Practices 

	Aspergillus flavus AF36, a non-aflatoxin-producing strain of A. flavus,
is proposed for application to corn to reduce the incidence of aflatoxin
producing strains of A. flavus and thereby reduce aflatoxin
contamination of corn.  Aspergillus flavus AF36 which does not produce
aflatoxin, competitively excludes aflatoxin producing A. flavus strains
without increasing A. flavus in the environment in the long term. 
Sterile wheat seed colonized with Aspergillus flavus AF36 is applied at
10 pounds of end-use product (total amount of active ingredient less
than 0.01 lb/acre) per acre.  The pesticide is currently being used in
certain counties in California and the States of Arizona and Texas in
cotton. The current submission proposes to establish a temporary
exemption from tolerance for residues of A. flavus AF36 on corn.

B.  Product Identity/Chemistry 

	1. Identity of the pesticide and corresponding residues.    The
pesticide and corresponding residues are identified as Aspergillus
flavus AF36.  

	2. Magnitude of residue at the time of harvest and method used to
determine the residue.   Aspergillus flavus AF36 is a naturally
occurring fungus that was originally isolated from cottonseed produced
in the Yuma Valley of Arizona.  Aspergillus flavus AF36 has been shown
to be naturally and consistently associated with corn grown in Texas. 
A. flavus is a widespread fungus.  It is particularly well adapted to
the hot regions of Texas where it is widespread in the environment.  The
communities of A. flavus in the desert and in agricultural fields are
naturally composed of both aflatoxin producing (toxigenic) and aflatoxin
non-producing (atoxigenic) strains.  Both atoxigenic and toxigenic
strains have been found on essentially all plant material and soils in
Texas.  The goal of applications is to increase the percent of the
Aspergillus flavus community composed of the strain AF36 and to decrease
the percent of A. flavus that produces aflatoxins on the crop and the
field.

	3. A statement of why an analytical method for detecting and measuring
the levels of the pesticide residue are not needed.   A temporary
exemption from the requirement of a

tolerance for residues of the microbial pesticide A. flavus AF36 in/on
corn is being proposed for corn in Texas.   All lots of the active
ingredient (A. flavus isolate AF36) and the formulated product are
monitored as part of a rigorous quality control program. Starter
cultures of A. flavus isolate AF36 used in the production of the end-use
product are always screened for aflatoxin production using TLC and
appropriate standards. Quality control standards are zero tolerance in
the starter cultures and in the formulated product for aflatoxin
production, and for A. flavus not identified as A. flavus isolate AF36. 
A. flavus AF36 has never been found to produce aflatoxin. Starter
cultures of A. flavus AF36 as well as end-use products containing this
active ingredient are also identified to isolate by vegetative
compatibility analysis. 

C.  Mammalian Toxicological Profile 

An acute oral toxicity test was performed whereby a single oral dose of
5,000 milligrams/kilogram (mg/kg) per animal of Aspergillus flavus AF36
colonized wheat seed was administered by gavage to five male and five
female Sprague Dawley rats.  The oral LD50 of Aspergillus flavus AF36
was determined to be greater than 5,000 mg/kg rat body weight.  No
clinical signs were observed during the 14 day study and no
abnormalities or adverse effects were observed in any of the rats upon
necropsy.

               An initial pulmonary rat study which resulted in
lethality in a significant number of animals treated with either the
live Aspergillus flavus AF36 in Tween 80 or heat killed Aspergillus
flavus AF36 in Tween 80.  Onset of symptoms was rapid after dosing with
all deaths occurring by day four of the study.  All rats surviving to
day four of the study recovered and all rats sacrificed (as scheduled)
on day 8 or day 15 of the study had totally eliminated viable
Aspergillus flavus AF36 from the lungs, caecal contents, and faeces. 
There was no evidence of infectivity.  The aetiology of deaths was
unclear.  It appeared that Aspergillus flavus AF36 prepared using Tween
80 caused a severe acute inflammatory response.  Retrospective
literature review and consultation with a toxicologist supported the
theory that the responses were a result of a synergism with Tween 80
and/or of Tween 80 breakdown products formed during preparation of the
spore suspension.

              A second rat study was therefore undertaken.  In the
second study the conidia were both washed from the wheat and suspended
in sterile physiological saline instead of Tween 80.  Animals (2 male
and 2 female for each treatment level) were dosed at 0, 105, 106, 107,
and 108 colony forming units per rat.  There were no clinical signs in
any of the treatment groups considered to be associated with the test
substance.  Rats were sacrificed at day 8 without treatment associated
mortality.  No abnormalities were observed in any of the animals at the
macroscopic examination at termination. 

	Based on these two mammalian studies, it was concluded that Aspergillus
flavus AF36 does not present either a toxicological or an infectious
risk to mammals.  

	Data waivers were requested for the toxicology studies listed below on
the following basis:(1) researchers and other workers have worked with
Aspergillus flavus AF36 at the Southern Regional Research Center for
over 10 years and in commercial fields (1996 to 1998) and in hand picked
field plots (1989 to 1994) without report of any adverse health effects;
(2) Aspergillus flavus AF36 is widely distributed in the environment and
its occurrence is natural; and (3) the label will require applicators
and other handlers to wear Personal Protective Equipment (PPE) such as
waterproof gloves, a dust/mist filtering respirator with the appropriate
NIOSH approval prefix N-95, P-95, or R-95, coveralls, long sleeved shirt
and long pants, and shoes plus socks, and goggles, so exposure should
not be a problem.  Applications will involve aerial application by
mixers/handlers who are licensed and trained to even handle restricted
materials. At the 10 lb/acre application rate of the formulated
material, the total amount of active ingredient is less than 0.01
lb/acre. Applications of AF36 do not significantly impact the total
amount of Aspergillus flavus in the soil or crop, but only change the
proportion of the AF36 strain in relation to the overall soil or crop
population. Since the product is applied as a granular formulation on
wheat,  exposure from drift is minimal.

	1. Acute dermal toxicity. This study was waived based upon the lack of
toxicity in animals dosed orally . While Aspergillus flavus has been
reported to be a dermal sensitizer, this testing is not warranted, since
the PPE required on the label will provide adequate protection from
dermal exposure to workers and pesticide handlers.

               

 	2. Primary dermal irritation. This study was waived based upon the
lack of toxicity in animals dosed orally.  Aerial applications will be
made by mixers/handlers and applicators, who are required to wear
appropriate PPE, and are licensed and trained to even handle restricted
materials. At the 10 lb/acre application rate of the formulated
material, the total amount of active ingredient is less than 0.01
lb/acre. Applications of AF36 do not significantly impact the total
amount of Aspergillus flavus in the soil or crop, but only change the
proportion of the AF36 strain in relation to the overall population.
Since the product is applied as a granular formulation on wheat, 
exposure from drift is minimal.

	3 Primary eye irritation.  This study was waived based upon the use of
appropriate PPE, including goggles, and other factors listed above. 
Since the product is applied aerially as a granular formulation on
wheat, exposure from drift is expected to be minimal.

	4. Acute  Intraperitoneal.  This study was waived based upon the lack
of toxicity in animals dosed orally and by intratracheal instillation. 
In addition, the factors listed above will mitigate exposure, which is
expected to be minimal.

	5.  Genotoxicity, reproductive and developmental toxicity, subchronic
toxicity and chronic toxicity testing were not performed, since no
adverse effects were observed in the acute toxicology study Tier 1
studies.   Tier II (885.3550) , subchronic toxicology study (USEPA OPPTS
885.3600) and chronic feeding studies (guideline 152-50) are only
required if triggered by adverse effects observed in Tier I studies. 

D.  Aggregate Exposure

	1. Dietary exposure. 

	i. Food.   Aspergillus flavus AF36 is a naturally occurring organism,
which does not produce aflatoxin and is thus safer than the
aflatoxin-producing A. flavus isolates that produce aflatoxin.  Proposed
uses and application rates will not result in increases in the total
population of A. flavus on the mature crop beyond naturally occurring
background levels.  Aspergillus flavus AF36, has been shown to
significantly reduce the levels of aflatoxin in corn at harvest without
changing the quantity of A. flavus on the crop.  Furthermore, the
proposed use and application rate will not increase exposure of humans
to A. flavus by dietary means.   Applications of Aspergillus flavus AF36
do not increase the indigenous populations of A. flavus associated with
the harvested crop.  The applications merely alter the composition of
the fungal community associated with the mature crop so that aflatoxin
producing strains are far less frequent.  The result is a much lower
incidence of aflatoxins in the crop and in the environment associated
with the developing and mature crop. 

	ii. Drinking water Aspergillus flavus AF36 is a naturally occurring
organism that is already widespread in the environment and is not
considered to be a risk to drinking water.  Both percolation through
soil and municipal treatment of drinking water would reduce the
possibility of exposure of A. flavus through the drinking water. 
Applications of Aspergillus flavus AF36 do not increase the long-term
populations of A. flavus in the environment, and thus are not expected
to influence the relationship of A. flavus to water sources. 
Applications merely change the composition of the A. flavus community so
that aflatoxins are less common in the environment.  

	2. Non-dietary exposure.   The potential for non-occupational,
non-dietary exposure to the general population is not expected to be
significant and is not expected to present any risk of adverse health
effects.]

E.  Cumulative Exposure  

	There are no other registered products containing Aspergillus flavus
AF36 or any other isolates (strains) of the microbial active ingredient.
 Data submitted show that the fungal metabolite of concern which is
aflatoxin is not produced by Aspergillus flavus AF36.   Aspergillus
flavus AF36 has been shown to exclude aflatoxin producing fungi
competitively from the developing crop and to reduce aflatoxin
contamination of corn.  Data show that the proposed use will not result
in appreciable increases in the long-term population of A. flavus on the
crop beyond naturally occurring levels.  Furthermore, there is no
expectation of cumulative effects with other pesticides.

F.  Safety Determination 

	1. U.S. population. Aspergillus flavus AF36 is a naturally occurring
organism. This isolate has low toxicity as demonstrated by the acute
oral toxicity study in rats.  A. flavus is ubiquitous throughout the hot
desert valleys in Arizona.  Studies have shown that treatment of cotton
fields just prior to flowering with sterile wheat seed colonized by
Aspergillus flavus AF36 at 10 lbs. per acre does not increase the
long-term populations of A. flavus either on the crop at maturity or in
the soil 1 year after application.  Based on this information, IR-4 is
of the opinion that the aggregate exposure to A. flavus over a lifetime
should not change with application of Aspergillus flavus AF36, and
exposure to both aflatoxin producing A. flavus strains and aflatoxin
should decrease.  This should be beneficial to human health.  Thus,
there is a reasonable certainty that no harm will result from aggregate
exposure to Aspergillus flavus AF36. 

	2. Infants and children.   Based on the lack of toxicity and natural
occurrence, there is reasonable certainty that no harm to infants,
children, or adults will result from aggregate exposure to Aspergillus
flavus AF36.  Exempting Aspergillus flavus AF36 from the requirement of
a tolerance should pose no significant risk to humans or the
environment. 

G.  Effects on the Immune and Endocrine Systems

	Aspergillus flavus AF36 is a naturally occurring organism, which does
not produce aflatoxin, and is thus safer than the A. flavus isolates
that produce aflatoxin.  To date there is no evidence to suggest that
Aspergillus flavus AF36 functions in a manner similar to any known
hormone, or that it acts as an endocrine disrupter. 

H.  Existing Tolerances

	The registrant is not aware of any existing tolerances or tolerance
exemptions for Aspergillus flavus AF36, other than the tolerance
exemption on cotton (40 CFR 180.1206)

I. Efficacy Data

            The following information was presented for a tolerance
exemption for AF36 on cotton.  The information is relevant for corn
because corn is grown in rotation with cotton. Therefore, the
farms(soil) which would be treated with AF36 in corn are already able to
be treated when those same fields are planted to cotton.  This request
is for a temporary exemption from the requirement of a tolerance for
AF36 on corn, as part of the EUP contained herein.  Existence of
aflatoxins in the environment is a public health hazard, and as such
data were submitted to demonstrate that proper use of Aspergillus flavus
AF36 results in reductions in the average aflatoxin producing potential
of fungi resident in treated areas and in reductions in the quantity of
aflatoxins in crops.  

	In field tests prior to 1996, the aflatoxin content of cottonseed was
shown to be inversely related to the proportion of the A. flavus
community on the crop composed of Aspergillus flavus AF36. Detailed
analyses of the aflatoxin content of commercial fields from 1996 through
1998 confirmed that reduced aflatoxin levels were associated with
displacement of aflatoxin producers by Aspergillus flavus AF36 from
treated crops and that treatments were associated with up to 90%
reductions in crop aflatoxin content.  Efficacy of applications of
Aspergillus flavus AF36 in displacing aflatoxin producers was
demonstrated for fungal communities both on cottonseed from treated
crops at harvest and in soils of treated fields one year after
treatment.  This included cotton crops treated in 1996 (112 acres
treated), 1997 (463 acres treated), 1998 (499 acres), 1999 (10,488
acres), 2000 (16,725 acres) and 2001 (19,975 acres treated).  

	Displacement of aflatoxin producers is indicated by the proportion of
A. flavus communities composed of Aspergillus flavus strain AF36.   In
1996 average incidence of AF36 on treated crops was 88.5% and in the
soil one year after treatment, 85.2%.  Incidences of AF36 on treated
crops were 78% and 67% in 1997 and 1998, respectively, and in the soil
one year after treatment incidences were 72% and 77%, respectively. 
Successful displacement was also observed as the acreage treated rapidly
expanded from 1999 to 2001 with average incidences of AF36 on treated
crops ranging from 57% in 1999 to 66% in 2001.  Applications were
particularly effective at displacing the high aflatoxin producing S
strain of A. flavus.  S strain isolates produce more aflatoxins than
other A. flavus isolates and are prominent in soils of cotton producing
areas of Arizona and South Texas.  

	The use of  Aspergillus flavus AF36 caused long-term reductions in the
aflatoxin producing potential of fungal communities in agricultural
fields.  Aspergillus flavus AF36 retained atoxigenicity (failure to
produce aflatoxins) upon repeated reisolation from treated fields one,
two, or three years after treatment. Field averages for aflatoxin
production by the displaced S strain isolates of A. flavus ranged from
7100-ppb aflatoxin to 22700 ppb aflatoxin. Thus, there was a long-term
reduction in the potential of fungal communities to produce aflatoxins
in treated areas.  

	There are preliminary data indicating that A. flavus AF36 is effective
in corn as well. In a study, corn  treated with AF36 reduced aflatoxin
contamination by 80 to 95 %.  The purpose of this temporary exemption
from tolerance is to cover the use of A. flavus under an Experimental
Use Permit so that additional efficacy data can be generated.  

J.  International Tolerances

There are no Codex maximum residue levels established for residues of
Aspergillus flavus AF36.  Aspergillus flavus AF36 containing products
are presently not registered for pest control outside of the United
States.  

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