## Request for Exemption from the Requirements of a Tolerance AF36 on Pistachios

EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBL ISHED IN THE FEDERAL REGISTER
EPA has received a pesticide petitio n from [Interregional Research Project Number 4 (IR

- 4), Rutgers University, 500 College Road East, Suite 201W, Princeton , NJ 08 540 on behalf of the Arizona Cotton Research and Protection Council] , [3721 East Wier Avenue Phoenix, Arizona 85040

- 2933] propo sing 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 180.120 6 to establish a tolerance exemption for the microbial pesticide [ Aspergillus flavus AF36] in or on Pistachios.

Pursu ant 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 08 540 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 08 540 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 uncle ar, 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 Univer sity, 500 College Road East, Suite 201W, Princeton , NJ 08 540 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 pro posed for application to pistachios to reduce the incidence of aflatoxin producing strains of A. flavus and thereby reduce aflat oxin contamination of pistachios. Aspergillus flavus AF3 6 which does not produce aflatoxin, competitively excludes aflatoxin producing A. flavus strains without2

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 e xemption from tolerance for residues of A. flavus AF 36 on pistachios. 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 AF 36 has been shown to be naturally

and consistently

associated with pistachios grown in California

. A. flavus is a widespread fungus. It is particularly well adapted to the hot regions of California 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 California. 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

. An exemption from the requirement of a tolerance for residues of the microbial pesticide A. flavus AF36 in/on pistachios is being proposed for pis tachios in California . 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 pro duct are always screened for aflatoxin production using TL C 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. Mammalia n 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 D awley rats. The oral LD 50 of Aspergillus flavus AF36 was determined to be greater than 5,000 mg/kg rat body weight. No3

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 i n 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 sy mptoms was rapid after dosing with all deaths occurring by day f our of the study

. A ll rats surviving to day f our of the study

recovered and all rats sacrificed (as scheduled) on day

8 o r day 1 5 of the study

h ad totally

e liminated viable Aspergillus flavus AF36 from the lungs, caecal contents, and faeces. There was no evidence of infectivity

. The aetiology o f 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 sy nergism with Tween 80 and/or of Tween 80 breakdown products formed during preparation of the spore suspension. A second rat study

w as 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, 10 5 , 10 6 , 10 7 , and 10 8 colony f orming units per rat. There were no clinical signs in any o f 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 anim als 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 toxico logy s tudies 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 ye ars and in commercial fields (1996 to 1998) and in hand picked field plots (1 989 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 be limited

. Application s will involve aerial application by m ixers/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. Applicati ons 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 w as 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.4

2. Primary

d ermal irritation. This study w as waived based upon the lack of toxicity in animals dosed orally

. A erial applications will be made by m ixers/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 activ e ingredient is less than 0.01 lb/acre. Applications of AF

- 36 do not significantly i mpact the total amount of Aspergillus flavus in the soil or crop, but only c hange the proportion of the AF

- 36 strain in relation to the overall population. Since the produc t is applied as a granular formulation on wheat, e xposure 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. S ince the product is applied aeri ally a s 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. G enotoxicity, reproductive and developmental toxicity, subchronic toxicity and chronic toxicity testing were not performed, since no adverse effects were observed in the acute toxic ology s tudy T ier 1 studies. T ier II (885.355 0) , subchronic toxicology s tudy (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 wil l not result in increases in the total population of A. flavus on the mature crop beyond naturally occurring background levels

. Aspergillus flavus AF36, when applied to the soil just prior to bloom, has been shown to significantly

reduce the levels of afl atoxin in pistachios 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 res ult 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 is5

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 in crease 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 comm on 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 E

xposure 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 Asper gillus flavus AF36 in the crop or in artificial media in the lab. Aspergillus flavus AF36 has been shown to exclude aflatoxin producing fungi competitively from the developing crop and to reduce aflatoxin contamination of pistachio . Data show that the p roposed 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

. Aspergi llus 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 app lication. 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 sh ould 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 occurrenc e, 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 o r the environment. G. Effects on the Immune and Endocrine Systems Aspergillus flavus AF36 is a naturally occurring organism, which does not produce6

aflatoxin, and is thus safer than the A. flavus isolates that produce aflatoxin. To date there is no e vidence 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 As pergillus flavus AF36, other than the tolerance exemption on cotton (40 CFR 180.1206 ) . There is a current temporary

exemption from tolerance on pistachio and corn I. Efficacy Data The atoxigenic Aspergillus flavus strain AF36 (the same product that is w idely used in Arizona cotton fields to substantially reduce aflatoxin contamination of cottonseed) was applied as a biocontrol agent in commercial pistachio orchards in California in 2008 and 2009. The AF36 strain was applied as a wheat

- AF36 product (the s ame product and same application rate as used in commercial cotton fields) as a single annual application to the soil surface of the orchards in late June or early

July . The level of the atoxigenic strain AF36 in the soil increased substantially in the tre ated orchards after applying the wheat

- AF36 product. Prior to applying the AF36 product, only

2% of the A. flavus isolates from the soil of these orchards belonged to the AF36 strain. But three months after the single application, 43% of the A. flavus isol ates in the soil belonged to the AF36 strain. In 2009 an additional application of the AF36 product was made in these orchards, and three months after this application 89% of the isolates were AF36. This demonstrates that the application of the wheat

- AF36 product was successful in substantially increasing the level of AF36 in the orchards. Nonetheless, application of the wheat

- AF36 product did not result in increased levels of kernel decay, and no cyclopiazonic acid was detected in nuts from treated orchard s. Furthermore, nuts from the orchards treated with the AF36 product were less likely to be contaminated with aflatoxin than those from nearby untreated orchards in 2008 and in 2009. Nut samples from the orchards treated with the AF36 product were less l ikely to be contaminated with aflatoxin than those from untreated orchards for both 2008 and 2009 harvests . Although sometimes pistachio orchards will have only a single harvest, in some cases there will be a second harvest or reshake. In 2009 the reshake s from the treated orchards had less aflatoxin contamination than those from untreated orchards . Although about 30% more samples collected from untreated orchards in 2009 were analy zed for aflatoxins, these samples had twice as high an incidence as those collected from AF36 treated orchards , suggesting that the AF36 treatment contributed to this reduction of aflatoxin contamination of pistachios. It is expected that with a further increase of AF36 in the orchards there will be a further decrease in aflato xin contamination as has been shown in other crops (i.e. cottonseed, corn , etc.). Application of the AF36 product did not increase the amount of cyclopiazonic acid (CPA) in the nuts. N o CPA was detected in any o f the nut samples from the treated orchards or from untreated7

orchards. Because application of AF36 did not increase CPA in the nuts nor increase nut decay, it is expected that treatment with AF36 will decrease aflatoxin contamination without any adverse effects to the nuts. 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.