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 PUBLISHED IN THE FEDERAL 

REGISTER 

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 tolerance 

exemption for the microbial pesticide [Aspergillus flavus AF36] in or on Pistachios.

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 pistachios to reduce the incidence of aflatoxin producing strains of A. flavus and 

thereby reduce aflatoxin contamination of pistachios. Aspergillus flavus AF36 which does not 

produce aflatoxin, competitively excludes aflatoxin producing A. flavus strains without 
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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 

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 AF36 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 nonproducing (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 pistachios 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 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 
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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 108colony 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 R95, coveralls, long sleeved shirt and long pants, and shoes plus socks, and goggles, so exposure 

should be limited. 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.

 
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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 AF-36 do not significantly impact the total amount of Aspergillus 

flavus in the soil or crop, but only change the proportion of the AF-36 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, when applied to the soil just prior to bloom, has been shown to 

significantly reduce the levels of aflatoxin 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 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 
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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 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 proposed use will not result in appreciable increases in the longterm 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 
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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). 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 widely 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 same 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 treated 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 isolates 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 orchards. 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 likely 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 reshakes 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 analyzed 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 aflatoxin 

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. No CPA was detected in any of the nut samples from the treated orchards or from untreated 
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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.