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EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY

NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL

REGISTER

EPA Biopesticides and Pollution Prevention Division contact: Driss Benmhend, 703-308-

9525, benmhend.driss@epa.gov.

INSTRUCTIONS: Please utilize this outline in preparing the pesticide petition. In cases

where the outline element does not apply, please insert "NA-Remove" and maintain the

outline. Please do not change the margins, font, or format in your pesticide petition. Simply

replace the instructions that appear in green, i.e., "[insert company name]," with the

information specific to your action.

SUBMISSION: E-mail the completed template to: hollis.linda@epa.gov.

TEMPLATE:

DSM Food Specialties B.V.

Petition number to be assigned.

EPA has received a pesticide petition (To be assigned) from DSM Food Specialties

B.V., Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands,

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.

(Options (pick one)

NA-Remove: 1. by establishing a tolerance for residues of

NA-Remove: 2. to establish an exemption from the requirement of a tolerance for

3. to establish an amendment/expansion of an existing tolerance exemption for the

(Options (pick one)

NA-Remove: 1. microbial pesticide [insert name of active ingredient]

2. biochemical pesticide (6,ll,28-Trioxatricyclo[22.3.1.05,7]octacosa-8,14,16,18,20-

pentaene-25-carboxylic acid, 22-[(3-amino-3,6-dideoxy-p~D-mannopyranosyl)oxy]-l,3,26-

trihydroxy-12-methyl-10-oxo-, (1R,3S,5R,7R,8E,I2R,14E,16E,18E,20E,22R,24S,25R,26S)-,

CAS Registry No. 7681-93-8.
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NA-Remove: 3. plant-pesticide [insert name of active ingredient] in or on [insert

commodity],

Pursuant to section 408(d)(2)(A)(i) of FFDCA, as amended, DSM Food Specialties B.V

has submitted the following summary of information, data, and arguments in support of their

pesticide petition. This summary was prepared by DSM Food Specialties B. V. 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. DSM Food Specialties B.V. Petition Summary

Petition number to be assigned.

A. Product Name and Proposed Use Practices

The Natamycin active ingredient (AI), a polyene macrolide antimycotic biochemical

substance, was discovered in a soil sample obtained near Pietermaritzburg in the state of

Natal in South Africa. This biochemical substance was found to have broad, potent

antifungal properties, but no antibacterial activity. The lack of antibacterial properties is a

distinctive advantage for a fungicidal pesticide because then there is no basis for concern

over development of resistance by human pathogenic bacteria.

During post-harvest pineapple packing, Natamycin will be used in a broad spectrum

liquid fungicidal formulation containing approximately 4% by weight of the technical

grade active ingredient (TGAI) as a stable aqueous suspension of crystals. This product,

ZivionTM P, is designed for the control of mold growth on pineapples during shipment and

storage. ZivionTM P can be applied as a dip or spray (pouring or cascade) with wax as a

post-harvest treatment.

B. Product Identity/Chemistry

\. Identity of the pesticide and corresponding residues.

Natamycin. No residues other than Natamycin were found.

2. Magnitude of residues at the time of harvest and method used to determine the residue.

The proposed use in the present application is for the control Fusarium oxysporum,

PeniciUiumfuniculosum, Rhizophus stolonifer, Aspergillus niger and Thielaviopsis paradoxa

on pineapple during shipment and storage. Zivion P and an alternative formulation of

ZivionTM P designated as ZivionTM A were applied post-harvest at a nominal concentration

of 400 ppm in the treatment solution with 0.25 L of Sta-Fresh 2981 food wax. To obtain

replicated data, trials were conducted with varieties MD-2 and Montelirio. ZivionTM P

maximum residues were 0.9352 mg/kg in whole pineapple, 0.0692 mg/kg in juice, 0.0130
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mg/kg in meat, 1.1644 mg/kg in rind and 0.1796 mg/kg in wet bran. No residues of

Natamycin were found in control pineapples. No metabolites nor degradation products of

Natamycin were found. LC-MS/MS methods were used for the analyses. Quantification of

Natamycin residues was accomplished relative to an external calibration curve. The limit of

Quantitation (LOQ) for the Natamycin residue method was 0.01 ppm in all pineapple

fractions. The limit of detection was assigned as the lowest concentration within the

calibration curve or 0.25ng/ml. The analytical method to detect and measure natamycin

residues and residues of metabolites, if they were present, was fully validated.

3. A statement of why an analytical method of detecting and measuring the levels of the pesticide

residue are not needed. A worst-case estimate of the Natamycin exposure in the diet were

calculated using the following: 1) Maximum pineapple consumption; 2) Use of the

consumption data of the most highly exposed population group; and 3) Use of the

maximum residue found in the trials in any treated juice or meat sample, rather than the

most probable residue. On this basis, calculations for exposure to Natamycin through

consumption of treated pineapple yielded a Margin of Exposure (MOE) of approximately

one million.

This very large worst-case MOE clearly establishes the safety of Natamycin for the

proposed use on pineapple.

Because the MOE is so large even when worst-case assumptions of exposure are

made, an exemption from tolerance rather than a numeric tolerance is appropriate, and an

analytical method for detecting and measuring pesticide residues in pineapple is not

needed.

C. Mammalian Toxicological Profile

Acute Toxicity Profile

ACUTE ORAL TOXICITY - Rat Toxicity Category III

The test substance, Natamycin, was evaluated for its acute oral toxicity potential in female

albino rats when administered as a gavage dose at a level of 2000 mg/kg. The study was

terminated following the stopping rules of this procedure. One of five animals died during

the study. There were no clinical signs of toxicity in survivors during the study; polyuria

was observed in the animal that died on test. There was no effect on body weight gain in

animals surviving to termination. Abnormal necropsy findings occurred only in the animal

that died on test, and pertained to anogenital area, lungs and contents of the intestines. The

acute oral LDSO was estimated to be greater than 2000 mg/kg.

ACUTE DERMAL TOXICITY -Rat Toxicity Category IV

The test substance, Natamycin, was evaluated for its dermal toxicity potential and relative

skin irritancy when a single dose moistened with 1.0 mL of deionized water/g test

substance, at a level of 5050 mg/kg, was applied to the intact skin of albino rats. No

mortality occurred during the study. There were no clinical signs of toxicity or signs of
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dermal irritation at any time throughout the study. There was no effect on body weight

gain, with the exception of one animal that failed to gain weight during the first week. The

gross necropsy conducted at termination of the study revealed no observable abnormalities.

The estimated LD50, as indicated by the data, was determined to be greater than 5050

mg/kg.

ACUTE INHALATION TOXICITY - Rat Toxicity Category IV

The test substance, Natamycin, was evaluated for its acute inhalation toxicity potential in

albino rats. Five males and five females were exposed for four hours to an aerosol

generated from the undiluted test substance (fine powder) at a level of 2.39 mg/L. There

was no mortality during the study. Clinical signs included activity decrease and

piloerection, which were no longer evident by Day 3. Body weights were unaffected by

exposure. The gross necropsy revealed no observable abnormalities. As indicated by the

data, the acute inhalation LC50 is greater than 2.39 mg/L.

EYE IRRITATION - Rabbit Toxicity Category IV

An acute eye irritation study was conducted on three albino rabbits using test substance

Natamycin. The test substance, 0.1 mL by volume (43.7 mg), was placed into the

conjunctiva] sac of the right eye of each animal selected for testing. All treated eyes were

washed with room temperature deionized water for one minute immediately after

recording the 24-hour observation.

A maximum average irritation score of 62.0, severely irritating, was obtained 1 hour after

treatment, but there were no positive effects exhibited in any eyes at 24 hours after

treatment. Hence, the initial irritation score could not be maintained and Natamycin TGAI

was assigned to Toxicity Category IV.

SKIN IRRTIATION - Rabbit Toxicity Category IV

A primary dermal irritation study was conducted on three albino rabbits using test

substance Natamycin. There was one intact test site per animal. Each test site was treated

with 500 mg of test substance moistened with 0.5 mL of deionized water and covered with a

semi-permeable dressing. The test substance was maintained in contact with the skin for 4

hours. Observations for dermal irritation and defects were made at 1, 24, 48 and 72 hours

after removal of the dressings.

Based on the PII of O.I, the test substance is rated slightly irritating. Based on the scores at

the 72- hour observation only, the test substance is assigned to Toxicity Category IV.

SKIN SENSITIZATION - Mice Not a Sensitizer

A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test

substance Natamycin possesses a significant potential to cause skin sensitization. Five

females were assigned to each of three groups, designated Groups I - III. The test groups
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were treated with an appropriate dilution (25% or 50%) in propylene glycol vehicle, or

undiluted test substance. Each animal received 25 uL to the dorsum of each ear. The

animals were treated once daily for three days. After a two-day rest period, all animals

were injected with tritiated methyl-thymidine in the tail vein. Five hours later, the animals

were sacrificed, and the draining auricular lymph nodes removed and prepared for cell

suspension and scintillation counting. A vehicle control group of five females was run

concurrently, treated in the same manner with vehicle only instead of test substance or

dilution. A positive control group of five females was also run concurrently, treated with

90% alpha-hexylcinnamaldehyde in acetonerolive oil.

The test substance produced a stimulation index of < 3 in all groups of test animals, and is

not therefore considered a sensitizer (defined as producing a positive response).

Genotoxicity.

NATAMYCIN was tested in the Salmonella typhimurium reverse mutation assay Avith four

histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98)

and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of

Escherichia coli WP2uvrA . The test was performed in two independent experiments in the

presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). Based on the

results of this study it is concluded that NATAMYCIN is not mutagenic in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay.

NATAMYCIN (88.9% purity) was tested to determine the number of chromosome

aberrations in cultured peripheral human lymphocytes in the presence and absence of a

metabolic activation system (Aroclor-1254 induced rat liver S9-mix) . NATAMYCIN did

not induce a statistically significant or biologically relevant increase in the number of cells

with chromosome aberrations in the absence and in the presence of S9-mix, in two

independently repeated experiments. NATAMYCIN is not clastogenic in human

lymphocytes under the experimental conditions described in this report.

Reproductive and Developmental Toxicity

Natamycin (as Delvocid 5%) was administered daily during days 6-18 of gestation by oral

intubation to groups of 15 to 19 female Dutch Belted rabbits at concentrations of 0, 5,15,

or 50 mg/kg-day . The administration of up to 50 mg/kg (body weight)-day of test material

for 13 consecutive days had no clearly discernible effect on nidation or on maternal or fetal

survival. The number of abnormalities seen in either soft or skeletal tissues of the test

groups did not differ from the number occurring spontaneously in the sham-treated

controls. From the results presented in this report the No Observed Adverse Effect Level

(NOAEL) for the teratology part of the study was established to be 50 mg/kg/day.

Subchronic Toxicity

In a 90 day study, Natamycin was administered daily via the diet to groups of 20 Wistar

rats (10 males and 10 females) at concentrations of 0, 125, 500 and 2000 ppm. Dietary
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exposure to Natamycin resulted in no treatment related findings in the 125 mg/kg/day and

500 mg/kg/day dose groups. The 2000 mg/kg/day dose group had reduced body weights

(males and females), increased alanine aminotransferase activity in individual males,

increased urea and inorganic phosphate values (males and females) and potassium values

(males), and reduced cholesterol and total protein values (females). From the results

presented in this report the No Observed Adverse Effect Level (NOAEL) was established to

be 500 ppm Natamycin, equivalent to approximately 38 and 43 mg/kg/day (corrected for

food scatter) for males and females respectively.

Toxicology Profile -- Existing U.S. Government findings of safety

Natamycin (pimaricin) has been approved by FDA in 21 CFR Part 172, Sec. 172.155 for

use as a direct food additive for use on cheese. "The additive may be applied on cheese, as

an antimycotic, in amounts not to exceed 20 milligrams per kilogram (20 parts per million)

in the finished product...". Natamycin as a 5% ophthalmic suspension is approved as an

antifungal drug for topical ophthalmic administration . In 21 CFR Part 573, Sec. 573.685

FDA has approved Natamycin for use in broiler chicken feeds at up to 10 grams per pound

of food .

Toxicology Profile —International Findings of Safety

The Food and Agricultural Organization of the United Nations (FAO) and the World

Health Organization (WHO) through the Joint FAO/WHO Expert Committee on Food

Additives (JECFA), evaluated Natamycin, most recently in 2002, for safety as a direct food

additive . The Committee concluded that "Although use of Natamycin as an antifungal

agent in food may result in exposure of the endogenous flora to trace quantities of

antimicrobial residues, bacteria in the human gastrointestinal tract are not affected by

polyenes, and the Committee concluded that disruption of the colonization barrier is not a

concern. Fungi are found in much smaller amounts than bacteria in the human <

gastrointestinal tract, and the negative results in studies of acquired resistance indicate that

selection of Natamycin-resistant fungi is not an issue. The Committee confirmed the

previously established ADI of 0-0.3 mg/kg bw for Natamycin, which was based on

observations of gastrointestinal effects in humans."

The Animal Metabolism of Natamycin

As described in the WHO evaluation , studies have been done to determine the fate of

Natamycin in rats and dogs. In rats, a series of studies showed that the material was not

absorbed and was eliminated in the feces and urine. The results of the autoradiographic

study indicate that Natamycin is minimally absorbed into the bloodstream and excreted

almost entirely in the feces. Other experiments gave similar results. In the stomach and

small intestine, Natamycin was mostly untransformed, as indicated by thin-layer

chromatography. Most degradation took place in the large intestine. The degradation

products were more hydrophobic than Natamycin and were found from about 4 h after

treatment. Most of the dose of 10 mg/kg was degraded about 8 h after treatment,

suggesting that elimination is relatively rapid. Biotransformation was attributed to the
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bacterial flora in the caecum and small intestine. Overall, no more than 5-7% of the total

radioactive dose was absorbed after oral administration of [14C]Natamycin, and

approximately 90% of the administered compound passed through the gastrointestinal

tract without resorption and was eliminated in the faeces . In dogs, after oral

administration, most of the radiolabel was eliminated in the faeces within 24 h, with less

than 4% of the total dose in urine. The WHO assessment concluded that there was very

little data on humans, but one study had shown that "No Natamycin (< 1 u,g/ml) could be

detected in the blood after ingestion of 500 mg by human subjects", indicating that it was

unlikely to be absorbed by humans.

D. Aggregate Exposure

1. Dietary exposure. Natamycin is currently permitted as an FDA food additive, and

is approved as an antimycotic for use in the finished cheese at a level of 20 ppm. (See 21

CFR § 172.155). It is also allowed by EPA for use on mushrooms without the requirement

for a tolerance. The lowest NOEL for Natamycin is 25 mg/kg bw/day or approximately

1.75 g/person per day. The proposed use in the present application is for the control of

Fusarium oxysporum, PeniciUlum funiculosum, Rhizophus stolonifer, Aspergillus niger and

Thielaviopsis paradoxa. on pineapple during shipment and storage. The application

produces Natamycin residue levels in the edible portions of the pineapple of 0.0692 mg/kg

in juice and 0.0130 mg/kg in meat.

1. Food. The highest annual pineapple consumption in the last decade was 14.16

pounds per person. The highest food consumption is for the 12-19 age group. Using the

highest annual pineapple consumption and the median weight in for females aged 12 - 19,

the average consumption would be 364 mg/kg.bw/day, resulting in a Margin of Exposure of

approximately one million.

ii. Drinking water. All uses of ZivionTM P will be post-harvest, and natamycin has

only very limited (-30-40 ppm at neutral pH) solubility in water. Hence, exposure through

drinking water is not expected.

2. Non-dietary exposure. The only non-dietary exposures from pesticidal uses of the

Natamycin will be occupational, i.e., commercial applicator/mixer loader exposures.

Occupational exposures are not included under the Federal Food, Drug and Cosmetic Act

(FFDCA) in the assessment of aggregate exposures for the purpose of establishing

tolerances and exemptions from tolerance.

E. Cumulative Effects. In assessing the cumulative effects of Natamycin it should be

recognized that it is one of a class of polyene antimycotics naturally present in the

environment. Others of the class are also typically obtained from some species of

Streptomyces bacteria. The amount of material likely to be present as residues of

Natamycin on pineapple results in so small an incremental exposure as to be negligible.

F. Safely Determination
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1. U.S. population. As discussed above, when expressed in pounds per day, per

capita food consumption is highest among men and women aged 12-19. When expressed on

a kilogram body weight basis, the intakes for all individuals are all within a factor of 2,

with the 12-19 age group females having the lower body weight.

2. Infants and children. On a unit bodyweight basis, there is no significant difference

in children's consumption from that of the average adult.

G. Effects on the Immune and Endocrine Systems

Because Natamycin is minimally absorbed into the bloodstream and rapidly

excreted almost entirely in the feces with minimal degradation, effects on the immune and

endocrine systems are not expected. The limited biotransformation found was attributed

to the bacterial flora in the caecum and small intestine, and the degradation products were

more hydrophobic than Natamycin.

H. Existing Tolerances •

There are no existing U.S. tolerances for Natamycin as a pesticide. EPA has granted

an Exemption from the Requirement of a Tolerance for Natamycin use on mushrooms.

/. International Tolerances

No international tolerances exist for use of Natamycin as a pesticide.