Document ID: EPA-HQ-OPP-2010-0727-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2011-04-22T04:00Z

MacroButton macIb01 i  

<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>

<	2. to establish an exemption from the requirement of a tolerance for>

	

<	NA-Remove: 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
(6,11,28-Trioxatricyclo[22.3.1.05,7]octacosa-8,14,16,18,20-pentaene-25-c
arboxylic acid,
22-[(3-amino-3,6-dideoxy-(-D-mannopyranosyl)oxy]-1,3,26-trihydroxy-12-me
thyl-10-oxo-, (1R,3S,5R,7R,8E,12R,14E,16E,18E,20E,22R,24S,25R,26S)-, CAS
Registry No. 7681-93-8.>

	

<	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 has submitted the following summary of information, data,
and arguments in support of their pesticide petition. This summary was
prepared by DSM Food Specialties 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 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 mushroom production, Natamycin will be used in a broad spectrum
liquid fungicidal formulation containing approximately 10% by weight of
the technical grade active ingredient (TGAI) as a stable aqueous
suspension of crystals.  This product, Natamycin L, has anti-germinating
properties and is designed for the control of moulds and yeasts in the
growth media of mushrooms.  Natamycin L can be applied to the growth
substrate up to four times during a typical production cycle for
mushrooms.  Pre-harvest intervals from a few days to more than 20 days
.>

<B. Product Identity/Chemistry>

<	1. 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 of Verticillum (Dry Bubble) disease and other mushroom
parasites in growing mushrooms.  The application produces Natamycin
residue levels in mushrooms in the 0.15-0.25 ppm range.  After rinsing,
the residues for the same mushrooms decrease to 0.022 - 0.076  ppm.  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 mushrooms. 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) Projected (and increased) mushroom intake several years
into the future; 2) Use of the mushroom consumption data of the most
highly exposed population group; and 3) Use of the maximum residue found
in the crop field trials in any treated mushroom, rather than the most
probable residue.  On this basis, calculations for exposure to Natamycin
through consumption of treated mushrooms 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 mushrooms.  Additionally, under normal
conditions mushrooms are rinsed prior to consuming and the actual MOE
will be substantially greater than a million.  

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 mushrooms 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 LD50 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
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 conjunctival 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 0.1, 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 were treated with an
appropriate dilution (25% or 50%) in propylene glycol vehicle, or
undiluted test substance. Each animal received 25 µL 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 acetone:olive 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 with 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 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
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 µg/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). 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
Verticillum disease and other mushroom fungal parasites in growing
mushrooms.  The application produces Natamycin residue levels in
mushrooms in the 0.15-0.25 ppm range.  After rinsing, the residues for
the same mushrooms decrease to 0.022 - 0.076 -ppm. 

<	i. Food.  The current U.S. annual per capita mushroom consumption is
4.00 pounds. The projected maximum, mushroom consumption for the current
decade to the year 2012 is 4.86 pounds.  The maximum daily intake of
Natamycin residues for the proposed use of Natamycin is conservatively
estimated to be 32 ng/kg.bw/day resulting in a Margin of Exposure of
approximately one million.  >

<	ii. Drinking water.  All uses of Natamycin L will be indoor, 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
mushrooms results in so small an incremental exposure as to be
negligible.>

<F. Safety Determination>

<	1. U.S. population.  As discussed above, when expressed in pounds per
day, per capita mushroom use is highest among men and women aged 20-39,
and lowest for children under the age of 12. When expressed on a
kilogram body weight basis, the intakes for all individuals are all
within a factor of 2, with the 29-39 age group still being slightly
higher consumers, particularly in females.>

<	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.>

<I. International Tolerances>

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

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