EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION

NOTICE OF FILING TEMPLATE FOR PESTICIDE PETITIONS PUBLISHED IN THE

FEDERAL REGISTER 

EPA Biopesticides and Pollution Prevention Division contact: [insert
name and telephone number with area code] 

EPA has received a pesticide petition [insert petition number] from
BioNext sprl, Passage des deportes, 2, B-5030 Gembloux, Belgium,
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 to
establish an exemption from the requirement of a tolerance for the
microbial pesticide Candida oleophila strain O in or on apples and
pears. 

Pursuant to section 408(d)(2)(A)(i) of the FFDCA, as amended, BioNext
sprl has submitted the following summary of information, data, and
arguments in support of their pesticide petition. This summary was
prepared by BioNext sprl 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. BioNext sprl Petition Summary 

[Insert petition number] 

A. Product name and Proposed Use Practices 

The microbial pest control agent Candida oleophila strain O, a yeast
antagagonist for control of pathogens (Botrytis cinerea and Pencicillium
sp.) that cause post harvest decay on Pome fruits. 

B. Product Identity/Chemistry 

Identity of the pesticide and corresponding residues. 

Phylum: Ascomycetes

Family: Saccharomycetaceae

Genus: Candida

Species, subspecies, strain: Candida oleophila strain O

Candida oleophila strain O is a single-celled budding organism,
approximately 1 (m in diameter.  It is a yeast found on plant tissues
(fruits, flowers, wood) and in water.  It has never been reported as a
pathogen of plants or animals (including humans). 

 

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

C. oleophila strain O naturally occurs on apples as it was originally
isolated in 1991 from the surface of apples cv. Golden Delicious.  On
harvested apples, population densities of white yeast can reach 1.5 x
103 cfu/cm².  This value includes C. oleophila natural population.
Consequently, it is expected that background levels of C. oleophila
strain O on apples are below 1.5 x 103 cfu/cm².  However, post-harvest
treatment with C.oleophila strain O leads to an increased level of the
yeast on apples. 

C. oleophila strain O is intended to be used against fruit decay caused
by

Penicillium expansum and Botrytis cinerea. Since the mode of action is
competition for nutrients, sufficient colonization of apples surfaces
has to be reached in order to ensure efficacy. The level of C. oleophila
strain O population ensuring efficacy is reached when application is
conducted at the recommended dose rate (1010  cfu/L). This application
dose rate leads to a residual C. oleophila strain O population density
estimated around 4x104 cfu/cm2 (105 cfu/apple). 

The oral acute toxicity study conducted on rats has shown that C.
oleophila strain O is neither

pathogenic nor infective when orally administered to the rat at > 1.1 x
109 cfu/kg/bw, a level which is 4 orders of magnitude higher than the
expected residue level.  

Since no toxin or metabolite production is expected, residue is defined
as viable and non viable cells.

In order to recover cells from food surface (such as apples), two steps
must be applied: extraction of

cells from apples surface followed by quantification of these cells.

1. Extraction of cells from apples surface

A KPB buffer [0.016M K2HPO4, 0.034 M KH2PO4 and 0.05 % (w/v) Tween 80;
pH 6.5] is 

produced as followed:

be adjusted with stock solutions if necessary. Finally, 500 μl of Tween
80 is added.  Four apples are washed in 500 ml of KPB buffer during 20
min. at 120 rpm. One milliter of washing is used for the quantification
step as described by Massart et al. (2005) (Ref. 1.10).

2. Quantification of cells within the extract

For quantification of viable cells of C. oleophila strain O, serial
dilution series are prepared and plated on semi-selective medium
HST-PDA. This medium is composed of PDA supplied with 12.5 mg/l
hygromycin B, 1.25 mg/L carbendazim, 1.25 mg/L diethofencarb, and 0.25
mg/L thiram.

Quantification of non-viable cells of C. oleophila strain O can be
achieved by PCR real-time as

described by Massart et al. (2005): this method allow quantification of
total cells (viable and nonviable cells). The non-viable cells level can
be reached by comparison between results obtained by numeration of
viable cells and quantification of total cells.

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

The acute toxicity and genotoxicity studies discussed in Section C of
this document are sufficient to show that there are no foreseeable human
or domestic health hazards likely to arise from the use of the product
to control post-harvest decay in pome fruit.  BioNext is requesting an
exemption from the requirement for a tolerance for any residues
remaining in/on pome fruit.  Since enforcement of residue levels would
not be needed, an enforcement analytical method is not required.

C. Mammalian Toxicological Profile 

Acute oral toxicity/pathogenicity- rat (OPPTS 885.3050)

Twelve male and 12 female Crl:CD rats (Charles River) were dosed orally
by gavage with viable C. oleophila strain O, at a rate of 108 CFU per
animal.  Three animals of each sex were sacrificed on days 4, 8, 15 and
22 (six animals per sacrifice).  All rats survived to the scheduled
sacrifice.  There was no effect of treatment on bodyweight gain or
post-dose temperature.  No abnormalities were observed in any animal at
the macroscopic examination.  Viable C. oleophila strain O was not
recovered from the organs, blood, stomach contents, seventh loop of the
small intestine or caecum contents of any treated or control rats over
the study period.  It was concluded that C. oleophila strain O showed no
evidence of toxicity or pathogenicity to rats following a single oral
dose of 2.3 – 3.8 x 108 viable CFU/rat.

Acute pulmonary toxicity/pathogenicity- rat (OPPTS 885.3150)

Seventeen male and 17 female Crl:CD rats (Charles River) were dosed by
intratracheal instillation with viable C. oleophila strain O, at a rate
of 108 CFU/rat.  Three animals of each sex were sacrificed on days 1, 4,
8, and 15 and five animals of each sex were sacrificed on day 22.  Two
animals receiving viable test substance showed transient low body
temperatures.  Abnormalities observed at the macroscopic examination
comprised enlarged, swollen or thickened lungs in animals sacrificed on
day 1 (1 hour after dosing). No abnormalities were observed in any other
animal at the macroscopic examination.  Viable C. oleophila strain O was
recovered from the lungs of all rats sacrificed at one hour after
dosing.  Viable C. oleophila strain O was not recovered from any other
samples from treated rats over the study period confirming that rapid
loss of viability of the test organism following dosing into rats.  It
was concluded that C. oleophila strain O showed no evidence of toxicity
or pathogenicity to rats following a single intratrachael administration
of 1.2 – 5.2 x 108 viable CFU/rat.

Acute dermal toxicity/pathogenicity- rat (OPPTS 885.3200)

Three male and 3 female Crl:CD rats (Charles River) were dosed with a
single subcutaneous injection of at least 107 CFU/rat.  There were no
unscheduled deaths, clinical signs, body temperature changes or
macroscopic abnormalities that were considered to be related to
treatment during the study.  There were no bodyweight changes that were
considered to be of toxicological significance during the study.  Viable
C. oleophila strain O was not recovered from the organs, blood or
injection site of any rats treated with the test substance over the
study period. Viable C. oleophila strain O was not recovered from the
stomach, seventh loop or caecum contents of any rat treated with the
test substance over the study period.  It was concluded that C.
oleophila strain O showed no evidence of toxicity or pathogenicity to
rats following a single subcutaneous administration of 1.1 – 2.0 x 107
CFU/rat.

Primary eye irritation (OPPTS 870.2500)

Four rabbits were each administered a single ocular dose of 100 mg of C.
oleophila strain O technical grade active ingredient and observed for up
to 15 days after instillation.  There were no signs of toxicity or ill
health in any rabbit during the observation period.  Injection of the
conjunctival blood vessels was evident during the first 24 hours after
instillation.  The highest mean score (Kay and Calandra (1962)) was 6.7
occurring at the one hour observation; accordingly under the criteria of
Kay and Calandra, C. oleophila strain O was classified as minimally
irritating to the eye.

Dermal irritation – rabbit (OPPTS 870.2500)

Three rabbits received a single four-hour, semi-occlusive, dermal
administration of approximately 0.5 grams of C. oleophila strain O
technical grade of the active ingredient and were observed for four
days.  No dermal reaction was observed in any animal throughout the
duration of the study.  The Primary Irritation Index was calculated to
be 0.0 therefore C. oleophila strain O was classified as a non-irritant.

Mutagenicity Test on Salmonella Typhimurium (Ames Assay)

C. oleophila strain O technical grade of active ingredient was tested
for mutagenic effect on five strains of Salmonella typhimurium.  The
assay was carried out both with and without metabolic activation using
hepatic microsomes from rat livers induced by Aroclor 1254 with an
incubation period of 48 hours.  Doses were prepared as an extract from
one gram of C. oleophila strain O containing 3 x 1010 CFU in 3 mL of
DMSO (1.1010 CFU/mL).  Actual doses were 0, 1, 3, 10, 30 and 100 uL of
initial extract/plate.  Under the experimental conditions of the test,
C. oleophila strain O induced no mutagenic activity in the five
Salmonella typhimurium strains tested.

Mutagenicity Test on L5178Y Mouse Lymphoma cells (TK Locus)

C. oleophila strain O technical grade of active ingredient was tested
for mutagenic effect on L5178Y mouse lymphoma cells.  The assay was
carried out both with and without metabolic activation using hepatic
microsomes from rat livers induced by Aroclor 1254.  Doses were prepared
as an extract containing 3 x 1010 CFU in 3 mL of DMSO (1.1010 CFU/mL). 
Actual doses were 0.63, 1.25, 2.5 and 5 uL/mL of the intial extract. 
Under the experimental conditions of the test, C. oleophila strain O
induced no mutagenic activity at the TK locus in L5178Y mouse lymphoma
cells.

D. Aggregate Exposure 

1. Dietary exposure.   C. oleophila strain O naturally occurs on apples
as it was originally isolated in 1991 from the surface of apples cv.
Golden Delicious.  On harvested apples, population densities of white
yeast can reach 1.5 x 103 cfu/cm².  This value includes C. oleophila
natural population. Consequently, it is expected that background levels
of C. oleophila strain O on apples are below 1.5 x 103 cfu/cm². 
However, post-harvest treatment with C.oleophila strain O leads to an
increased level of the yeast on apples. C. oleophila strain O is
intended to be used against fruit decay caused by Penicillium expansum
and Botrytis cinerea. Since the mode of action is competition for
nutrients, sufficient colonization of apples surfaces has to be reached
in order to ensure efficacy. The level of C. oleophila strain O
population ensuring efficacy is reached when application is conducted at
the recommended dose rate (1010  cfu/L). This application dose rate
leads to a residual C. oleophila strain O population density estimated
around 4x104 cfu/cm2 (105 cfu/apple). 

i. Food.  C. oleophila is a yeast naturally occurring on plant tissues,
which means it is also found in/on food.  The table below gives an
overview of the plant origin of C. oleophila strains found in important
micro-organisms collections.

 Origin of strains from micro-organisms collections

Strain reference	Origin

CBS2219

CBS4371

CBS7419

CBS8269

IGC4709

ATCC MYA1208	Olives, Olea europaea

Cider

Oil-pipes in food-processing plant

Strawberries

Fermenting grapes

Tomatos

Moreover, from open literature it appears that species of the genus
Candida are ubiquitous as they have been recovered in a high number of
foods including fruits, fruit juices, soft drinks, alcoholic beverages
and grains, salted and acid preserved foods, dairy products, and meat
and meat-derived products.

. Drinking water. No drinking water exposure is anticipated because of
the use pattern and use sites.  There are no aquatic use sites permitted
for this pesticide, so exposure to drinking water is not expected. 
Further, there is no evidence of adverse effects from exposure to this
organism.  Exposure from the proposed use of C. oleophila strain O is
not likely to pose any incremental risk via consumption of drinking
water to adult humans, infants and children. 

2. Non-dietary exposure. Since C. oleophila is a yeast that is naturally
occurring on plant tissues, there may be non-dietary exposure from these
naturally occurring background levels.   However, it is not expected
that the pesticidal use of C. oleophila will significantly increase the
non-dietary and non-occupational exposure to the yeast.

E. Cumulative Exposure 

Because C. oleophila strain O is non-toxic and non-pathogenic in
mammals, no mechanism of toxicity or pathogenicity has been identified. 
Therefore no cumulative effects from the residues of this product with
other related microbial pesticides are anticipated. 

F. Determination of Safety for U.S. population, Infants and Children 

There is reasonable certainty that no harm will result to the U.S.
population, including infants and children, from aggregate exposures to
residues of Candida oleophila strain O, as a result of its proposed
uses.  This includes all anticipated dietary exposures and all other
exposures for which there is reliable information.  As discussed
previously, there appears to be no potential for harm, from this yeast
in its use as a microbial pesticide in apple and pear post harvest
treatments.  Furthermore, the organism is naturally occurring in food
items, and non-toxic and non-pathogenic to animals and humans.  This
conclusion is based on the results of the mammalian toxicity tests
discussed in Section C.  Moreover, potential non-occupational inhalation
or dermal exposure is not expected to pose any adverse effects to
exposed populations via aggregate and cumulative exposure.

G. Effects on the Immune and Endocrine Systems 

There is no information to indicate that Candida oleophila strain O
would have an effect in humans similar to an effect produced by a
naturally-occurring estrogen or other endocrine effects.  There is no
known metabolite produced by this yeast that acts as an endocrine
disruptor.  The submitted toxicity/pathogenicity studies in rodents
indicate that following dosing that no viable yeast were found in organs
or tissues of test animals.  It is concluded that there will be no
incremental adverse effects to the endocrine system.

H. Existing Tolerances 

EPA has established an exemption from the requirement of a tolerance for
Candida oleophila isolate I-182 at 40 CFR Sec. 180.1144.  The exemption
is for:

“Sec. 180.1144  Candida oleophila isolate I-182, when used as a
post-harvest biological fungicide, is exempted from the requirement of a
tolerance in or on all raw agricultural commodities.”

 

I. International Tolerances 

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