Document ID: EPA-HQ-OPP-2007-0810-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2007-09-28T04:00Z

<EPA REGISTRATION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE
PETITIONS PUBLISHED IN THE FEDERAL REGISTER  >

<EPA Registration Division contact: Mr. James Tompkins, 703-305-5697>

 

<Cutting Edge Formulations, Inc.>

<7F7225>

<	EPA has received a pesticide petition (7F7225) from Cutting Edge
Formulations, Inc., 5106 Bristol Industrial Way, Suite 400, Buford,
Georgia. 30518 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.>

<>

<	1.  to establish an exemption from the requirement of a tolerance for
>

<	d-Limonene in or on tree, vine and berry crops, vegetable crops,
alfalfa, rice, cotton, herbs and spices.  EPA has determined that the
petition contains data or information regarding the elements set forth
in section 408 (d)(2) of  FDDCA; however, EPA has not fully evaluated
the sufficiency of the submitted data at this time or whether the data
supports granting of the petition. Additional data may be needed before
EPA rules on the petition.>

<A. Residue Chemistry>

<	1. Plant metabolism.>

<	2. Analytical method. This petition is to establish an exemption from
the requirement of a tolerance.  Therefore, practical methods for
detecting and measuring residues are not applicable. >

<	3. Magnitude of residues. This petition is to establish an exemption
from the requirement of a tolerance.  Therefore, analysis of the
magnitude of residues is not applicable. >

<B. Toxicological Profile>

<	1. Acute toxicity.  The acute toxicity of d-limonene in rodents is
fairly low after oral, intraperitoneal, subcutaneous, and intravenous
administration. >The oral LD50 of d-limonene in mice is 5600 mg/kg
(male) and 6600 mg/kg (female.)  In rats, the oral LD50 in rats is 4400
mg/kg (male) and 5100 mg/kg (female).  The dermal LD50 is >5000 mg/kg. 
Irritation to rabbit eyes have been observed.  Dermal irritation studies
have been conducted on guinea pigs, rabbits and rat with low or moderate
irritation observed.  D-Limonene did not cause dermal sensitization.  No
studies on the acute inhalation of d-limonene in animals have been
identified (an inhalation study on humans has been reported.)  An
intraperitoneal study on rats resulted in an LD50 of 3600 mg/kg (males)
and 4500 mg/kg (females).  Subcutaneous intravenous studies in mice
resulted in LD50s of >41.5 g/kg (single application) and >21.5 g/kg (7
days). 

<	2. Genotoxicty. On the basis of available data, there is no evidence
that >d-limonene or its metabolites are genotoxic or mutagenic. 
Limonene and its epoxides were not mutagenic when tested at
concentrations of 0.3-3333 µg/plate in in vitro assays using different
strains of Salmonella typhimurium, in the presence or absence of
metabolic activation (Florin et al., 1980; Watabe et al., 1981; Haworth
et al., 1983; Connor et al., 1985; NTP, 1990).   d-Limonene did not
increase the frequency of forward mutation at the TK+/- locus in mouse
L5178Y cells (NTP, 1990), induce cytogenetic damage in Chinese hamster
ovary cells (Anderson et al., 1990), or malignantly transform Syrian
hamster embryo cells (Pienta, 1980).  In one in vitro study, following
exposure with benzo(a)pyrene, d-limonene (21.9 µmol/litre) inhibited
the formation of transformed cell colonies in tracheal epithelium
isolated from rats (Steele et al., 1990).  No evidence of mutagenicity
was reported in an in vivo spot test with mice, involving the
intraperitoneal injection of limonene at 215 mg/kg body weight per day
on days 9-11 during gestation (Fahrig, 1984).

<	3. Reproductive and developmental toxicity. Various study data in the
rat, mouse and rabbit, indicate that there is no evidence that limonene
has teratogenic or embryotoxic effects in the absence of maternal
effects. In addition, a rat developmental toxicity study was reviewed in
the RED (1994), and based on the data presented, it is concluded that
d-limonene is not a developmental toxicant.  The NOAEL was determined to
be 250 mg/kg/day for both maternal and developmental toxicity. There
were small decrements in maternal body weight gain at 500 mg/kg/day, and
there were slight, but statistically significant and dose-dependent
increases in the number of litters and fetuses with 14 ribs, instead of
13 ribs, at 500 mg/kg/day. The RED considered these effects to be
variations in skeletal formation, not accompanied by other effects, and
were secondary to the maternal toxicity, so the RED concluded these
effects do not represent a concern for the developmental toxicity of
limonene.>

<	4. Subchronic toxicity. Several subchronic feeding studies have been
conducted on mice and rats with d-limonene the results of which are
detailed in the EPA TRED.  A 16-day oral gavage with mice resulted in
deaths at 3300 and 6600 mg/kg bw/d, but no compound-related clinical
signs were observed in mice in 1650 mg/kg-bw dose group that lived to
the end of dosing, plus no compound related histological effects. The
NOAEL was 1650 mg/kg-bw/d and the LOAEL 3300 mg/kg-bw/d (NTP, 1990).  A
13-week oral gavage study with mice reduced body weights and death, plus
rough hair coats and decreased activity.  The NOAEL was 500 mg/kg-bw/d
and the LOAEL, 1000 mg/kg-bw/d (NTP, 1990).  A 16-day feeding study with
rats observed deaths at 3300 and 6600 mg/kg-bw/d, but no clinical signs
in 1650 mg/kg-bw dose group or lower, and no compound-related
histopathological effects seen in any rats.  The NOAEL was 1650
mg/kg-bw/d and the LOAEL 3300 mg/kg-bw/d (NTP, 1990).  A 26-day oral
gavage study was completed with rats (males only).  At the lowest dose
tested, effects were observed on kidneys of male rats.  In addition,
increased relative kidney and liver weights, measured in 300 mg/kg-bw/d
dosed group, but not in 150 mg/kg-bw/d group.  In light microscopy,
kidneys showed dose-related hyaline droplet formation, as well as
granular casts in outer zone of medulla and chronic nephrosis, but no
evident alterations observed in liver sections, even at highest dose
tested. The observed NOAEL was 150 mg/kg-bw/d (liver); the LOAEL was 75
mg/kg-bw/d (kidney) and 300 mg/kg-bw/d (liver) (Kanerva et al., 1987).  
A 30-day oral gavage was conducted with male rats at 400 mg/kg-bw/d.  At
only dose tested, 20-30% increase in amount and activity of different
liver enzymes, increase in relative liver weight, and decrease in
cholesterol levels; no histopathological examinations conducted. The
LOAEL was 400 mg/kg-bw/d (males only) (Ariyoshi et al., 1975).  A
13-week oral gavage study was completed on males rats.  The NOAEL was 5
mg/kg-bw/d (kidney) and 30 mg/kg-bw/d (liver); the LOAEL was 30
mg/kg-bw/d (kidney) and 75 mg/kg-bw/d (liver).  For kidneys,
“incidence and severity of these lesions increased slightly at 10 mg
d-limonene/kg body weight although ... a clear 91-day lowest-observable
effect (LOEL) was produced at 30 mg d-limonene/kg body weight (P <
0.01)”. For liver, no differences in absolute liver weights, and
increase in relative liver weights statistically significant only at 75
mg/kg-bw/d, but no histopathological effects observed (Webb et al.,
1989).  A 13-week oral gavage study was completed on rats.  The NOAEL
was undetermined in male, and 600 mg/kg-bw/d in female.  The LOAEL was
150 mg/kg-bw/d in male, 1200 mg/kg-bw/d in female. In males, kidneys
showed nephropathy at all doses, with severity dose related. In females,
at 1200 mg/kg-bw, rough hair coats, lethargy, and excessive lacrimation
(NTP, 1990).

>

<	5. Chronic toxicity. Two chronic feeding studies were conducted, one
with mice and the other rats.  The 103-week oral gavage study with mice
used dose rates of 0, 250, and 500 mg/kg-bw/d for males, and 0, 500, and
1000 mg/kg-bw/d for females. The NOAEL was 250 mg/kg-bw/d (male) and 500
mg/kg-bw/d (female).  The LOAEL was 500 mg/kg-bw/d (male) and 1000
mg/kg-bw/d (female).  In males dosed at 500 mg/kg-bw/d, livers exhibited
presence of cells with abnormal numbers of nuclei and cytomegaly. In
females, decreased survival and lower body weights (5 - 15%) in highest
dose tested, 1000 mg/kg-bw, but no treatment related clinical signs at
any dose tested (NTP, 1990).

A chronic oral gavage study was also conducted with rats.  Males were
administered 0, 75, and 150 mg/kg-bw/d; females: 0, 300, 600 mg/kg-bw/d.
   The NOAEL was undetermined for the male kidney, 150 mg/kg-bw/d (male;
other than kidney, no adverse effects at highest dose tested), and 300
mg/kg/bw/d in female.  The LOAEL was 75 mg/kg-bw/d (male: kidney),
undetermined (male; other than kidney with no other adverse effects at
highest dose tested), and 600 mg/kg-bw/d in female.  In males, effects
in kidneys included dose-related increases in incidences of
mineralization and epithelial hyperplasia, even at lowest dose tested;
however, no other significant adverse effects were observed by NTP in
males dosed at 150 mg/kg-bw/d, other than on kidneys. In females, at 600
mg/kg-bw/d, reduced survival, but no adverse effects in low dose group,
300 mg/kg-bw/day (NTP, 1990)>

<	6. Animal metabolism. >

<	7. Metabolite toxicology. The biotransformation of d-limonene has been
studied in many species, with several possible pathways of metabolism. 
Metabolic differences between species have been observed with respect to
the metabolites present in both plasma and urine.  About 25-30% of an
oral dose of  d-limonene in humans was found in urine as
d-limonene-8,9-diol and its glucuronide; about 7-11% was eliminated as
perillic acid (4-(1-methylethenyl)-1-cyclohexene-1-carboxylic acid) and
its metabolites (Smith et al., 1969; Kodama et al., 1976). 
d-Limonene-8,9-diol is probably formed via  d-limonene-8,9-epoxide
(Kodama et al., 1976; Watabe et al., 1981).  In another study, perillic
acid was reported to be the principal metabolite in plasma in both rats
and humans (Crowell et al., 1992).  Other reported pathways of limonene
metabolism involve ring hydroxylation and oxidation of the methyl group
(Kodama et al., 1976).

Falk-Filipsson et al. (1993) reported on a study to assess the
toxicokinetics of d-limonene in human volunteers, exposed by inhalation
for 2 hours each, in an exposure chamber. The exposures were at
concentrations of approximately 10, 225, or 450 mg/m3 d-limonene. The
relative pulmonary uptake of the d-limonene was high, about 70% of the
amount supplied. The blood clearance (1.1 L/kg/hr) indicates that the
d-limonene is readily metabolized, although a long half-timer in the
blood was observed during the slow elimination phase, suggesting some
accumulation in adipose tissues. After the end of the exposure, about 1%
of the total d-limonene uptake was eliminated unchanged in the expired
air, while 0.003% was eliminated in the urine.  It was observed that
there was a decrease in the vital capacity after exposure, in those
exposed at the highest dose, but that none of the subjects experienced
any irritative symptoms or any symptoms related to the central nervous
system. >

<	8. Endocrine disruption. >

<C. Aggregate Exposure>

<	1. Dietary exposure. d-Limonene occurs naturally in citrus and certain
fruits, vegetables, meats and spices. And, d-limonene is classified by
the US FDA as a GRAS food additive. It is present in baked goods, ice
cream products, gelatins, puddings, mouthwash and chewing gum at levels
ranging from 68 to 2300 ppm from the direct food additive use. 

Given the physical/chemical properties of d-limonene, it is unlikely
that d-limonene will occur in drinking water sources. 

As an inert ingredient, d-limonene can presently be applied to all
agricultural crops.  In the TRED, the Agency’s screening level dietary
assessment was performed based on a use pattern that considered uses on
all crops and thus the calculations in the TRED are appropriate for the
proposed exemption from tolerance when d-limonene is used as an active
ingredient on raw agricultural commodities. 

Based on the vapor pressure of 2 mm Hg, thus indicative of significant
evaporation, (1) it is unlikely that any significant amount of residues
of a volatile chemical would remain on the surface of the plant or
edible commodity and (2) it is also unlikely that residues of such a
volatile chemical would be incorporated into the raw agricultural
commodity that is eventually harvested. 

Generally, the Agency has advocated a position that if a pesticide
chemical is used on a food crop, residues of that chemical substance are
assumed to be present unless there is compelling data to the contrary.
Such data could be a radiolabelled uptake study of sufficient
sensitivity to ascertain whether or not the residues exist in the edible
commodity. The Agency is unaware of such data for a chemical such as
d-limonene. Therefore, the generic dietary exposure estimates used in
this assessment are overly conservative for a chemical such as
d-limonene, and the estimated MOEs could be even larger. 

For this assessment of d-limonene, there are no dermal or inhalation
toxicological studies in animals and no dermal absorption studies
available in the existing literature. Therefore, to assess short-term
dermal and inhalation exposures, an oral LOAEL was used. The dermal dose
was conservatively converted to an equivalent oral dose using a 100%
dermal and inhalation absorption factor. The oral toxicological LOAEL
endpoint of 400 mg/kg-day was used (Ariyoshi et al. 1975). This LOAEL
was based on liver effects (increased enzymes and liver weights)
observed in a 30-day rat oral (gavage) study (WHO, 1998). Since this
endpoint is based on a LOAEL, an additional safety factor of 3X was
applied to the uncertainty factor of 100 (10 for interspecies
extrapolation and 10 for intraspecies variation). A Margin of Exposure
(MOE) of 300 or greater is protective for these short-term risk
assessments. 

The calculated dietary MOE is 290 which is less than the target MOE of
300.  However, as previously discussed the dietary exposures are
considered to be much over-estimated due to the volatile nature of
d-limonene. If the dietary exposure is divided by 2 (0.12/2 = 0.06),
then the MOE is 305. >

<	i. Food. >

<	ii. Drinking water. d-Limonene is only somewhat soluble in water (13.8
mg/L) and has an estimated octanol/water partition coefficient of 4.2.
d-Limonene is expected to rapidly volatilize from water to the
atmosphere, with an estimated half-life for volatilization from a model
river of 3.4 hr, although adsorption to sediment and suspended organic
matter may attenuate the rate of this process. Based on these data, it
is unlikely that d-limonene will occur in drinking water sources
resulting from any of the registered and proposed uses as an active
ingredient or when used as an inert ingredient. 

>

<	2. Non-dietary exposure. >

<D. Cumulative Effects>

<	Section 408(b)(2)(D)(v) of the FFDCA requires that, when considering
whether to establish, modify, or revoke a tolerance, the Agency consider
“available information” concerning the cumulative effects of a
particular pesticide’s residues and “other substances that have a
common mechanism of toxicity.” Unlike other pesticides for which EPA
has followed a cumulative risk approach based on a common mechanism of
toxicity, EPA has not made a common mechanism of toxicity finding as to
d-limonene and any other substances, and d-limonene does not appear to
produce a toxic metabolite produced by other substances. 

For the purposes of this tolerance action, therefore, EPA has not
assumed that d-limonene has a common mechanism of toxicity with other
substances. 

>

<E. Safety Determination>

<	1. U.S. population. >

<	2. Infants and children. At this time, there is no concern for
potential sensitivity to infants and children. Based on the data from
the study reviewed in the RED (EPA, 1994) and the various teratogenicity
and embryotoxicity studies reviewed in the WHO study (1998), it is now
again concluded that limonene is not a developmental toxicant. 

Calculated MOE for all population subgroups from infants (< 1 year) to
youths (13 - 19 years) range from 360 to 1500.  >

<F. International Tolerances>

<	The WHO (1998) reported that “the establishment of an acceptable
daily intake expressed in numerical form was not deemed necessary.” >

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