ATTACHMENT I:  The unsigned DRAFT HED Chapter of the Nicotine and
derivatives Reregistration Eligibility Decision Document presented to
the HED Risk Assessment Review Committee (RARC2) on August 22, 2007 for
consideration.  79 p.  [Note:  This document includes minor editorial
modifications made after August 22, 2007.]



UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C.  20460

OFFICE OF

PREVENTION, PESTICIDES

AND TOXIC SUBSTANCES

August xx, 2007

MEMORANDUM

SUBJECT:	Nicotine and derivatives:  HED Chapter of the Reregistration
Eligibility Decision Document (RED).  PC Code:  056702.  Case #:  2460. 
DP Barcode:  D341246.

Regulatory Action:  Phase 1 Reregistration

Risk Assessment Type:  Single Chemical Aggregate

FROM:	Bonnie Cropp-Kohlligian, Environmental Scientist

Reregistration Branch 4

Health Effects Division (7509P)

AND

Abdallah Khasawinah, Toxicologist

Reregistration Branch 4

Health Effects Division (7509P)

THROUGH:	Susan V. Hummel, Chemist/Senior Scientist

Reregistration Branch 4

Health Effects Division (7509P)

TO:	Jill Bloom, Chemical Review Manager

Reregistration Branch 2

Special Review and Reregistration Division (7508P)

Attached is the Health Effects Division’s Chapter to the Nicotine and
derivatives Reregistration Eligibility Decision (RED) Document
addressing the Human Health Risk Assessment for nicotine (PC 056702) or
nicotine alkaloid or nicotine as a naturally occurring component of
tobacco dust.  Listed below are the companion disciplinary chapters to
this risk assessment:

	Occupational and Residential Exposure/Risk Assessment for the Nicotine
and derivatives Reregistration Eligibility Decision (RED) Addressing the
Fulex Nicotine Greenhouse Fumigator, B. Cropp-Kohlligian, D341249,
08/xx/2007.

	Occupational and Residential Exposure/Risk Assessment for the Nicotine
and derivatives Reregistration Eligibility Decision (RED) Addressing the
Bonide Dog and Rabbit Repellant, B. Cropp-Kohlligian, D341897,
08/xx/2007.

	Review of Nicotine Incident Reports, J. Blondell and M. Spann, D276938,
08/10/2001.

1.0	Executive Summary

A Human Health Risk Assessment is being conducted for Nicotine and
derivatives (List B Reregistration Case #2460).  Of the nicotine and
derivatives listed as active ingredients in the Office of Pesticide
Programs Information Network (OPPIN), which include nicotine (PC
056702), nicotine sulfate (PC 056703), and tobacco dust (PC 056704),
only nicotine (PC 056702) is present as an active ingredient in
currently registered products.  Therefore, the active ingredients
nicotine sulfate (PC 056703) and tobacco dust (PC 056704) will not be
addressed further.

Nicotine or nicotine alkaloid or nicotine as a naturally occurring
component of tobacco dust is present as an active ingredient (PC 056702)
in three currently registered end-use products.  Only two of these
end-use products are being supported under reregistration.  Fuller
System, Inc. and Bonide Products, Inc. have recently informed the Agency
(Use Closure for Nicotine RED, J. Bloom, 06/18/2007) that they intend to
support the reregistrations of Fulex Nicotine Fumigator (EPA Reg. No.
1327-41) and Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465),
respectively.  [Note:  According to OPPIN Bonide® Rabbit & Dog Chaser
(EPA Reg. No. 4-465) was transferred from Faesy & Besthoff (EPA Reg. No.
779-29) on 08/16/2006.]  Bonide Products, Inc. does not intend to
support the reregistration of Bonide® Tobacco Dust (EPA Reg. No. 4-340)
and has requested voluntary cancellation of this end-use product; hence
this end-use product will not be discussed further.

In previous phases of the reregistration process for Nicotine and
derivatives, Fuller Systems, Inc. and Bonide Products, Inc. held
registrations but were not the registrants primarily responsible for
generating data in support of this List B Reregistration Case (#2460). 
Phase 4 of the reregistration process was completed in 1990 and
according to SRRD outstanding data requirements for nicotine (PC
056702), including toxicology and occupational and residential exposure
(ORE) data, were identified in Data Call-Ins (DCIs), however, details
concerning the communication of previously identified reregistration
data requirements to Fuller Systems, Inc. and Bonide Products, Inc. are
unknown to HED.  Hence, HED defers to SRRD on the topic of regulatory
history for nicotine.

Neither of the two end-use products which are being supported under
reregistration are registered for use on food or feed; hence, any
remaining tolerances listed under 40 CFR §180.167 are not being
supported under reregistration and should be revoked.  Also, HED notes
that no Codex MRLs have been established for nicotine (PC 056702),
nicotine sulfate (056703), or tobacco dust (PC 056704).  This topic will
not be discussed further.

HED notes that under 40 CFR §152.175, which concerns pesticides
classified for restricted use, there is a regulation concerning all
formulations of nicotine applied to cranberries; however, nicotine is
not currently registered for use on cranberries and this regulation
should be revoked.  This topic will not be discussed further.

After considering the remaining uses of nicotine which are being
supported under reregistration, the Health Effects Division (HED) and
the Environmental Fate and Effects Division (EFED) have agreed that
conducting a drinking water risk assessment is not appropriate.  This
topic will not be discussed further.

Since neither of the two end-use products which are being supported
under reregistration are registered for use on food or feed and given
that HED and EFED have agreed that conducting a drinking water risk
assessment is not appropriate, dietary (food + water) and aggregate
(food + water + residential exposure) risk assessments were not
conducted.

HED notes that since Nicotine and derivatives is a List B Reregistration
Case, the Product Chemistry Chapter for the Nicotine and derivatives
Reregistration Eligibility Decision (RED) is the responsibility of the
Registration Division.  This topic will not be discussed further.

Hazard Profile

There are no guideline toxicity studies available on nicotine and while
there are numerous published studies in which nicotine was administered
subcutaneously or intravenously, the utility of these studies to
characterize the nicotine toxicity for this risk assessment is very
limited.  There are, however, a few studies in which nicotine or a salt
of nicotine was administered by the oral or inhalation routes and these
studies have been used to characterize the nicotine toxicity for this
risk assessment.  No dermal toxicity or dermal absorption studies using
nicotine have been identified.

Nicotine is acutely toxic (Category I) by all routes of exposure (oral,
dermal, and inhalation).  The LD50 of nicotine is 50 mg/kg for rats and
3 mg/kg for mice.  A dose of 40–60 mg can be a lethal dosage for adult
human beings and doses as low as 1-4 mg can be associated with toxic
effects in some individuals.  Based on data collected with a 40%
nicotine formulation, nicotine is only a slight dermal irritant
(Category IV).  It is reported that nicotine causes dermatitis (skin
sensitization) in humans although data collected with a 40% nicotine
formulation was not a skin sensitizer in the guinea pig.

Nicotine is an agonist at nicotinic receptors in the peripheral and
central nervous system.  It inhibits the function of acetylcholine
receptors located at the neuromuscular junctions.  In general terms, it
causes stimulation of the ganglions in low doses but causes blockade at
higher concentrations.  

Nicotine in subchronic amounts administered to animals resulted in
increased pancreatic biosynthesis and accumulation of digestive enzymes
within the pancreas.  It also enhances synthesis of cholesterol,
triglycerides, phospholipids and free fatty acids in the liver and
testes and lowers serum testosterone and estradiole levels suggesting
gonadotoxic effects.  Nicotine is hepatotoxic in some animal tests.  It
also adversely affects bone formation and decreases body storage of
vitamin D.  

Experimental data in male rats suggest that nicotine ingested
chronically alters metabolic and   endocrine factors that may be
responsible, at least in part, for the development of gastrointestinal
ulcers and pancreatitis.  Chronic inhalation exposure to low level of
nicotine (500 μg/m3) for two years produced slight weight reduction and
did not result in other adverse effects in rats.

Nicotine is an animal and human teratogen according to numerous studies.
 It has detrimental effect on general growth and development as well as
on palatogenesis and ossification in mice fetuses prenatally exposed
during gestation.  It is also a developmental neurotoxicant.  It
produces biochemical changes in the fetal brain that result in abnormal
behavior in the offspring of exposed animals.  Experimental data in rats
suggests that exposure to a high dose of nicotine in utero might cause a
predisposition to diseases related to a dopaminergic dysfunction in the
frontal cortex.

In some tests nicotine and its metabolites did not cause bacterial
mutations nor did they increase the frequency of sister chromatid
exchanges. In some other tests nicotine was found to induce chromosomal
aberration.

According to the International Program on Chemical Safety (IPCS) review,
literature reports indicate that nicotine is neither an initiator nor a
promoter of tumors in mice. There is inconclusive evidence to suggest
that cotinine, an oxidized metabolite of nicotine, may be carcinogenic
in the rat (  HYPERLINK
"http://www.inchem.org/documents/pims/chemical/nicotine.htm" 
http://www.inchem.org/documents/pims/chemical/nicotine.htm ).

Nicotine is extensively metabolized to a number of metabolites by the
liver.  Quantitatively the most important metabolite of nicotine in most
mammalian species is the lactam derivative cotinine.

Tests in animals suggest that nicotine may adversely affect the immune
system.

mole/L (8.76, 17.52 mg/L; equivalent to 1.25 and 2.5 mg/kg/day
assuming a rat consumes 50 mL of water per day) resulted in mild fatty
change, mild focal necrosis and mild dark cell change (containing
numerous prominent pore annuli in the nuclear membranes and the
mitochondria appeared decreased in size with a decrease in mitochondrial
granules and loss of aristae) in a dose proportional manner.  
Histopathological changes seen at the lower dose (1.25 mg/kg/day) were
not statistically significant and this was considered to be a NOAEL and
the LOAEL was 2.5 mg/kg/day.  A margin of exposure of 1000 is applied to
account for inter-species extrapolation (10X), intra-species variability
(10X) and database uncertainty (10X).

Since the dermal and inhalation endpoints are based on the same
toxicological effects, dermal and inhalation risks may be combined.

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41)

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41) is a ready-to-use
formulation containing nicotine or nicotine alkaloid, as the sole active
ingredient, and is used as an insecticide to control aphids and most
thrips on ornamental plants grown in greenhouses.  The product is
formulated as a smoke generator in which nicotine is intended for
release as a vapor.  Based on the currently registered end-use product
label (EPA Reg. No. 1327-41; dated 08/10/2005) and information provided
by the registrant (Use Closure for Nicotine RED, J. Bloom, 06/18/2007),
non-dietary exposures for this use are expected to be short-term (1-30
days) and intermediate-term (1-6 months) in duration.   Although there
is the potential for nicotine to be applied year-round in greenhouses,
daily long-term (>6 months) worker exposure is deemed unlikely.  

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41; label date 08/10/2005)
is a restricted use pesticide and applications may be made by or under
the direct supervision of a certified applicator (40 CFR §152.175). 
According to the currently registered label, applicators are required to
wear:  (1) coveralls over long-sleeve shirt and long pants, (2)
waterproof gloves, (3) chemical-resistant footwear plus socks, (4)
protective eyewear, (5) chemical resistant headgear for overhead
exposure, and (6) a respirator with either an organic vapor-removing
cartridge with a prefilter approved for pesticides (MSHA/NIOSH approval
number prefix TC-23C) or a canister approved for pesticides (MSHA/NIOSH
approval number prefix TC-14G).  Note:  The currently registered label
does not specify if the respirator is a half-face or full-face mask.

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41; label date 08/10/2005)
contains 13.4% nicotine as a smoke fumigator available in 12 ounce and 6
ounce ready-to-use screw-capped canisters packaged with wire igniters
(sparklers).  Each 12 ounce canister contains 0.10 lbs a.i. and treats
20,000 ft3.  Each 6 ounce canister contains 0.05 lbs a.i. and treats
10,000 ft3.  

Fumigations are typically conducted overnight.  Prior to fumigations,
greenhouse vents are closed.  The canisters are shaken and set in place
with the screw caps removed.  The wire igniters are lit and inserted
into the open canisters for the purpose of burning the inert ingredients
to produce smoke and vaporizing the nicotine.  Sometime later,
greenhouse vents are re-opened by handlers and canisters are
re-collected.  Canisters that did not ignite are re-capped and stored
for later use.  Post-fumigation re-entry is governed by Worker
Protection Standards (WPS) ventilation requirements specified under 40
CFR 170.110(c)(3).

No nature of the residue data are available for this unique application
method which alters nicotine's physical state and almost certainly form
degradates.  During fumigation, nicotine may be present as a vapor and
possibly bound or adsorbed to particulate matter.  Given the lack of
sophistication of the ignition device (sparklers), without nature of the
residue data, it is difficult to determine if nicotine is only vaporized
and to some extent adsorbed to particulate matter or the extent to which
it is decomposed as a result of the application method.  Formation of
significant compounds of interest such as N-nitrosonornicotine (NNN) and
4-(N-methyl-N-nitrosamino)-1-(3-pyridil)-1-butanone (NNK) cannot be
excluded without supporting data.  However, since exposure estimates in
this risk assessment are based on maximum theoretical air concentration
and surface residue data/calculations and default assumptions, all
potential degradates of nicotine from the Fulex Nicotine [Greenhouse]
Fumigator use have been included in the exposure estimates of this risk
assessment and the de facto hazard assumption is equivalent toxicity
with nicotine.

The American Conference and Governmental Industrial Hygienists (ACGIH)
have established a threshold limit value (TLV) as an 8-hour
time-weighted average (TWA) of 0.5 mg/m3 for nicotine.  There is a skin
notation; however, sufficient data were not available to recommend a
Senisitizer (SEN) notation or carcinogenicity notation or a TLV as a
short-term exposure limit (STEL).  The Occupational Safety and Health
Agency (OSHA) has established a permissible exposure limit (PEL) as an
8-hour time-weighted average (TWA) of 0.5 mg/m3 for nicotine consistent
with the ACGIH TLV and The National Institute for Occupational Safety
and Health (NIOSH) concurs with the OSHA PEL and has established a value
of 5 mg/m3 for nicotine as a level that is immediately dangerous to life
or health (IDLH).

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41) Residential
(Non-Occupational) Exposure/Risk Assessment

No residential (non-occupational) handler exposure scenarios have been
identified for the Fulex Nicotine [Greenhouse] Fumigator use; however,
residential (non-occupational) postapplication exposure scenarios via
inhalation and dermal routes have been identified.  These exposures are
expected to be short-term in duration.

The Fulex Nicotine Fumigator (EPA Reg. No. 1327-41) is a restricted use
product for use by and under direct supervision of a certified
applicator (40 CFR §152.175); however, the current label does not
prohibit application to privately owned greenhouses in residential
settings by certified commercial applicators where it is possible for
residents to be exposed following application and according to
information provided by the registrant (Use Closure for Nicotine RED, J.
Bloom, 06/18/2007), the product is used in retail greenhouses where it
is considered possible for non-occupational members of the general
public to be exposed following application.  These residential
(non-occupational) postapplication exposures are addressed by the
occupational postapplication risk assessment for this end-use product.

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41) Occupational Handler
Exposure/Risk Assessment

Occupational handler exposure scenarios via the inhalation route have
been identified for the Fulex Nicotine [Greenhouse] Fumigator use and
are expected to be short-term (1-30 days) and intermediate-term (1-6
months) in duration.  Although there is the potential for nicotine to be
applied year-round in greenhouses, daily long-term (>6 months) worker
exposure is deemed unlikely.  

Since short-term and intermediate-term risk estimates will be based on
the same endpoint (1.25 mg/kg/day) and level of concern (1000), only
short-term risks have been calculated.

Two major handler use patterns were identified:  (1) opening/lighting of
canisters, and (2) reentering after canisters are deployed but before
the WPS ventilation requirements are met to open greenhouses vents and
dispose of canisters (as specified under 40 CFR §170.3 Handler
(1)(vii).  These two activities were considered as a single exposure
scenario.  The available data in the Pesticide Handlers Exposure
Database (PHED) do not reflect these Fulex Nicotine [Greenhouse]
Fumigator use patterns.  

No mixing/loading methods are necessary for the Fulex Nicotine
[Greenhouse] Fumigator use. Therefore, a mixing/loading exposure
assessment was not performed. 

Dermal exposures from a smoke formulation to handlers are assumed to be
minimal relative to the exposures and risks from inhalation.  Therefore,
a dermal exposure assessment for handlers was not performed.

Since the duration of handler exposure is relative to the number of
canisters needed for treatment based on the size of the individual
greenhouse structure to be treated and the number of individual
greenhouse structures to be treated per day at any given facility,
exposure periods of 30 minutes, representing smaller greenhouse
facilities and 60 minutes, representing larger greenhouse facilities,
were used as estimations of exposure periods for handlers.  

Inhalation MOEs greater than 1000 are not of concern to HED.  Based on
the currently registered end-use product label (EPA Reg. No. 1327-41;
dated 08/10/2005), information provided by the registrant (Use Closure
for Nicotine RED, J. Bloom, 06/18/2007), theoretical air concentration
data/calculations, and default assumptions, inhalation risks of concern
(i.e., MOEs less than 1000) were identified for individuals performing
application activities both with and without respiratory protection
(e.g., half- and full-face respirators with chemical and particulate
filter cartridges), except for those individuals using a self-contained
breathing apparatus (SCBA).  Moreover, inhalation risks of concern
(i.e., MOEs less than 1000) were identified for individuals using half-
and full-face respirators with chemical and particulate filter
cartridges for exposure periods of 1 and >3 minutes, respectively. 
These risk estimates may be considered conservative since they are based
on theoretical air concentration data/calculations and default
assumptions in the absence of acceptable chemical-specific data.

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41) Occupational
Postapplication Exposure/Risk Assessment

Postapplication exposure scenarios via inhalation and dermal routes have
been identified for the Fulex Nicotine [Greenhouse] Fumigator use and
are expected to be short-term (1-30 days) and intermediate-term (1-6
months) in duration.  Although there is the potential for nicotine to be
applied year-round in greenhouses, daily long-term (>6 months) worker
exposure is deemed unlikely.  

Since short-term and intermediate-term risk estimates will be based on
the same endpoint (1.25 mg/kg/day) and level of concern (1000), only
short-term risks have been calculated.

Inhalation and dermal MOEs greater than 1000 are not of concern to HED. 
All occupational postapplication exposure scenarios had dermal risks of
concern (i.e., MOEs less than 1000) at day-zero; however, inhalation
risks were not of concern (i.e., MOEs greater than 1000).  For dermal
risks, Restricted Entry Intervals of 40+ days would be required to
achieve acceptable MOEs.  This risk estimate may be considered
conservative since it is based on theoretical air concentration and
surface residue data/calculations and default assumptions, including
100% dermal absorption, in the absence of acceptable chemical-specific
data.

Note:  The technical grade of the active ingredient nicotine is
classified as a Category I toxicant based on acute oral toxicity data
which would, under the Worker Protection Standard (WPS), require a
minimum restricted entry interval (REI) of 48-hours (40 CFR
§156.208(c)); however, this criteria for determining the REI does not
apply to any product that is a fumigant (40 CFR §156.208(d)).  Hence,
the REI for the Fulex Nicotine [Greenhouse] Fumigator, if it is
determined to be a fumigant, will be governed by the WPS ventilation
criteria (40 CFR §170.110(c)(3)) and product-specific REI calculations.
 In the absence product-specific data collected in accordance with 40
CFR §158.390, these calculations have been based on theoretical
data/calculations and default assumptions.

Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465)

Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) a ready-to-use
formulation containing nicotine as a naturally occurring component of
tobacco dust and is used as an animal repellant in outdoor settings. 
Based on the currently registered Bonide® Rabbit & Dog Chaser end-use
product label (EPA Reg. No. 4-465; label date 12/28/2006), small
packaging sizes (available in 1- and 3-pound packages), and information
provided by the registrant (Use Closure for Nicotine RED, J. Bloom,
06/18/2007), this product is intended for use by homeowners as a
repellent via a barrier/perimeter treatment to prevent eastern
cottontail rabbits (Sylvilagus floridanus) from eating and defecating on
ornamental plants and domestic dogs (Canis l. familiaris) from
defecating on ornamentals, including lawns.  It may also be used around
the perimeter of vegetable gardens.  Bonide® Rabbit & Dog Chaser (EPA
Reg. No. 4-465; label date 12/28/2006) contains 0.35% nicotine (as a
naturally occurring component of tobacco dust), as well as two other
active ingredients, dried blood and naphthalene.  [NOTE:  The nicotine
content of tobacco dust in the end-use product is substantially less
than the content normally found in dried tobacco leaves (up to 8% by
weight according to the Merck Index) and in the tobacco found in
cigarettes (on average, 1.5% by weight according to the 1988 report of
the Surgeon General, entitled, “The Health Consequences of
Smoking”).]  Given that the nicotine in this product is contained in
tobacco dust, the formulation is considered a dust for this risk
assessment.  Based on the currently registered Bonide® Rabbit & Dog
Chaser end-use product label (EPA Reg. No. 4-465; label date 12/28/2006)
and information provided by the registrant (Use Closure for Nicotine
RED, J. Bloom, 06/18/2007), non-dietary exposures to this product are
expected to be short-term in duration.

HED notes that under 40 CFR §152.175, which concerns pesticides
classified for restricted use, nicotine (alkaloid) as a liquid or dry
formulations containing 1.5% and less is unclassified for all uses. 
Toxicity data provided for the Bonide® Rabbit & Dog Chaser has shown
low acute toxicity by the oral, dermal, and inhalation routes (Category
IV).  It is a weak eye or dermal irritant (Category IV) and does not
cause dermal sensitization in the guenea pig.  See Table A.2.1b.  

Maximum use rates for this product are not specified on the currently
registered Bonide® Rabbit & Dog Chaser end-use product label (EPA Reg.
No. 4-465; label date 12/28/2006) and are difficult to determine.  The
product is available in 1 pound and 3 pound ready-to-use packages. 
According to the label, a 3-pound package of product will produce a band
of product 1 inch wide and 85 feet long and a 1-pound package of product
will produce a band of product 1 inch wide and 28 feet long.  Both the
3-pound and 1-pound packages provide the same use directions.  The
product is sprinkled directly from the package in 2 or more inch-wide
bands around areas to be treated.  The product may be used throughout
the year and repeat applications are as needed.

The label does not provide any distinction in use directions for
professional (occupational) and non-professional (residential homeowner)
applicators and no occupational postapplication scenarios have been
identified.  Therefore, only the residential handler exposures and risks
have been assessed which should be protective of occupational handlers.

Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) Residential
(Non-Occupational) Handler Exposure/Risk Assessment

It has been determined that there is a potential for dermal and
inhalation exposure in residential settings during the application of
Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465; label date
12/28/2006).  Based on the currently registered Bonide® Rabbit & Dog
Chaser end-use product label (EPA Reg. No. 4-465; label date 12/28/2006)
and information provided by the registrant (Use Closure for Nicotine
RED, J. Bloom, 06/18/2007), non-dietary exposures to this product are
expected to be short-term in duration.

Residential MOEs equal to or greater than 1000 are not of concern to
HED.  The short-term combined dermal and inhalation MOE for residential
handlers who apply one three-pound package of the ready-to-use Bonide®
Rabbit & Dog Chaser (EPA Reg. No. 4-465) was 1875 and therefore not of
concern to HED.  However, application of two or more three-pound
packages would be of concern, since this is estimated to result in a
combined dermal and inhalation MOE less than 1000.  The concerns are
primarily for dermal exposure/risk and HED notes that the
inhalation/dermal risk estimates may be considered conservative since,
in the absence of chemical-specific data, they are based on theoretical
data/calculations and default assumptions, including 100%
inhalation/dermal absorption, for this product containing nicotine as a
naturally occurring component of tobacco.  

Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) Residential
(Non-Occupational) Postapplication Exposure/Risk Assessment

Because the use pattern results in applications to areas not frequented
by children or in areas where maintenance would result in significant
exposure, dermal and incidental oral assessments were not conducted. 
However, episodic ingestion of the material is considered reasonable due
to packages which are not child resistant and which could be accessible
to children prior to application or due to the potential for children to
come in contact with the product postapplication.

Residential MOEs equal to or greater than 1000 are not of concern to
HED.  The short-term oral MOE for episodic ingestion of the ready-to-use
Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) containing nicotine
(as a naturally occurring component of tobacco dust) was 1 and therefore
is of concern to HED.  Moreover, ingestion of more than 0.002 teaspoons
of product by a toddler (15 kg) exceeds the level of concern (MOE less
than 1000); however, this type of ingestion is considered an episodic
event and not a routine behavior.  Because HED does not believe that
this would occur on a regular basis, our concern for human health is
related to acute poisoning rather than short -term residue exposure.

2.0	Ingredient Profile  TC \l1 "2.0	Ingredient Profile 

2.1	Summary of Registered/Proposed Uses   TC \l2 "2.1	Summary of
Registered/Proposed Uses 

2.1.1	Fulex Nicotine Fumigator (EPA Reg. No. 1327-41)

See:  Occupational and Residential Exposure/Risk Assessment for the
Nicotine and derivatives Reregistration Eligibility Decision (RED)
Addressing the Fulex Nicotine Greenhouse Fumigator, B. Cropp-Kohlligian,
D341249, 08/xx/2007.

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41; label date 08/10/2005)
contains 13.4% nicotine as a smoke fumigator available in 12 ounce and 6
ounce ready-to-use screw-capped canisters packaged with wire igniters
(sparklers).  Each 12 ounce canister contains 0.10 lbs a.i. and treats
20,000 ft3.  Each 6 ounce canister contains 0.05 lbs a.i. and treats
10,000 ft3.  Since both the 12 ounce and 6 ounce canisters provide the
same maximum application rate, hereafter, only the 12 ounce product will
be use in exposure estimate calculations.  Table 2.1.1 summarizes the
use pattern and formulation specified in the end-use product used in
greenhouses containing nicotine.

	Table 2.1.1.  Use Patterns and Formulations for the Fulex Nicotine
[Greenhouse] Fumigator use of Nicotine.

Formulation	Method of Application	Use Sites	Application Rates	Timing of
Application and Restrictions

Fulex Nicotine Fumigator

Insecticide (13.4% ai)

EPA Reg. No. 

1327-41

Note:  Available in ready-to-use 6- and 12-oz. screw-capped steel
canisters with wire igniters which look and act like sparklers.	Smoke
fumigator	Greenhouses on ornamental plants (to control aphids and
thrips)	1 canister (12 oz)/

20,000 ft3

or 

1 canister (6 oz)/

10,000 ft3

Note:  Both canister sizes provide the same application rate.	At
fumigation, greenhouse temperature between 70-90 F. Foliage and blossoms
must be dry. Avoid rainy or windy days. Close all vents prior to use. 
Repeat treatment in one week if necessary.

Note:  According to information provided by the registrant (Use Closure
for Nicotine RED, J. Bloom, 06/18/2007), in contradiction to the label
restrictions, applications are typically made every 3-12 days.  The
actual minimum re-treatment interval (RTI) is therefore assumed to be 3
days.



Given the lack of sophistication of the ignition device (sparklers), it
is difficult to determine if nicotine is only vaporized (and to some
extent decomposed) as a result of the application method producing
nicotine vapor and possibly nicotine bound or adsorbed onto particulate
matter or burned during the application method producing pyrolysis
products of nicotine including N-nitrosonornicotine (NNN) and
4-(N-methyl-N-nitrosamino)-1-(3-pyridil)-1-butanone (NNK).  However,
since exposure estimates in this risk assessment are based on maximum
theoretical air concentration data/calculations and default assumptions,
all potential degradates of nicotine  from the Fulex Nicotine
[Greenhouse] Fumigator use have been included in the exposure estimates
of this risk assessment and the de facto hazard assumption is equivalent
toxicity with nicotine.

2.1.2	Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465)

See:  Occupational and Residential Exposure/Risk Assessment for the
Nicotine and derivatives Reregistration Eligibility Decision (RED)
Addressing the Bonide Dog and Rabbit Repellant, B. Cropp-Kohlligian,
D341897, 08/xx/2007.

Based on the currently registered Bonide® Rabbit & Dog Chaser end-use
product label (EPA Reg. No. 4-465; label date 12/28/2006), small
packaging sizes (available in 1- and 3-pound packages), and information
provided by the registrant (Use Closure for Nicotine RED, J. Bloom,
06/18/2007), this product is intended for use by homeowners as a
repellent via a barrier/perimeter treatment to prevent eastern
cottontail rabbits (Sylvilagus floridanus) from eating and defecating on
ornamental plants and domestic dogs (Canis l. familiaris) from
defecating on ornamentals, including lawns.  It may also be used around
the perimeter of vegetable gardens.  Bonide® Rabbit & Dog Chaser (EPA
Reg. No. 4-465; label date 12/28/2006) contains 0.35% nicotine (as a
naturally occurring component of tobacco dust), as well as two other
active ingredients, dried blood and naphthalene.  [NOTE:  The nicotine
content of tobacco dust in the end-use product is substantially less
than the content normally found in dried tobacco leaves (up to 8% by
weight according to the Merck Index) and in the tobacco found in
cigarettes (on average, 1.5% by weight according to the 1988 report of
the Surgeon General, entitled, “The Health Consequences of
Smoking”).]  Given that the nicotine in this product is present as a
naturally occurring component of tobacco dust, the formulation is
considered a dust for this risk assessment.  The product is available in
1 pound and 3 pound ready-to-use packages; applications are the same.  A
3-pound package of product will produce a band of product 1 inch wide
and 85 feet long.  The product is sprinkled directly from the package in
2 or more inch-wide bands around areas to be treated.  Maximum use rates
in terms of lb ai/A/application are not specified on the product label;
the reviewer estimates that a 3-pound package of product will provide a
barrier/perimeter treatment around an area encompassing at most 144 ft2.
 The product may be used throughout the year and repeat applications are
as needed.  The maximum number of treatments per season is not specified
on the product label.  Table 2.1.2 summarizes the use pattern and
formulation specified in the end-use product containing nicotine (as a
naturally occurring component of tobacco dust).

	Table 2.1.2.  Use Patterns and Formulations for the BonideRabbit and
Dog Repellant.

Formulation	Method of Application	Use Sites	Application Rates, Timing of
Application, and Restrictions

Bonide® Rabbit & Dog Chaser EPA Reg. No. 4-465

(The product contains 0.35% nicotine and is present in this product as a
naturally occurring component of tobacco dust; hence, the formulation is
considered a dust for this risk assessment.)

Note:  Marketed in ready-to-use in 1 and 3-pound poly bags.

	Rabbit and dog repellant applied by hand.  The product is sprinkled
onto treated area directly from the package. 	Barrier/perimeter
treatments for use by homeowners around residential lawns, flower
gardens, vegetable gardens, ornamentals, trees and shrubs.	According to
the label, a 3-pound package of product will produce a band of product 1
inch wide and 85 feet long and a 1-pound package of product will produce
a band of product 1 inch wide and 28 feet long.  The product is applied
in 2 or more inch-wide bands around areas to be protected.

The reviewer estimates that a 3-pound package of product will provide a
barrier/perimeter treatment around ≤144 ft2 area as follows:

A 3-pound package of product will provide 42.5 linear feet of 2
inch-wide bands of barrier/perimeter treatment.  If this product is
applied in a closed circle it will encompass at most a 144 ft2 of area
(Area=πr 2 ) with a circumference (Circumference = 2πr) of 42.5 ft.

According to the label, before treatment, try to remove all traces of
animal droppings and urine from the areas to be protected.  Be careful
not to apply product directly to foliage or stems.  Repeat treatment as
needed.  More frequent applications will be needed if heavy rains, heavy
snowfalls, hot weather, or high winds occur.  Applications may be made
throughout the year.



2.2	Structure and Nomenclature  TC \l2 "2.2	Structure and Nomenclature 

 

Common name	Nicotine

IUPAC name	(S)-3-(1-methylpyrrolidin-2-yl)pyridine (according to
allanwood.net)

CAS name	(S)-3-(1-Methyl-2-pyrrolidinyl)pyridine (according to the Merck
Index)

CAS registry number	54-11-5

End-use products (EP)	Fulex Nicotine Fumigator (13.4% nicotine): EPA
Reg. No. 1327-41)

Bonide Rabbit & Dog Chaser (0.35% nicotine as a naturally occurring
component of tobacco dust); EPA Reg. No. 4-465)





2.3	Physical and Chemical Properties  TC \l2 "2.3	Physical and Chemical
Properties 

Nicotine is a colorless to pale yellow oily liquid, which slowly turns
brown when exposed to air or light.  It is hygroscopic and forms
water-soluble salts including the hydrochloride, sulfate, and tartrate. 
Nicotine decomposes slightly at its boiling point.  

Nicotine is a tertiary amine composed of a pyridine and a pyrrolidine
ring.  Nicotine may exist in two stereoisomers.  Tobacco contains only
(S)-nicotine, which is the more pharmacologically active form. 
Racemization is expected with combustion.

According to Merck Index, Nicotine comes from the dried leaves of
Nicotiana tabacum and N. rustica where is occurs to the extent of 2 to
8%, combined with citric and malic acids.  Commercial nicotine is
entirely a byproduct of the tobacco industry.

According to a report of the Surgeon General (1988) entitled, “The
Health Consequences of Smoking”, on average, nicotine content of the
tobacco used in American cigarettes is 1.5% (by weight).

Table 2.3.  Physicochemical Properties of Nicotine. 

Parameter	Value	Reference

Boiling point/range	247°C at 745 mmHg (partial decomposition)	Merck
Index

pH



Density	specific gravity 1.0097 at 20° referred to water at 4°	Merck
Index

Water solubility



Solvent solubility	Soluble in water, chloroform, alcohol, ether,
kerosene, and oils	Merck Index

Vapor pressure	4.25 x 10-2 torr at 20°C

	Dissociation constant, pKa



Octanol/water partition coefficient, Log(KOW)



UV/visible absorption spectrum



3.0	Hazard Characterization/Assessment  TC \l1 "3.0	Hazard
Characterization/Assessment 

Nicotine toxicity has been investigated extensively.  This review is
based on published literature studies to identify hazards from exposure
to nicotine that will aid in assessing the risks from its limited
pesticidal uses.  None of the studies reviewed followed EPA guidelines.

Nicotine is a potent toxicant.  It is an agonist at nicotinic receptors
in the peripheral and central nervous system. It inhibits the function
of acetylcholine receptors located at the neuromuscular junctions.  In
general terms, it causes stimulation of the ganglions in low doses but
causes blockade at higher concentrations.  The oral LD50 of nicotine is
50 mg/kg for rats and 3 mg/kg for mice.  A dose of 40–60 mg can be a
lethal dosage for adult human beings.  Nicotine is readily absorbed by
the skin making it equally toxic by the dermal route.  Its high
solubility both in polar and non-polar solvents and its low molecular
weight make it an efficient skin penetrant.  Workers handling and
harvesting green tobacco develop “green-tobacco sickness” due to the
dermal absorption of nicotine from the leaves. The average blood
concentration in lethal cases is 29 μg/mL (0.18 mM). 

It is reported that nicotine causes contact dermatitis (skin
sensitization) in humans although a 40% nicotine formulation was not a
skin sensitizer in the guinea pig.

Nicotine in subchronic amounts administered to animals resulted in
increased pancreatic biosynthesis and accumulation of digestive enzymes
within the pancreas.  It also enhances synthesis of cholesterol,
triglycerides, phospholipids and free fatty acids in the liver and
testes and lowers serum testosterone and estradiole levels suggesting
gonadotoxic effects.  Nicotine is hepatotoxic in some animal tests.  It
also adversely affects bone formation and decreases body storage of
vitamin D.  

of nicotine (500 μg/m3) for two years produced slight weight reduction
and did not result in other adverse effects in rats.

Nicotine is an animal and human teratogen according to numerous studies.
 It has detrimental effect on general growth and development as well as
on palatogenesis and ossification in mice fetuses prenatally exposed
during gestation.  It is also a developmental neurotoxicant.  It
produces biochemical changes in the fetal brain that result in abnormal
behavior in the offspring of exposed animals.  Experimental data in rats
suggests that exposure to a high dose of nicotine in utero might cause a
predisposition to diseases related to a dopaminergic dysfunction in the
frontal cortex.

In some tests nicotine and its metabolites did not cause bacterial
mutations nor did they increase the frequency of sister chromatid
exchanges. In some other tests nicotine was found to induce chromosomal
aberration.

According to the International Program on Chemical Safety (IPCS) review,
literature reports indicate that nicotine is neither an initiator nor a
promoter of tumors in mice. There is inconclusive evidence to suggest
that cotinine, an oxidized metabolite of nicotine, may be carcinogenic
in the rat (  HYPERLINK
"http://www.inchem.org/documents/pims/chemical/nicotine.htm" 
http://www.inchem.org/documents/pims/chemical/nicotine.htm ).

Tests in animals suggest that nicotine may adversely affect the immune
system.

3.1	Hazard and Dose-Response Characterization  TC \l2 "3.1	Hazard and
Dose-Response Characterization 

3.1.1	Database Summary

There are no guideline toxicity studies available on nicotine.  There
are numerous published studies where nicotine was administered
subcutaneously or intravenously. The utility of such studies in hazard
characterization for the risk assessment of nicotine exposure is very
limited.   However, few studies were available where nicotine was
administered orally or by inhalation.  These (oral/inhalation) studies
were used to characterize the nicotine toxicity for this current risk
assessment.  Only key studies used to derive or support endpoints of
toxicity for the hazard characterization of nicotine are discussed
below. All other studies are discussed in Appendix A.3

3.1.1.1	Studies available and considered (animal, human, general
literature)  TC \l4 "3.1.1.1	Studies available and considered (animal,
human, general literature) 

Acute Toxicity

The acute toxicity of nicotine is summarized in Appendix A Table 2.1. 
Nicotine is highly toxic. It is reported that as low as 60 mg ingested
by an adult human being are fatal and that doses as low as 1-4 mg can be
associated with toxic effects in some individuals (Saxena and Scheman,
1985).  The oral LD50 of nicotine is 50 mg/kg for rats and 3 mg/kg for
mice (Other published studies summarized in the tox profile in Appendix
A.2. showed that mice tolerated higher doses without mortality).  It is
equally toxic by the oral and dermal routes. Workers handling and
harvesting green tobacco absorb through their skins nicotine doses
oozing from the wet leaves to produce frequently toxic symptoms of what
is known as “green-tobacco sickness” (Gehlbach et al, 1974 and 1975;
Ghosh et al, 1985; Jones and Goldy, 1998; Hipke, 1993; Boylan et al
1993; Quandt et al, 2000).  A 40% formulation of nicotine known as Black
Leaf 40 produced slight dermal irritation to the rabbit skin and was not
a skin sensitizer in the guinea pig (MRID 41521101 & 41521102). 
However, nicotine is reported to cause contact dermatitis (skin
sensitization) in humans (Bircher et al, 1991; Vincenzi et al, 1993).

Nicotine dermal patches applied topically (1 to 2 mg/kg/24 h) or orally
(2.8 to 13.4 mg/kg over 25-57 h, with mean maximal plasma levels of 36 -
73 ng/mL) to dogs produced minimal clinical symptoms of excess
salivation, emesis and vomiting.  The acute oral LD50 for nicotine in
dogs is at least 10-12 mg/kg (Matsushima et el, 1995).

Data derived from five fatal case reports indicated that the average
blood concentration of nicotine was 29 ug/mL equivalent to a 0.18 mM
mean lethal concentration (Baselt and Cravey, 1977 as cited in Jover et
al, 1994).

Subacute/Subchronic Toxicity 

In a study by Yuen et al, 1995, nicotine hydrogen tartrate was
administered to pregnant (10±1 day pregnant, 16 rats/dose) and
non-pregnant female (72±3 day old, 24 rats/dose) Sprague Dawley rats in
the drinking water for 10 days at 0, 54 or 108 mole/L (8.76, 17.52
mg/L; equivalent to 1.25 and 2.5 mg/kg/day assuming a rat consumes 50 mL
of water per day).  On the 10th day before sacrifice, each group was
subdivided into two subgroups. One subgroup was sacrificed by cervical
dislocation without further treatment. The second subgroup of rats was
administered subcutaneous dose of carbon tetrachloride at 6 g/kg, 4
hours before they were killed.  Nicotine treatment resulted in mild
fatty change, mild focal necrosis and mild dark cell change (containing
numerous prominent pore annuli in the nuclear membranes and the
mitochondria appeared decreased in size with a decrease in mitochondrial
granules and loss of aristae) in a dose proportional manner. In the
non-pregnant rats fed with 54 mole/L of nicotine, there was a slight
increase (not statistically significant) in the number of rats showing
mild fatty change and mild necrosis compared with the control group. 
The livers of the 108 mole/L non-pregnant rats showed significant
pathological changes in all parameters assessed in comparison to the
control rats with increases in degree of fatty change, dark cell change,
and of focal and confluent necrosis. The latter three parameters were
also significantly more severe when compared with the low dose group. 
Similar pathological changes were seen in the pregnant rats treated with
nicotine, but were less severe than the effects seen in the non-pregnant
rats. Thus in the low dose group, no increased hepatic changes were seen
compared to controls. Pretreatment with nicotine aggravated the
hepatotoxicity of carbon tetrachloride.  Pregnant rats were more
resistant to the hepatotoxicity of both materials.  This study
demonstrated the hepatoxicity of nicotine.  A LOAEL of 108 mole/L of
nicotine (2.5 mg/kg/day) in drinking water is suggested by this study
based on the histopathological changes produced and the NOAEL is 54
mole/L (1.25 mg/kg/day).

In another study nicotine tartrate, administered orally to 10 New
Zealand white male rabbits (2.4 mg/kg/day) in the drinking water for 25
weeks produced in vivo morphologic effect on endothelial cells in the
aortic arch (Booyse et al, 1981).  Fasting serum levels of glucose,
triglycerides, total cholesterol, and LDL-cholesterol were significantly
(<0.001) elevated in nicotine-treated rabbits.  Endothelial cells from
nicotine-treated arched areas (Evans-blue-stained) showed extensive
changes such as increased cytoplasmic silver disposition, increased
formation of microvilli, and numerous focal areas of  “ruffled”
endothelium (projections on cell surfaces).

Chronic Toxicity

g/ m3 of >99% pure nicotine/m3 (68 rats),  20 h/day, 5 days/week for
2 years resulted in slightly depressed body weight (Waldum et al, 1996).
 Rats were weighed weekly and the body weight reduction in the nicotine
treated rats was less than 6% compared to controls.  Body weight gains
in the treated rats were initially less than the controls (70% of the
controls at 4 months) and became almost comparable at 24 months (91% of
the controls).  Rats were housed to a maximum of 8 rats in a cage. To
avoid oral intake, food and water were available ad libitum only during
non exposure periods. Rats from each cage were weighed in a group once
weekly.  Nicotine air concentration in the inhalation chambers was
determined once or twice weekly throughout the two year study period and
was fairly constant. The administered dose of 501±151 g/m3 is
equivalent to 0.34 ± 0.1 mg/kg/day (based on inhalation rate of 0.29
m3/day and 350 g average rat weight).  Nicotine concentration in the
blood was determined 5 days after exposure, and at 6, 12, 18 and 24
months of exposure. Also at those intervals except for the 5 day
interval, specified number of rats were examined grossly for tumors in
the brain, lungs, gastrointestinal tract, live, kidney and ovaries and
further examined histopathologically.  Rats exhibiting unhealthy signs
(bristling fur, emaciation, and shiny eyes) were withdrawn (7 controls
(22%) and 10 nicotine exposed (16%) rats) and these were examined
wherever possible.  At the end of the study, the remaining rats-7
controls and 22 nicotine exposed- were sacrificed and examined for
tumors and atherosclerosis.  Nicotine plasma concentration after 5 days
was 108.4 ± 55.1 ng/mL and remained fairly constant and after 24 months
it was 129.8±43.0 ng/mL.  All parameters measured were comparable to
the controls except for elevated adenomas of pituitary gland (4/59)
which was attributed to the neuroendocrine action of nicotine.  There
was no increase in mortality, in atherosclerosis or frequency of tumors
in these rats compared with controls.  Particularly, there were no
microscopic or macroscopic lung tumors or any increase in pulmonary
neuroendocrine cells.  Throughout the study, however, the body weight of
the nicotine exposed rats was reduced as compared with controls.  Based
on this study there was no indication of any harmful effect of nicotine
when given in its pure form by inhalation and the NOAEL is 0.3
mg/kg/day.  Other potential effects of nicotine inhalation in this study
were published separately and no negative effects on bone mineral
density, ultimate bending moment, ultimate energy absorption, stiffness,
or deflection of the femurs examined were found except for depressed
body weights (Syversen et al, 1999).

Developmental Toxicity

Nicotine (12 mg/kg/day) or nicotine plus caffeine (125 mg/kg/day)
administered by intubation by water during gestation days 6-18 to female
mice (7/group) had minimal ossification effects on the fetuses as
measured by staging and measuring craniofacial bones, and counting
ossification centra in sternbrae and in cervical and sacrococcygeal
vertebrae (Leblebicioglu-Bekcioglu et al, 1995).  Caffeine had a
significantly greater effect on fetal growth and ossification than
nicotine.  In this study overnight bred ICR female mice weighing 25-26 g
(20/group) were intubated three times a day (4 hours apart) with 125
mg/kg caffeine, 12 mg/kg nicotine or 125 mg/kg caffeine plus 12 mg/kg
nicotine on gestation days 6-18.  A control group of similar number was
administered distilled water.  These doses were selected on the basis of
a range finding study where groups of female mice (7/group) were
intubated for 12 consecutive days (three times a day to sustain stable
daily blood levels) with 85 mg/kg caffeine plus 8 mg/kg nicotine, or 100
mg/kg caffeine plus 10 mg/kg nicotine or 125 mg/kg caffeine plus 12
mg/kg nicotine.  Nicotine blood levels ranged from 6.1 -30.2 ng/mL (mean
20.1), 31.0 – 41.6 ng/mL (mean 35.2), 52.1 – 87.7 ng/mL (mean 70.4)
at the 8, 10 and 12 mg/kg nicotine dose, respectively.  Nicotine blood
levels peaked within three days and remained steady during exposure
indicating the rapid elimination. In the main study, all pregnant rats
were sacrificed on day 18, and fetuses collected and subjected to
detailed examination.  No compound related mortality was reported.
Nicotine intubated mice experienced hyperactivity for nearly 10 minutes
after intubation, during the initial 2-3 days.  This was especially
noted after the third dose and this effect subsided after the third day.
 Three mice in the caffeine plus nicotine group and two mice from the
nicotine group died during the first three days.  These deaths were
attributed to the nicotine treatment.  None of the caffeine treated mice
died. 

Mean fetal body fat was significantly increased in fetuses of rats
administered nicotine (2.46 ± 0.18 mg/kg/day in drinking water) during
pregnancy throughout gestation day 20 (Williams & Kanagasabai, 1984). 
Rate for maternal lypolysis were higher in the nicotine treated animals.
 Maternal body weights gains were significantly lower (77.2% of
controls, p < 0.001).  

Nicotine - delivering transdermal patches applied on the back of
pregnant female rats resulted in 100% pregnancy failure in 2 animals
treated with 3.5 mg/day during the entire pregnancy (GD 2-19) and 50% in
8 animals exposed to the same amount during the first trimester (GD 2-7)
and 55% in 13 animals exposed to 1.75 mg/day during the entire pregnancy
(Witschi et al, 1994).  Litter size and pup weights were not affected by
the nicotine treatment.  Nicotine and cotinine plasma levels in the
sacrificed animals were not detected in animals that had carried a patch
during the first trimester of pregnancy.  In animals exposed the entire
pregnancy at 1.75 mg/day patches, 3 pregnant animals out of six had
measurable nicotine levels (43 ± 22 ng/mL) and all had cotinine levels
(100 ± 48 ng/mL).  The non pregnant females of the 1.75 mg/day patches
had 70 ± 57 ng/mL of plasma nicotine and 231 ± 84 ng/mL of plasma
cotinine.  The two non-pregnant animals exposed to 3.5 mg/day patches
had nicotine plasma levels of 241 ± 51 ng/mL and cotinine levels of 302
± 94 ng/mL. 

Many studies have demonstrated that nicotine penetrates the fetal brain
to cause various biochemical changes with behavioral consequences on the
offspring.  These studies are listed in the tox profile in Appendix A.2.
 In all of these studies, nicotine was administered subcutaneously or
intraperitonealy.  Performance deficits in both learned and innate
behavioral measures throughout development and adulthood in offspring of
animals exposed to nicotine during gestation have been reported.  Doses
as low as 0.25 mg/kg/day produced behavioral changes.

Genotoxicity

g/total body weight injected to young mice (Bishun et al, 1972). 
Cotinine (a metabolite of nicotine and a biological monitoring marker of
nicotine absorption in humans) was positive in the presence or absence
of S9 metabolic activation in the bacterial luminescence genotoxicity
test at 1.25 - 2.5 mg/mL (9- 30 h incubation) while, nicotine was not
positive to 20 mg/mL concentrations for up to 40 hours of incubation
(Yim & Hee 1995).  Nicotine has been shown to cause concomitant
genotoxic and antiapoptotic effect in human gingival fibroblasts (HGFs)
in the cytokinesis-block micronucleus (CBMN) test (Argentin and
Cicchetti, 2004).  Recent data indicate that nicotine exerts significant
direct genotoxic effects in human lymphocytes in vitro (Kleinsasser et
al, 2005).  These tests are presented further in Appendix A.3. of this
review.

3.1.1.2	Mode of action, metabolism, toxicokinetic data

“Nicotine is an agonist at nicotinic receptors in the peripheral and
central nervous system.  In man, as in animals, nicotine has been shown
to produce both behavioral stimulation and depression.  Pharmacodynamic
studies indicate a complex dose response relationship, due both to
complexity of intrinsic pharmacological actions and to rapid development
of tolerance.”

(  HYPERLINK
"http://www.inchem.org/documents/pims/chemical/nicotine.html" 
http://www.inchem.org/documents/pims/chemical/nicotine.html )

“Nicotine inhibits the function of acetylcholine receptors located at
the neuromuscular junctions.  In general terms, it causes stimulation of
the ganglions in low doses but causes blockade at higher concentrations.
 The nicotinic acetylcholine receptors (named for their interaction with
nicotine, and not to be confused with the muscarinic acetylcholine
receptor) are 270kD proteins with 4 subunits located in the CNS.  Under
normal conditions, a change in calcium ion concentration releases
acetylcholine from storage vesicles.  Acetylcholine then crosses the
synaptic cleft and binds to the alpha subunit of the nicotinic receptor
causing conformational changes which opens an ion channel, allowing the
passage of cations.  This depolarizes the postsynaptic membrane
initiating an action potential in the adjacent membrane, and thus a
signal is transmitted.  Nicotine stimulates, and then blocks the
acetylcholine receptor, locking the ion channels in the open position
and impairing signaling ability. 

The metabolism and disposition kinetics of nicotine with focus on humans
has been extensively reviewed by Hukkanen et al, 2005.  In this review,
evidence is presented to the rapid absorption of nicotine from the
inhaled cigarette smoke and its rapid distribution via the blood stream
to various tissues including the brain.  However, nicotine is poorly
absorbed from the stomach because it is protonated in the acidic gastric
fluid, but is well absorbed in the small intestine, which has a more
alkaline pH and a large surface area.  Various formulations of nicotine
replacement therapy (NRT) to aid in smoking cessation, such as nicotine
gum, transdermal patch, nasal spray, inhaler, sublingual tablets, and
lozenges, are buffered to alkaline pH to facilitate the absorption of
nicotine through cell membranes.  Absorption of nicotine from all NRTs
is slower and the increase in nicotine blood levels more gradual than
from smoking.  Only nasal spray provides a rapid delivery of nicotine
that is closer to the rate of nicotine delivery achieved with smoking. 
Following the administration of nicotine capsules or nicotine in
solution, peak concentrations in the blood are reached in about 1 hour. 
Oral bioavailability is incomplete because of the hepatic first-pass
metabolism.

Binding to plasma proteins is minimal (<5%).  Nicotine is distributed
extensively to body tissues.  With the highest affinity for nicotine is
in the liver, kidney, spleen, and lung and the lowest affinity in
adipose tissue.  In poisoning cases, nicotine was found to bind to brain
tissues with high affinity, and the receptor binding capacity is
increased in smokers compared with nonsmokers.  The increase in the
binding is caused by a higher number of nicotinic cholinergic receptors
in the brain of the smokers.  Nicotine accumulates markedly in gastric
juice and saliva. Nicotine also accumulates in breast milk (milk/plasma
ratio 2.9).  Nicotine crosses the placental barrier easily, and there is
evidence for the accumulation of nicotine in fetal serum and amnionic
fluid in slightly higher concentrations than in maternal serum.  The
plasma half-life of nicotine after intravenous infusion or cigarette
smoking averages about 2 h.  However, when half-life is determined using
the time course of urinary excretion of nicotine, which is more
sensitive in detecting lower levels of nicotine in the body, the
terminal half-life averages 11 h (Hukkanen et al, 2005).

Nicotine is extensively metabolized to a number of metabolites by the
liver.  Six primary metabolites of nicotine have been identified. 
Quantitatively, the most important metabolite of nicotine in most
mammalian species is the lactam derivative cotinine.  In humans, about
70 to 80% of nicotine is converted to cotinine.  Other pathways of
nicotine metabolism are oxidation of the pyrrolidine ring, methylation
of the pyridine nitrogen giving nicotine isomethonium ion (also called
N-methylnicotinium ion) and glucuronidation.  About 3 to 5% of nicotine
is converted to nicotine glucuronide and excreted in urine in humans. 
Oxidative N-demethylation is a minor pathway in the metabolism of
nicotine.  Conversion of nicotine to nornicotine in

humans has been demonstrated (Hukkanen et al, 2005).  

A new cytochrome P450 mediated metabolic pathway for nicotine metabolism
was recently reported by Hecht et al. (2000) and summarized by Hukkanen
et al, 2005. 

3.1.1.3	Sufficiency of studies/data  TC \l4 "3.1.1.3	Sufficiency of
studies/data 

Although there are no guideline studies, there are few studies suitable
for the characterization of risks from human exposure to nicotine.

  TC \l2 "3.4	Safety Factor for Infants and Children 

3.2	Hazard Identification and Toxicity Endpoint Selection  TC \l2 "3.5
Hazard Identification and Toxicity Endpoint Selection 

3.2.1	Acute Reference Dose (aRfD) and Chronic Reference Dose (cRfD)

No acute or chronic dietary (food and drinking water) exposure to
nicotine is expected based on the use patterns.  Therefore no acute or
chronic reference dose was selected for this assessment.

3.2.2	Incidental Oral (Short-Term)   TC \l3 "3.5.4	Incidental Oral
(Short- and Intermediate-Term) 

The endpoint selected for this risk assessment is derived from
subchronic oral rat toxicity study (Yuen et al.  1995) discussed below. 
In this study, histopathological changes seen at the lower dose (1.25
mg/kg/day expressed as nicotine (alkaloid) were not statistically
significant and this was considered to be a NOAEL.  The LOAEL was 2.5
mg/kg/day, expressed as nicotine (alkaloid).  A margin of exposure of
1000 is applied for assessment of this type to account for inter-species
extrapolation (10X), intra-species variability (10X), and database
uncertainty (10X).

3.2.3	Dermal Absorption  TC \l3 "3.5.5	Dermal Absorption 

There are no dermal absorption studies available with nicotine. 
Numerous studies indicate that nicotine is absorbed readily through the
skin.  Therefore, 100% dermal absorption factor is assumed.

When free nicotine was applied to the backs of cats weighing 1.8-4.5 kg
(treated area, 5-7 cm, in diameter was clipped with scissors and not
shaven) at 2- 10 mL per cat, nicotine was fatal within a few minutes. 
On the other hand when three cats were exposed to 10 mL of nicotine
sulfate they did not succumb suggesting that the sulfate is not readily
absorbed by the cat.  When one of the cats was treated with 10 mL of
free nicotine, it immediately succumbed (Faulkner, 1933).  In other
tests with cats receiving dermal doses of 200 mg of nicotine or nicotine
sulfate per cat, 81% of the cats receiving the nicotine base died within
21-195 minutes (Travell, 1960). Poisoning occurred very rapidly within
1-4 minutes including vomiting, salivation, swallowing difficulty, and
increased rate of respiration.  The remaining nicotine treated cats were
moribund after 4 hours of exposure.  Symptoms of the nicotine sulfate
treated cats were milder and none of the animals died.  This indicated
the nicotine base is absorbed completely from the skin, while nicotine
sulfate which is ionized is absorbed slightly.  In experiments with dogs
using nicotine dermal patches, nicotine was absorbed and produced
clinical signs in 15% of the treated dogs with plasma concentrations
reaching 43 ng/mL (Matsushima et al, 1995).  Nicotine’s high
solubility in both polar and non polar solvents (log Kw = 1.17) and its
low molecular weight (162.2 g/mole) make it theoretically efficient
penetrant (ZORIN et al, 1999).

3.2.4	Dermal Exposure (Short-, Intermediate- and Long-Term)  TC \l3
"3.5.6	Dermal Exposure (Short-, Intermediate- and Long-Term) 

No nicotine dermal toxicity studies were identified.  The Yuen et al,
1995 oral toxicity study, discussed above was selected as a basis for
estimating the dermal toxicity endpoint.  In this study nicotine
hydrogen tartrate was administered to pregnant and non-pregnant female
rats in the drinking water for 10 days at 54 or 108 mmole/L (8.76, 17.52
mg/L; equivalent to 1.25 and 2.5 mg/kg/day expressed as nicotine
(alkaloid) and assuming a rat consumes 50 mL of water per day) resulted
in mild fatty change, mild focal necrosis and mild dark cell change
(containing numerous prominent pore annuli in the nuclear membranes and
the mitochondria appeared decreased in size with a decrease in
mitochondrial granules and loss of aristae) in a dose proportional
manner.  Histopathological changes seen at the lower dose (1.25
mg/kg/day expressed as nicotine (alkaloid) were not statistically
significant and this was considered to be a NOAEL.  The LOAEL was 2.5
mg/kg/day, expressed as nicotine (alkaloid).  A margin of exposure of
1000 is applied for assessment of this type to account for inter-species
extrapolation (10X), intra-species variability (10X), and database
uncertainty (10X).

The selected endpoint for this risk assessment is supported by another
study where nicotine tartrate, administered orally to 10 New Zealand
white male rabbits (2.4 mg/kg/day) in drinking water for 25 weeks
produced in vivo morphologic effect on endothelial cells in the aortic
arch (Booyse et al, 1981).  Fasting serum levels of glucose,
triglycerides, total cholesterol, and LDL-cholesterol were significantly
(<0.001) elevated in nicotine-treated rabbits.  Endothelial cells from
nicotine-treated arched areas (Evans-blue-stained) showed extensive
changes such as increased cytoplasmic silver disposition, increased
formation of microvilli, and numerous focal areas of “ruffled”
endothelium (projections on cell surfaces).

The selected endpoint is applicable to all exposure durations, since
other studies have shown that nicotine is eliminated quickly from the
body and its serum levels or its metabolites do not accumulate with
continuous dosing suggesting that its toxicity is more dependent on the
dose than the exposure period.  

3.2.5	Inhalation Exposure (Short-, Intermediate- and Long-Term)

The endpoint selected for this risk assessment is derived from the
subchronic oral rat toxicity study (Yuen et al.  1995) discussed above. 
In this study, histopathological changes seen at the lower dose
(1.25mg/kg/day expressed as nicotine (alkaloid) were not statistically
significant and this was considered to be a NOAEL.  The LOAEL was 2.5
mg/kg/day, expressed as nicotine (alkaloid).  A margin of exposure of
1000 is applied for assessment of this type to account for inter-species
extrapolation (10X), intra-species variability (10X), and database
uncertainty (10X).  This selected endpoint is applicable to all exposure
durations, since other studies have shown that nicotine is eliminated
quickly from the body and its serum levels or its metabolites do not
accumulate with continuous dosing suggesting that its toxicity is more
dependent on the dose than the exposure period.

The Waldum et al 1996 inhalation study discussed earlier suggested a
NOAEL of 0.3 mg/kg/day based on lack of adverse effects. Although, this
study was well conducted, it used one dose only which limits its use of
selecting this NOAEL as a Point of Departure (POD) for toxicity. The
effects seen were minimal reduced weight gain during the long exposure
duration of the study.

3.2.6	Level of Concern for Margin of Exposure  TC \l3 "3.5.8	Level of
Concern for Margin of Exposure 

Table 3.2.6  Summary of Levels of Concern for Risk Assessment of
Nicotine.

Route	Short-Term

(1 - 30 Days)	Intermediate-Term

(1 - 6 Months)	Long-Term

(> 6 Months)

Occupational (Worker) Exposure

Dermal	1000	1000	NA

Inhalation	1000	1000	NA

Residential Exposure

Dermal	1000	NA	NA

Inhalation	1000	NA	NA

Incidental Oral	1000	NA	NA



3.2.7	Classification of Carcinogenic Potential  TC \l3 "3.5.10
Classification of Carcinogenic Potential 

According to the International Program on Chemical Safety (IPCS) review,
literature reports indicate that nicotine is neither an initiator nor a
promoter of tumors in mice.  There is inconclusive evidence to suggest
that cotinine, an oxidized metabolite of nicotine, may be carcinogenic
in the rat (  HYPERLINK
"http://www.inchem.org/documents/pims/chemical/nicotine.htm" 
http://www.inchem.org/documents/pims/chemical/nicotine.htm ).  Nicotine
has not been evaluated by the NCI National Toxicology Program, the
International Agency for Research on Cancer and the EPA Integrated Risk
Information System for its potential carcinogenicity. 

3.2.8	Summary of Toxicological Doses and Endpoints for Use in Human
Risk Assessments  TC \l3 "3.5.11	Summary of Toxicological Doses and
Endpoints for Use in Human Risk Assessments 

Table 3.2.8.  Summary of Toxicological Doses and Endpoints for Nicotine
for Use in Occupational and Residential Human Health Risk Assessments

Exposure/

Scenario	Point of Departure	Uncertainty Factors	Level of Concern for
Risk Assessment	Study and Toxicological Effects

Incidental Oral, short-term exposure (1-30 days)	NOAEL=1.25 mg nicotine
alkaloid /kg/day	UFA=10x

UFH=10x

UFDB=10x	Residential LOC for MOE = 1000	Yuen et al.  1995: oral study in
drinking water

LOAEL = 2.5 mg nicotine alkaloid/kg/day based on hepatotoxicity: mild
fatty change, mild focal necrosis and mild dark cell change (containing
numerous prominent pore annuli in the nuclear membranes and the
mitochondria appeared decreased in size with a decrease in mitochondrial
granules and loss of aristae) were seen in both pregnant and
non-pregnant rats, but more severe in the non-pregnant rats.

Dermal, Short-Term (1-30 days) and Intermediate-Term (1-6 months)
NOAEL=1.25 mg nicotine alkaloid /kg/day	UFA=10x

UFH=10x

UFDB=10x	Occupational and Residential LOC for MOE = 1000	Yuen et al. 
1995: oral study in drinking water



Inhalation, Short-Term (1-30 days) and Intermediate-Term (1-6 months)
NOAEL=1.25 mg nicotine alkaloid /kg/day	UFA=10x

UFH=10x

UFDB=10x	Occupational and Residential LOC for MOE = 1000	Yuen et al. 
1995: oral study in drinking water



Cancer (oral, dermal, inhalation)	Classification:  Nicotine has not been
evaluated by the NCI National Toxicology Program, the International
Agency for Research on Cancer and the EPA Integrated Risk Information
System for its potential carcinogenicity. It has not been tested in
rodents exempt for the Waldum et al, 1996 inhalation study which tested
one dose only.  According to the International Program on Chemical
Safety (IPCS) review, literature reports indicate that nicotine is
neither an initiator nor a promoter of tumors in mice. There is
inconclusive evidence to suggest that cotinine, an oxidized metabolite
of nicotine, may be carcinogenic in the rat.  

Point of Departure (POD) = A data point or an estimated point that is
derived from observed dose-response data and  used to mark the beginning
of extrapolation to determine risk associated with lower environmentally
relevant human exposures.  NOAEL = no observed adverse effect level. 
LOAEL = lowest observed adverse effect level.  UF = uncertainty factor. 
UFA = extrapolation from animal to human (interspecies).  UFH =
potential variation in sensitivity among members of the human population
(intraspecies).  UFL = use of a LOAEL to extrapolate a NOAEL.  UFS = use
of a short-term study for long-term risk assessment.  UFDB = to account
for the absence of key date (i.e., lack of a critical study).  MOE =
margin of exposure.  LOC = level of concern.  N/A = not applicable.

3.3	Endocrine disruption  TC \l2 "3.6	Endocrine disruption 	

EPA is required under the FFDCA, as amended by FQPA, to develop a
screening program to determine whether certain substances (including all
pesticide active and other ingredients) “may have an effect in humans
that is similar to an effect produced by a naturally occurring estrogen,
or other such endocrine effects as the Administrator may designate.” 
Following recommendations of its Endocrine Disruptor and Testing
Advisory Committee (EDSTAC), EPA determined that there was a scientific
basis for including, as part of the program, the androgen and thyroid
hormone systems, in addition to the estrogen hormone system.  EPA also
adopted EDSTAC’s recommendation that the Program include evaluations
of potential effects in wildlife.  For pesticide chemicals, EPA will use
FIFRA and, to the extent that effects in wildlife may help determine
whether a substance may have an effect in humans, FFDCA authority to
require the wildlife evaluations.  As the science develops and resources
allow, screening of additional hormone systems may be added to the
Endocrine Disruptor Screening Program (EDSP).

When additional appropriate screening and/or testing protocols being
considered under the Agency’s EDSP have been developed, nicotine may
be subjected to further screening and/or testing to better characterize
effects related to endocrine disruption.

There is experimental evidence suggesting endocrine effects by nicotine.
 A few of these studies are discussed below.  Other studies are listed
in the toxicity profile.

Nicotine inhibited ovulation, estradiol production, and fertilization
both in vivo and in vitro in rat models of ovulation when pregnant
mare’s serum gonadotropin (PMSG) - primed and human chorionic
gonadotropin (hCG) - triggered rat ovaries were exposed to nicotine (ip
injection of 6.25 ng/g animal weight) (Blackburn et al, 1994).  A dose
dependent reduction in oocytes within the fallopian tube was noted in
nicotine treated rats (p<0.001).  On the other hand cotinine, the
primary nicotine metabolite, did not affect ovulation, estradiol
production or fertilization in those tests.

oduced alterations in catecholamines (CA) release, tyrosine hydroxylase
(TH), dopamine β-hydroxylase (DBH), and the ability of isolated storage
vesicles to incorporate 3H-epinephrine in the adrenal glands and these
alterations persisted when nicotine administration was discontinued
(Slotkin and Seidler, 1975).  

Prenatal exposure to nicotine can interfere with the development of the
male gonadal axis and with the organization of sexually dimorphic
behavior (Lichtensteiger and Schlumpf 1985).  Time-pregnant Sprague
Dawley rats were implanted on gestational day (GD) 12 with an osmotic
minipump containing either nicotine tartrate (delivered at a rate of 25
ug/100 g x hr), tartaric acid or saline. Others were sham-operated on GD
12 or left untreated.  Male fetuses of all control groups displayed the
characteristic rise in plasma testosterone at GD 18 (as compared to GD
17 and 19); this was abolished by nicotine.  Adult offspring of
untreated or tartaric acid-treated dams exhibited a marked sexual
dimorphism in their preference for saccharin-containing drinking water
at 0.06-0.25%.  No such sex difference was seen in offspring of
nicotine-treated rats.  In controls, the sexes differed with respect to
the proportion of rats with high saccharin preference.  In the group of
males prenatally exposed to nicotine, the proportion of animals with
high preference increased to the female level. 

4.0	Public Health and Pesticide Epidemiology Data  TC \l1 "4.0	Public
Health and Pesticide Epidemiology Data 

4.1	Incident Reports  TC \l2 "4.1	Incident Reports 

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41)

See:  Review of Nicotine Incident Reports, J. Blondell and M. Spann,
D276938, 08/10/2001.

The following databases were searched by HED for poisoning incident data
on the active ingredients nicotine (PC Code 056702) and nicotine sulfate
(PC Code 056703):  (1) OPP Incident Data System (IDS); (2) Poison
Control Centers; (3) California Department of Pesticide Regulation; and
(4) National Pesticide Telecommunications Network (NPTN).

Three nicotine related incident reports were identified in IDS.  A
pesticide incident occurred in 1994 (Incident #2796-52), when an
individual, who was not wearing gloves, reported nausea and convulsions
three hours after handling nicotine shreds.  The individual was treated
overnight in the hospital.  A second pesticide incident occurred in 1998
(Incident #7981-1), when the owner of a home misused the smoke generator
product to control roaches in the home.  A woman and her children were
exposed to the product and one of the children (a 10-year old male)
reported coughing.  A third pesticide incident occurred in 1999
(Incident #9175-1), when a pest control operator illegally used a
nicotine-based bug bomb in a small apartment.  A woman and her four
children reported chest tightness, nausea, and coughing.

Results from the years 1993 through 1998 were acquired for 45 exposures
to nicotine reported to Poison Control Centers.  Cases involving
exposures to multiple products were excluded.  Only 12 cases were
reported among children under six years of age and no cases among older
children and adults exposed at their workplace.  This was too few cases
to warrant a detailed analysis.  None of these cases reported a serious
or even a moderate outcome.  There were 31 non-occupational exposure
cases among older children and adults.  Of these cases, 16 had outcomes
determined of which none were moderate or major cases.  

Detailed descriptions of 20 cases submitted to the California Pesticide
Illness Surveillance Program (1982-1999) were reviewed.  In 12 of these
cases, nicotine was used alone or was judged to be responsible for the
health effects.  Only cases with a definite, probable or possible
relationship were reviewed.  None of the individuals required
hospitalization and two of the twelve individuals were reported to have
taken 1 or 2 days off from work.  Work activities included applicator,
drift, field residue, and routine or unknown occupational activity. 
Drift was associated with more exposures than any other work activity. 
Illnesses included symptoms of difficulty breathing, throat tightness,
headache, nausea, and eye irritation.  Nicotine ranked 81st as a cause
of systemic poisoning in California based on data for 1982 through 1994.

On the list of top 200 chemicals for which NPTN received calls from
1984-1991 inclusively, nicotine was ranked 178th with 14 incidents in
humans reported and 1 incident in animals.

4.1.2	Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465)

The OPP Incident Data System (IDS) was searched by HED (email
communication with N. Spurling, OPP/ITRMD/ISB, 07/25/2007) for poisoning
incident data on the active end-use products EPA Reg. No. 4-465
(Bonide® Rabbit & Dog Chaser) and EPA Reg. No. 779-29 (according to
OPPIN, transferred from Faesy & Bestoff to Bonide® Rabbit & Dog Chaser
08/16/2001).  No incidents were reported in IDS for these end-use
products.

5.0	Dietary Exposure/Risk Characterization  TC \l1 "5.0	Dietary
Exposure/Risk Characterization 

A dietary (food + water) risk assessment was not conducted.  Neither of
the two end-use products which are being supported under reregistration
are registered for use on food or feed and HED and EFED have agreed that
conducting a drinking water risk assessment is not appropriate.

6.0	Residential (Non-Occupational) Exposure/Risk Characterization  TC
\l1 "6.0	Residential (Non-Occupational) Exposure/Risk Characterization 

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41)

See:  Occupational and Residential Exposure/Risk Assessment for the
Nicotine and derivatives Reregistration Eligibility Decision (RED)
Addressing the Fulex Nicotine Greenhouse Fumigator, B. Cropp-Kohlligian,
D341249, 08/xx/2007.

NOTE: 40 CFR 157.21(e) defines "residential use" as follows: 
"Residential use means use of a pesticide or device:  (1) directly on
humans or pets; (2) In, on, or around any structure, vehicle, article,
surface, or area associated with the household, including but not
limited to areas such as non-agricultural outbuildings, non-commercial
greenhouses, pleasure boats and recreational vehicles; or (3) in or
around any preschool or daycare facility."

No residential (non-occupational) handler exposure scenarios have been
identified for the Fulex Nicotine [Greenhouse] Fumigator use; however,
residential (non-occupational) postapplication exposure scenarios via
inhalation and dermal routes have been identified.  These exposures are
expected to be short-term in duration.

The Fulex Nicotine Fumigator (EPA Reg. No. 1327-41) is a restricted use
product for use by and under direct supervision of a certified
applicator (40 CFR §152.175); however, the current label does not
prohibit application to privately owned greenhouses in residential
settings by certified commercial applicators where it is possible for
residents to be exposed following application and according to
information provided by the registrant (Use Closure for Nicotine RED, J.
Bloom, 06/18/2007), the product is used in retail greenhouses where it
is considered possible for non-occupational members of the general
public to be exposed following application.  These non-commercial
postapplication exposures are addressed by the occupational
postapplication risk assessment for this end-use product.  (See Section
9.1.3)

6.2	Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465)

See:  Occupational and Residential Exposure/Risk Assessment for the
Nicotine and derivatives Reregistration Eligibility Decision (RED)
Addressing the Bonide Dog and Rabbit Repellant, B. Cropp-Kohlligian,
D341897, 08/xx/2007.

Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) is a ready-to-use
animal repellant for use in outdoor residential settings containing
nicotine (as a naturally occurring component of tobacco dust).  It is
intended for use by homeowners as a repellent via a barrier/perimeter
treatment to prevent eastern cottontail rabbits (Sylvilagus floridanus)
from eating and defecating on ornamental plants and domestic dogs (Canis
l. familiaris) from defecating on ornamentals, including lawns.  It may
also be used around the perimeter of vegetable gardens.

Based on use patterns, residential (homeowner) handlers may be exposed
to nicotine (as a naturally occurring component of tobacco dust) while
applying the product by hand.  No postapplication scenarios have been
identified for homeowners with the exception of potential concern for
availability to homeowners in packages which are not child resistant and
could be accessible to children prior to application or potential for
children to come in contact with the product postapplication; hence, an
episodic ingestion is also being assessed.

The registrant has not provided any chemical-specific data to assess the
exposure for this risk assessment.

6.2.1	Residential (Homeowner) Handler Risks TC \l2 "6.1	Residential
(Homeowner) Handler (Risks  

It has been determined that there is a potential for dermal and
inhalation exposure in residential settings during the application of
Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465; label date
12/28/2006).  Based on the currently registered Bonide® Rabbit & Dog
Chaser end-use product label (EPA Reg. No. 4-465; label date 12/28/2006)
and information provided by the registrant (Use Closure for Nicotine
RED, J. Bloom, 06/18/2007), non-dietary exposures to this product are
expected to be short-term in duration.

Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465; label date 12/28/2006)
contains 0.35% nicotine (as a naturally occurring component of tobacco
dust).  The product is sprinkled directly from the package in 2 or more
inch-wide bands around areas to be treated.  A 3-pound package of
product will produce a band of product 1 inch wide and 85 feet long. 
The reviewer estimates that a 3-pound package of product will provide a
barrier/perimeter around a 144 ft2 area at most.  

Residential MOEs equal to or greater than 1000 are not of concern to
HED.  The short-term combined dermal and inhalation MOE for residential
handlers who apply one three-pound package of the ready-to-use Bonide®
Rabbit & Dog Chaser (EPA Reg. No. 4-465) was 1875 and therefore not of
concern to HED.  However, application of two or more three-pound
packages would be of concern, since this is estimated to result in a
combined dermal and inhalation MOE less than 1000.  The concerns are
primarily for dermal exposure/risk and HED notes that the
inhalation/dermal risk estimates may be considered conservative since,
in the absence of chemical-specific data, they are based on theoretical
data/calculations and default assumptions, including 100% dermal
absorption, for this product containing nicotine as a naturally
occurring component of tobacco.  See Table 6.2.1.

Data and Assumptions for Handler Exposure Scenarios

The quantitative exposure/risk assessment developed for residential
handlers (applicators only) is based on the following exposure scenario
in the PHED Surrogate Exposure Guide, as a surrogate for application of
this product:

Applicator:  Standard Operating Procedures (SOPs) for Residential
Exposure Assessments (PHED Version 1.1)  Wettable Powder, Open Mixing
and Loading

Assumptions and Factors:

Based on the currently registered Bonide® Rabbit & Dog Chaser end-use
product label (EPA Reg. No. 4-465; label date 12/28/2006) and
information provided by the registrant (Use Closure for Nicotine RED, J.
Bloom, 06/18/2007), no mixing/loading methods are necessary for this
ready-to-use end-use product.  Therefore, a mixing/loading exposure
assessment was not performed.

HED used the Standard Operating Procedures (SOPs) for Residential
Exposure Assessments (PHED Version 1.1)  Wettable Powder, Open Mixing
and Loading to assess residential handler exposure.  This scenario which
considers a wettable powder formulation is deemed best suited to
represent a residential handler applying a dust formulation by hand
while wearing short pants, a short-sleeved shirt, and no gloves.

≤144 ft2 area.

Body Weight:  An average adult body weight of 70 kg was used for
short-term calculations, since the dermal and inhalation endpoints were
based on effects that were not sex-specific.

Equations and Calculations:

Average Daily Dose (ADD):  Daily dose (inhalation or dermal) was
calculated by normalizing the daily dermal or inhalation exposure value
by body weight and accounting for dermal or inhalation absorption. 
Dermal and inhalation absorption factors of 100% were assumed.  Daily
dose was calculated using the following formula:

Where,

	ADD		= 	Average daily dose absorbed dose received from exposure to a
pesticide in a given scenario (mg pesticide active ingredient/kg body
weight/day)

	Daily Exposure	= 	Amount (mg ai/day) deposited on the surface of the
skin that is available for dermal absorption or amount inhaled that is
available for inhalation absorption;

	Absorption Factor= 	A measure of the amount of chemical that crosses a
biological boundary such as the skin or lungs 

	Body Weight = 		Body weight determined to represent the population of
interest in a risk assessment.

Margin of Exposure (MOE): the calculations of daily dermal dose and
daily inhalation dose received by handlers were then compared to the
appropriate endpoint (i.e., NOAEL) to assess the total risk to handlers
for each exposure route within the scenarios. All MOE values were
calculated separately for dermal and inhalation exposure levels using
the following formula:

	MOE = 	______NOAEL (mg/kg/day)____      

			ADD (mg/kg/day)

Where:

	MOE 		= Margin of exposure value used by HED to represent risk or how
close a chemical exposure is to being a concern (unitless)

	ADD 		= Average daily dose is absorbed dose received from exposure to
pesticide

	NOAEL 	= Dose level in a toxicity study, where no observed adverse
effects occurred in the study

Total MOE:  When the dermal and inhalation endpoints, effects and routes
of exposure are the same the doses may be added together to determine a
total dose and MOE using the following formula:

	TOTAL MOE				NOAEL (mg/kg/day) 

				Dermal Dose (mg/kg/day) + Inhalation Dose (mg/kg/day)

Table 6.2.1.  Residential Handler (Applicator Only) Short-term Exposure
and Risk for Nicotine (as a naturally occurring component of tobacco
dust).

Scenario	Use Site	Dermal

Unit

Exposure (mg/lb) 1	Inhalation

Unit

Exposure (mg/lb) 1	Application

Rate 2

(lb ai/site/day)	Area

Treated	Dermal

Dose 3 

(mg/kg/day)	Dermal

MOE 5	Inhalation

Dose 4 (mg/kg/day)	Inhalation

MOE 6	Total

MOE 7

Applicator

Nicotine (0.35% a.i) dust

Reg.# 4-465	In and around homes as a barrier/perimeter treatment to
repel rabbits and dogs	4.4	0.0434	0.0105

(1 3-pound package)	42.5 linear feet; could provide a barrier/perimeter
around a 144 ft2 area (or 0.0033 acres)

42.5 ft/2π = 6.77 ft

area = π(6.77 ft)2 = 144 ft2	6.6E-4	1894	6.5E-6	1.9E5	1875





0.0210

(2 3-pound packages)	288 ft2 area (or 0.0066 acres)	1.32E-3	947	1.3E-5
9.6E4	938













1  Standard Operating Procedures (SOPs) for Residential Exposure
Assessments (PHED Version 1.1)  Wettable Powder, Open Mixing and
Loading; Dermal unit exposure (assumes short pants, short sleeved shirt
and no gloves) = 4.4 mg/lb; Inhalation Unit Exposure = 0.0434 mg/lb.

2  Application Rate based on currently registered Bonide Rabbit & Dog
Chaser end-use product label (EPA Reg. No. 4-465) and assuming 1, 5, or
10 3-pound packages of product are used per day.  One 3-pound package of
product contains 0.35% nicotine (as the alkaloid) or 0.0105 lb nicotine
(as the alkaloid).

3  Short-term Dermal Dose  (mg/kg/day)  = [ Rate (lb ai/A/day) x  UE (mg
/lb ai ) / BW (70 kg)

4  Short-term  Inhalation Dose  (mg/kg/day)  = [ Rate (lb ai/A/day) x UE
(mg /lb ai ) / BW (70 kg)

5  Short-term Dermal MOE = [Dermal NOAEL (1.25 mg/kg/day)]/ Dermal Dose
(mg/kg/day)

6  Short-term Inhalation MOE = [Inhalation NOAEL (1.25 mg/kg/day)] /
Inhalation Dose (mg/kg/day) 

7  Total MOE = NOAEL (1.25 mg/kg/day) / Dermal Dose (mg/kg/day) +
Inhalation Dose (mg/kg/day)

	Note:  Dermal and Inhalation LOC is 1000.

6.2.2	Residential (Homeowner) Postapplication Exposure TC \l2 "6.2.
Residential  Postapplication Exposure 

Because the use pattern results in applications to areas not frequented
by children or in areas where maintenance would result in significant
exposure, dermal and incidental oral assessments were not conducted. 
However, episodic ingestion of the material is considered reasonable due
to packages which are not child resistant and which could be accessible
to children prior to application or due to the potential for children to
come in contact with the product postapplication.

Residential MOEs equal to or greater than 1000 are not of concern to
HED.  The short-term oral MOE for episodic ingestion of the ready-to-use
Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) containing nicotine
(as a naturally occurring component of tobacco dust) was 1 and therefore
is of concern to HED.

On average, the bulk density of the ready-to-use Bonide® Rabbit & Dog
Chaser (EPA Reg. No. 4-465) is 35 lb/ft3.  This is equivalent to
approximately 8.3 grams of product per tablespoon or 2.8 grams of
product per teaspoon calculated as follows:

	35 lb/ft3 x 454 g/lb x 1ft3/1915 tablespoons = 8.3 g/tablespoon or 2.8
g/teaspoon

Ingestion of more than 0.002 teaspoons of product by a toddler (15 kg)
exceeds the LOC for the MOE (1000); however, this type of ingestion is
considered an episodic event and not a routine behavior.  Because HED
does not believe that this would occur on a regular basis, our concern
for human health is related to acute poisoning rather than short -term
residue exposure.  See Table 6.2.2.

6.2.2.1		Data and Assumptions for Episodic Oral Ingestion

This scenario was assessed using the residential SOP 2.3.1,
Postapplication Potential Among Toddlers from Ingestion of Pesticide
Granules from Treated Areas.  This SOP provides a standard method for
estimating postapplication doses among toddlers from incidental
ingestion of pesticide granules.

Assumptions and Factors:

Ingestion rate for dry pesticide formulation (granules) is 5 gram/day. 
This is based on poison specialist estimate used in the Rodenticide
Cluster RED (1998) which assumed that a one year old child weighing 10
kg could consume approximately 5 grams of a granular formulation in one
swallow.  Note:  This estimated ingestion rate (5 grams of product/day)
is equivalent to approximately 1% (by weight) of a 1-pound package of
Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) which may be applied
as multiple one inch bands of product around the perimeter of lawns and
gardens.

Average weight of toddler is 15 kg

Equations and Calculations:

Potential Daily Dose (PDD):  Potential daily dose (oral) was calculated
by normalizing the amount of chemical that could be ingested by body
weight.  Daily dose was calculated using the following formula:

	PDD	=	IgR x F x CF1 ( BW

	Where,

	PDD	=	Potential Daily Dose

	IgR	= 	ingestion rate of dry pesticide formulation (grams/day)

	F	=	fraction of ai in dry formulation (%)

	CF1	=	weight unit conversion factor to convert g units to mg for daily
exposure (1,000 mg/g)

	BW	=	Body Weight (15kg)

Margin of Exposure (MOE):  The calculations of potential daily dose were
then compared to the appropriate endpoint (i.e., NOAEL) to assess the
risk using the following equation:

	Short-term Oral MOE =NOAEL (1.25 mg/kg/day) ( Potential Daily Dose
(PDD)

Table 6.2.2.  Postapplication Exposure and Risk  for Episodic Ingestion

Scenario	IgR (g/day)	F 	CF1 (mg/g)	Dose 1 (mg/kg/day)	MOE 2

Episodic Ingestion 	5	0.35%	1000	1.17	1

	0.005

(0.002 tsp)

	0.00117	1000

1  Dose = IgR x F x CF1 ( BW

	Where,

	IgR	=	ingestion rate of dry pesticide formulation (grams/day)

	F	=	fraction of ai in dry formulation (%)

	CF1	=	weight unit conversion factor to convert g units to mg for daily
exposure (mg/g)

	BW	=	Body Weight (15kg)

2  MOE = NOAEL (1.25 mg/kg/day)/Dose 

Note:  LOC is 1000.

6.3	Other (Spray Drift, etc.) TC \l2 "6.3	Other (Spray Drift, etc.) 

Spray drift is always a potential source of exposure to residents nearby
to spraying operations.  This is particularly the case with aerial
application, but, to a lesser extent, could also be a potential source
of exposure from the ground application method employed for nicotine. 
The Agency has been working with the Spray Drift Task Force, EPA
Regional Offices and State Lead Agencies for pesticide regulation and
other parties to develop the best spray drift management practices.  On
a chemical by chemical basis, the Agency is now requiring interim
mitigation measures for aerial applications that must be placed on
product labels/labeling.  The Agency has completed its evaluation of the
new database submitted by the Spray Drift Task Force, a membership of
U.S. pesticide registrants, and is developing a policy on how to
appropriately apply the data and the AgDRIFT computer model to its risk
assessments for pesticides applied by air, orchard airblast and ground
hydraulic methods.  After the policy is in place, the Agency may impose
further refinements in spray drift management practices to reduce
off-target drift with specific products with significant risks
associated with drift.

7.0	Aggregate Risk Assessments and Risk Characterization  TC \l1 "7.0
Aggregate Risk Assessments and Risk Characterization 

An aggregate (food + water + residential exposure) risk assessment was
not conducted.  Neither of the two end-use products which are being
supported under reregistration are registered for use on food or feed
and HED and EFED have agreed that conducting a drinking water risk
assessment is not appropriate.

8.0	Cumulative Risk Characterization/Assessment  TC \l1 "8.0	Cumulative
Risk Characterization/Assessment 

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 nicotine and any other
substances and nicotine 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 nicotine has a common mechanism of
toxicity with other substances. For information regarding EPA’s
efforts to determine which chemicals have a common mechanism of toxicity
and to evaluate the cumulative effects of such chemicals, see the policy
statements released by EPA’s Office of Pesticide Programs concerning
common mechanism determinations and procedures for cumulating effects
from substances found to have a common mechanism on EPA’s website at  
HYPERLINK http://www.epa.gov/pesticides/cumulative/.
http://www.epa.gov/pesticides/cumulative/. 

9.0	Occupational Exposure/Risk Pathway  TC \l1 "9.0	Occupational
Exposure/Risk Pathway 

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41)

See:  Occupational and Residential Exposure/Risk Assessment for the
Nicotine and derivatives Reregistration Eligibility Decision (RED)
Addressing the Fulex Nicotine Greenhouse Fumigator, B. Cropp-Kohlligian,
D341249, 08/xx/2007.

No nature of the residue data are available for this unique application
method which alters nicotine's physical state and has the potential to
form degradates.  During fumigation, nicotine may be present as a vapor
and possibly bound or adsorbed to particulate matter.

A nicotine pyrolysis study has not been submitted but according to the
1981 Report of the Surgeon General entitled, “The Health Consequences
of Smoking;  The Changing Cigarette”, during tobacco curing,
fermentation, and burning, nicotine gives rise to N-nitrosonornicotine
(NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridil)-1-butanone (NNK). 
See structures below.

NNN

 	NNK

 



Given the lack of sophistication of the ignition device (sparklers),
without nature of the residue data, it is difficult to determine if
nicotine is only vaporized (and to some extent adsorbed to particulate
matter) or decomposed as a result of the application method.  If
actually burned, formation of NNN and NNK cannot be excluded without
supporting data.  

Exposure estimates in this risk assessment are based on maximum
theoretical air concentration data/calculations and default assumptions,
all potential degradates of nicotine from the Fulex Nicotine
[Greenhouse] Fumigator use have been included in the exposure estimates
of this risk assessment and the de facto hazard assumption is equivalent
toxicity with nicotine.

9.1.1	Regulatory Standards Fulex Nicotine Fumigator (EPA Reg. No.
1327-41)  TC \l2 "9.1.1	Regulatory Standards 

The American Conference and Governmental Industrial Hygienists (ACGIH)
have established a threshold limit value (TLV) as an 8-hour
time-weighted average (TWA) of 0.5 mg/m3 for nicotine.(1)  This value is
intended to minimize the potential for gastrointestinal disturbances,
cardiovascular effects, and adverse central nervous system effects.  A
skin notation is based on the well-documented percutaneous absorption of
nicotine and resultant systemic toxicity from handling uncured tobacco
leaves(2) and in the manufacture(3) and application(4) of nicotine
insecticides.  Sufficient data were not available to recommend a
Senisitizer (SEN) notation or carcinogenicity notation or a TLV as a
short-term exposure limit (STEL).

The 8-hour intake of nicotine received by inhalation was calculated as
0.07 mg/kg/day; (5) 

based on the metabolism and controlled dosing of human volunteers(5,6) 
and on the NOAEL level of 1.14 mg/kg/day found in chronic studies in
rodents.(7)  In a chronic rat study (Wenzel and Richards), rats given
1.14 or 4.56 mg nicotine/kg/day for 34 weeks in their drinking water
produced subtle biochemical changes (increased isocitric dehydrogenase
and β-glucuronidase activities) in myocardiums only at the high dose.

The Occupational Safety and Health Agency (OSHA) has established a
permissible exposure limit (PEL) as an 8-hour time-weighted average
(TWA) of 0.5 mg/m3 for nicotine(1)  consistent with the ACGIH TLV and
The National Institute for Occupational Safety and Health (NIOSH)
concurs with the OSHA PEL.(1) 

NIOSH has established a value of 5 mg/m3 for nicotine as a level that is
immediately dangerous to life or health (IDLH).(1)  No inhalation
toxicity data were identified on which to base and IDLH for nicotine. 
Therefore, the IDLH is based on acute oral toxicity data in humans(8) 
and animals.(8,9)

________________________________________________________________________
___________________

References

1.	American Conference of Governmental Industrial Hygienists.  (2004)
1995-1996 Threshold Limit Values (TLVs) for Chemical Substances and
Physical Agents and Biological Exposure Indices (BEIs).

2.	Gehlback, S.H.; Williams, W.A.; Perry, L.D.; et al.:  Green-Tobacco
Sickness; An Illness of Tobacco Harvesters.  JAMA 229:1880-1883 (1974).

3.	Lockhart, L.P.:  Nicotine Poisoning.  Br. Med. J. 1:246-274 (1933).

4.	Haines, K.A.; Davis, L.G.:  Protection of Workers Applying Nicotine
alkaloid as a Concentrated Mist Spray. J. Econ. Entomol. 4:513 (1948).

5	Hayes, Jr., W.J.:  Pesticides Studied in Man, pp. 86-91.  Williams &
Wilkins, Baltimore (1982).

6	Henningfield, J.E.; Miyasato, K.; Jesinski, D.R.:  Abuse Liability and
Pharmacodynamic Characteristics of Intravenous and Inhaled Nicotine.  J.
Pharmacol. Exp. Ther.234:1-12 (1985).

7	Wenzel, D.G.; Richards, M.H.:  Effects of Chronic Nicotine, Acute,
Hypoxia and Their Interactions on Myocardial Enzymes.  Toxicol. Appl.
Pharmacol. 16:656-667 (1970).

8	Lazutka, F.A.; Vasilyauskene, A.D.; Gefen, S.C.:  Toxicological
Evaluation of the Insecticide nicotine sulfate.  Gig. Sanit. 34(5):3033
(1969).

9	Franke, F.E.; Thomas, J.E.:  A note on the minimal fatal dose of
nicotine for unanesthetized dogs.  Proc. Soc. Exp. Biol. Med.
29:1177-1179 (1932).

9.1.2	Short-/Intermediate-/Long-Term (Non-Cancer) Handler Risk  TC \l2
"9.2	Short-/Intermediate-/Long-Term (Non-Cancer) Handler Risk 

Since short-term and intermediate-term risk estimates will be based on
the same endpoint (1.25 mg/kg/day) and level of concern (1000), only
short-term risks have been calculated.  A summary of the short-term
(non-cancer) inhalation risks for handlers is included in Table 9.1.2.  

Inhalation MOEs greater than 1000 are not of concern.  The short-term
inhalation risk estimate resulted in a MOE less than 1000 for all
individuals performing application activities except those using a
self-contained breathing apparatus (SCBA) which is estimated to provide
99.99% protection (PF10,000).  Moreover, inhalation risks of concern
(i.e., MOEs less than 1000) were identified for individuals using half-
and full-face respirators with chemical and particulate filter
cartridges for exposure periods of 1 and >3 minutes, respectively.  This
risk estimate may be considered conservative since it is based on
theoretical air concentration data/calculations and default assumptions.

Fulex Nicotine Fumigator (EPA Registration No. 1327-41) contains 13.4%
nicotine as a smoke fumigator for use as an insecticide in greenhouses
for the prevention and/or treatment of aphids and most thrips.  The
application rate is one 12 ounce canister per 20,000 ft3 or one 6 ounce
canister per 10,000 ft3.  Each ready-to-use 12 ounce canister contains
0.10 lbs ai.

The application method is as follows:

close all vents;

shake each canister;

distribute the canisters spaced equidistant in the center aisle of an
ordinary greenhouse;

remove top covers;

starting with canister farthest from exit, light the sparkler at the
bottom near the handle;

insert lighted sparkler deeply into the contents of the canister; 

proceed to next canister, until all canisters are ignited.

Based on the number of applications indicated on the currently
registered end-use product label (EPA Reg. No. 1327-41; dated
08/10/2005) and information provided by the registrant (Use Closure for
Nicotine RED, J. Bloom, 06/18/2007), handler exposures are expected to
be short-term and intermediate-term in duration.

9.1.2.1		Data and Assumptions for Handler Exposure Scenarios

The quantitative exposure/risk assessment developed for commercial
handlers is based on the following exposure scenario:

Applicator: Smoke Generator Canister

The assumptions, parameters and factors used for the exposure
calculations include:

Based on the currently registered end-use product label (EPA Reg. No.
1327-41; dated 08/10/2005) and information provided by the registrant
(Use Closure for Nicotine RED, J. Bloom, 06/18/2007), no mixing/loading
methods are necessary for these ready-to-use end-use products.
Therefore, a mixing/loading exposure assessment was not performed.

The application rate for this product was based on the currently
registered end-use product label (EPA Reg. No. 1327-41; dated
08/10/2005).  The application rate was adjusted to pounds of active
ingredient per 12 ounce canister. The application rate for a 12 ounce
canister is 0.10 lb ai per 20,000 cubic feet, using the following
equation:  

(12 ounces/canister)(1canister/20,000 ft3)(1 lb/16 ounces)(0.134 ai) =
0.10 lb ai/20,000 ft3

The air concentration was calculated at the maximum application rate.
The application rate was adjusted to an air concentration of 80.1 mg
ai/m3 (milligrams of active ingredient per cubic meters). The air
concentration was calculated using the following equation:

(0.10 lb ai/20,000 ft3)(35.3 ft3/m3)(454,000 mg/lb) = 80.1 mg ai/m3

The same nicotine  air concentration is assumed to be encountered by
handlers when they apply/light smoke canisters and when they enter the
treated greenhouse to open vents and dispose of canisters.  These two
activities are considered as a single exposure scenario.

A single handler could treat multiple greenhouses per day.  The exposure
period for handlers would depend on the number of canisters used to
treat an individual greenhouse and the number of individual greenhouses
treated per day.  Therefore, handler exposure periods per day are
estimated at 30 minutes or less for smaller greenhouse facilities and up
to an hour for larger facilities.

For handlers (i.e., those who ignite the canisters as well as those who
enter before the WPS ventilation criteria are met to open vents and
collect spent canisters), dermal exposures are assumed to be negligible
relative to the exposures and risks from inhalation.  This assumption is
based on the use pattern where potential dermal exposure is limited to
possible contact with nicotine :  (1) while opening the canisters and
inserting the sparkler, (2) an accidental spill during lighting of a
canister or retrieval of an unlit canister, and (3) possible contact
with residue on the outside of a spent canister.  These dermal exposures
are expected to be relatively infrequent and of relatively short
duration in comparison with the estimated inhalation exposure times. 
Therefore, a dermal exposure assessment for handlers was not performed.

Handler exposure assessments for inhalation routes of exposure were
completed by HED using no respirator (baseline), 10PF respirator, 50 PF
respirator, and 10,000PF respirator.  While the currently registered
end-use product label specifies that handlers must use a respirator with
either an organic vapor-removing cartridge with a prefilter approved for
pesticides (MSHA/NIOSH approval number prefix TC-23C) or a canister
approved for pesticides (MSHA/NIOSH approval number prefix TC-14G), the
label does not specify if the respirator is a half-face or full-face
mask.

Body Weight:  An average adult body weight of 70 kg was used since the
dermal and inhalation endpoints were based on effects that were not
sex-specific.

The maximum air concentration levels potentially encountered by handlers
assumes that during fumigation all of the active ingredient in the smoke
canister enters the greenhouse air at the maximum label application
rate.

Air Concentration Equations: 

Inhalation Dose (mg/kg/day) = (Ca  x  BR  x  ET)/BW

Where,

Ca	= air concentration at time of application (time = 0)

BR	= adult breathing rate (1 m3/hr)

ET	= exposure time; assumed to comprise the time to ignite canisters and
re-enter after the fumigation but before the WPS ventilation criteria
are met to open vents and dispose of canisters

	(smaller greenhouse 30 min/day = 0.50 hr/day)

	(larger greenhouse 60 min/day = 1.0 hr/day)

BW 	= body weight (70 kg)

Margin of Exposure (MOE) Equation:

	MOE = Inhalation NOAEL (mg/kg/day)/ Inhalation Dose (mg/kg/day)

Where,

Short-term and Intermediate-term Inhalation NOAEL = 1.25 mg/kg/day

Note:  Inhalation LOC is 1000.

Table 9.1.2.  Short-Term Occupational (Non-cancer) Handler Inhalation
Exposure and Risk for Nicotine Used in Greenhouses. 1

Mitigation Level	Ca

(mg ai/m3)	BR

(m3/hr)	ET

(hr/day)	BW

(kg)	Inhalation Dose 1 (mg/kg/day)	Inhalation MOE 2

Baseline

No Respirator	80.1	1	0.5	70	0.57	2



	1.0

1.14	1

PF10 Respirator

Half-face organic-vapor-removing respirator providing 90% protection
8.01	1	0.5	70	0.057	22



	1.0

0.114	11

	8.01	1	0.011

(<1 minutes)	70	0.00125	1000

PF50 Full-face organic-vapor-removing respirator providing 98%
protection	1.6	1	0.5	70	0.01	125



	1.0

0.02	63

	1.6	1	0.055

(~3 minutes)	70	0.00125	1000

PF10,000 Self-contained breathing apparatus (SCBA) providing 99.99%
protection	0.008	1	1.0	70	0.0001	12500

Note:  Shaded areas provide estimates of exposure time with PPE at which
the estimated Inhalation MOE is at or above the LOC (1000).

1  Inhalation Dose (mg/kg/day) = [Ca (mg ai/m3) x BR (m3/hr) x ET
(hr/day)] / BW (70 kg)

Where,

Ca	= air concentration at time of application (time = 0)

BR	= adult breathing rate (1 m3/hr)

ET	= exposure time; assumed to comprise the time to ignite canisters and
re-enter after the fumigation but before the WPS ventilation criteria
are met to open vents and dispose of canisters.

(smaller greenhouse 30 min/day = 0.50 hr/day)

(larger greenhouse 60 min/day = 1.0 hr/day)

BW = body weight (70 kg)

	

2  MOE = Inhalation NOAEL (mg/kg/day) / Inhalation Dose (mg/kg/day) 

Where,

Short-and Intermediate-term Inhalation NOAEL = 1.25 mg/kg/day

Note:  Inhalation LOC is 1000.

9.1.3	Short-/Intermediate-/Long-Term (Non-Cancer) Postapplication Risk 
TC \l2 "9.3	Short-/Intermediate-/Long-Term (Non-Cancer) Postapplication
Risk 

A summary of the dermal exposure and risk during postapplication
activities is included in Table 9.1.3.1.  Based on the currently
registered end-use product label (EPA Reg. No. 1327-41; dated
08/10/2005) and information provided by the registrant (Use Closure for
Nicotine RED, J. Bloom, 06/18/2007), postapplication exposures are
expected to be short-term and intermediate-term in duration.  Short-term
dermal postapplication MOEs were below 1000 (the level of concern) on
the day of treatment.  This risk estimate may be considered conservative
since it is based on theoretical surface residue levels/calculations and
default assumptions, including 100% dermal absorption, in the absence of
acceptable chemical-specific data.  For dermal risks, a Restricted Entry
Interval of 40+ days is required to achieve acceptable MOEs.

A summary of the inhalation exposure and risk during postapplication
activities is included in Table 9.1.3.2.  The MOE after 10 air changes
(a ventilation option from the Worker Protection Standard (WPS)) is 3049
and does not exceed the level of concern (MOE is greater than 1000). 
This risk estimate may be considered conservative since it is based on
theoretical air concentration data/calculations and default assumptions
in the absence of acceptable chemical-specific data.  While the other
WPS ventilation options are not quantifiable without assumptions for
greenhouse size and ventilation rates, it is assumed, because the WPS
ventilation options are considered equally protective, that the
resulting exposure/risk is equal for all six ventilation options.

9.1.3.1		Data and Assumptions for Dermal Postapplication Exposure
Scenarios

Exposures during postapplication activities were estimated using dermal
transfer coefficients from the Science Advisory Council For Exposure:
Agricultural Reentry Task Force (ARTF) Ornamental Plants Transfer
Coefficients, April 2002, summarized in Table 9.1.3.1.1, and the
following assumptions:

Application Rate = 2.178 lb ai/A 

The application rate for this product was based on the currently
registered end-use product label (EPA Reg. No. 1327-41; dated
08/10/2005).  The application rate was adjusted to pounds of active
ingredient per acre. The application rate for an assumed 10 foot
greenhouse ceiling is 2.178 lb ai/acre, using the following equation:  

(0.10 lb ai/canister)(1 canister/20,000 ft3)(43,560 ft2/acre)(10 ft
ceiling) = 2.178 lb ai/acre 

Exposure Duration = 8 hours per day

Body Weight = 70 kg

Fraction of a.i. retained on foliage is assumed to be 20% (0.2) on day
zero (= % dislodgeable foliar residue, DFR, after initial treatment). 
This fraction is assumed to further dissipate at the rate of 10% (0.1)
per day on following days.  These are standards values established by
HED’s Science Advisory Council (SAC) for Exposure.

Table 9.1.3.1.1.  Anticipated Postapplication Activities and Dermal
Transfer Coefficients.

Crops	Transfer Coefficients (cm2/hr)	Activities	Reference  

Ornamentals	175	greenhouse hand pinching ornamentals; nurseries
activities	MRID 453445-01; ARTF Study No. ART039

	400	harvest (workers moved plants to trucks and reorganized the gallon
pots or containers)	MRID 454695-02; ARTF Study No. ART044

	5100	hand harvesting cut flowers	ExpoSAC Meeting minutes 01/26/2006-

The information in the table is based on proprietary and non-proprietary
data.

Equations/Calculations:

The following equations were used to calculate dermal risks for workers
performing postapplication activities:

Disoldgeable Foliar Residue (DFR)

DFRt (ug/cm2)	= Application Rate (lb ai/acre) x F x (1-D)t x 4.54E8
µg/lb x 2.47E-8 acre/cm2

Where:	

DFRt 		=	dislodgeable foliage residue on day "t" (ug/cm2)

F		=	fraction of ai retained on foliage (0.2 unitless)

D		=	fraction of residue that dissipates daily (0.1 unitless)

Dermal Dose t = DFRt (µg/cm2) x 1E-3 mg/µg x Tc (cm2/hr) x ET (hrs)			
					BW (kg)

Where,

t		=	number of days after application day (days)

DFRt 		=	dislodgeable foliage residue on day "t" (ug/cm2)

Tc		=	transfer coefficient (cm2/hr)	

ET		=	exposure time (hr/day)

BW		=	body weight (kg)

MOE = Dermal NOAEL (mg/kg/day)/ Dermal Dose (mg/kg/day)

Where,

Short-and Intermediate-term Dermal NOAEL = 1.25 mg/kg/day

Note:  Dermal LOC is 1000.

9.1.3.2		Data and Assumptions for Inhalation Postapplication Exposure
Scenarios

According to the Worker Protection Standard (40 CFR Parts 170.110)
greenhouse re-entry following application of a pesticide applied as a
smoke is governed by one of six ventilation options when no inhalation
exposure level is specified on the label:

10 air changes; or

24 hours with no ventilation; or

2 hours mechanical ventilation; or

4 hours passive ventilation; or

11 hours no ventilation with 1 hour mechanical ventilation; or

11 hours no ventilation with 2 hours passive ventilation.

Postapplication inhalation exposure and risk were calculated for
re-entry based on the assumption that the area air concentration will
follow pseudo-first order kinetics upon ventilation (i.e., air changes
or ventilation rate) [ExpoSAC Meeting Minutes, 01/26/2006].  The
following equation was used for this calculation:

CAC = Ci  x  0.5 (# ACH/0.693)

Where,

CAC	= air concentration after number of air changes of interest (mg
ai/m3)

Ci 		= initial air concentration at time of application (80.1 mg ai/m3)

# ACH	= number of air changes (10)

Inhalation Dose (mg/kg/day) = (CAC  x  BR  x  ET)/BW

Where,

CAC= air concentration after number of air changes of interest

BR	= adult breathing rate (1 m3/hr)

ET	= exposure time (8 hr/day)

BW= body weight (70 kg)

MOE = Inhalation NOAEL (mg/kg/day)/ Inhalation Dose (mg/kg/day)

Where,

Short- and Intermediate-term Inhalation NOAEL = 1.25 mg/kg/day

Note:  Inhalation LOC is 1000.

Table 9.1.3.1.  Non-cancer Dermal Postapplication Exposure and Risk for
Nicotine  Used in Greenhouses.

Crops	DAT1	DFR2 (ug/cm2)	Dermal Dose3 (mg/kg/day)	Dermal MOE4



	Low

(Tc=175)	High

(Tc= 5100)	Low	High

Ornamentals, Baseline	0	4.88	0.0976	2.844	13	<1

	3 (RTI5 )	3.56	0.0712	2.075	18	<1

	40	0.0721	0.00144	0.042	868	30









1  DAT = Days after treatment

2  DFR = Dislodgeable Foliar Residue

3  Dermal Dose = [DFR (ug/cm2) x Tc (cm2/hr) x 0.001 mg/ug x 8 hrs/day]
( Body Weight (70 kg)

4  Dermal MOE = NOAEL (1.25 mg/kg/day)/Dermal Dose

Note:  Dermal LOC is 1000.

5  RTI = Minimum Retreatment Interval

Table 9.1.3.2.  Non-cancer Inhalation Postapplication Exposure and Risk
for Nicotine  Used in Greenhouses.

Exposure Scenario (Scenario #)	Ci

(mg/ai/m3)	#ACH	CAC 1

(mg ai/m3)	BR

(m3/hr)	ET

(hr/day)	BW

(kg)	Inhalation Dose 2 (mg/kg/day)	Inhalation MOE 3



Greenhouse Re-entry	80.1	10	0.0036	1	8	70	0.00041	3049

1  CAC = Ci  x  0.5 (# ACH/0.693)

Where,

CAC	= air concentration after number of air changes of interest (mg
ai/m3)

Ci 	= initial air concentration at time of application (80.1 mg ai/m3)

# ACH	= number of air changes (10)

2  Inhalation Dose  (mg/kg/day)  = [CAC (mg ai/m3) x BR (m3/hr) x ET
(hr/day)] / BW

Where,

Ca=  air concentration at time of application (time = 0)

BR=  adult breathing rate (1 m3/hr)

ET=  exposure time (8 hr/day)

BW = body weight (70 kg)

3  Inhalation MOE = Inhalation NOAEL (1.25 mg/kg/day) / Inhalation Dose
(mg/kg/day)

Note:  Inhalation LOC is 1000.



Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465)

See:  Occupational and Residential Exposure/Risk Assessment for the
Nicotine and derivatives Reregistration Eligibility Decision (RED)
Addressing the Bonide® Dog & Rabbit Chaser, B. Cropp-Kohlligian,
D341897, 08/xx/2007.

The Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465) end-use product
label does not provide any distinction in use directions for
professional (occupational) and non-professional (residential homeowner)
applicators and no occupational postapplication scenarios have been
identified.  The residential handler exposures and risks have been
assessed (see Section 6.2.1) which should be protective of occupational
handlers.

10.0	Data Needs and Label Recommendations  TC \l1 "10.0	Data Needs and
Label Recommendations 

10.1	Toxicology  TC \l2 "10.1	Toxicology 			

870.1100	Acute Oral Toxicity

870.1200	Acute Dermal Toxicity

870.1300	Acute Inhalation Toxicity

870.2400	Primary Eye Irritation

870.3200	21-day dermal toxicity study in rats

870.3465	90-day inhalation study-duration reduced to 21 days

870.3700a	Prenatal developmental - rodent

870.3800	Reproduction and fertility effects

10.2	Occupational and Residential Exposure  TC \l2 "10.2	Occupational
and Residential Exposure 

Fulex Nicotine Fumigator (EPA Reg. No. 1327-41)

1.  In accordance with 40 CFR 156.212(e), the Personal Protective
Equipment (PPE) should be based on acute toxicity of the end-use
product.  HED notes that the label recommends use of waterproof gloves
for applicators.  HED recommends that this be revised to
chemical-resistant gloves.

2.  Label(s) must specify respirator type (half-face, full-face, other).

NOTE:  The technical grade of the active ingredient nicotine is
classified as a Category I toxicant based on acute oral toxicity data
which would, under the Worker Protection Standard (WPS), require a
minimum restricted entry interval (REI) of 48-hours (40 CFR
§156.208(c)); however, this criteria for determining the REI does not
apply to any product that is a fumigant (40 CFR §156.208(d)).  Hence,
the REI for the Fulex Nicotine [Greenhouse] Fumigator, if it is
determined to be a fumigant, will be governed by the WPS ventilation
criteria (40 CFR §170.110(c)(3)) and product-specific REI calculations.
 In the absence product-specific data collected in accordance with 40
CFR §158.390, these calculations have been based on theoretical
data/calculations and default assumptions.

NOTE:  Although not a data requirement, the registrant should be aware
that refinements to the exposure estimates would be possible with
acceptable nature of the residue data to determine the residues of
concern and area air monitoring and foliar dislodgeable residue data to
provide better estimates of the level(s) of exposure to the residue(s)
of concern.  It is expected that none of these types of studies require
the participation of human subjects; however, protocols for these
studies must be evaluated by the HSRB before studies are initiated.

Bonide® Rabbit & Dog Chaser (EPA Reg. No. 4-465)

1.  The product contains 0.35% nicotine (as a naturally occurring
component of tobacco dust).  The nicotine content of tobacco dust in the
end-use product is substantially less than the content normally found in
dried tobacco leaves (up to 8% by weight according to the Merck Index)
and in the tobacco found in cigarettes (on average, 1.5% by weight
according to the 1988 report of the Surgeon General, entitled, “The
Health Consequences of Smoking”).  The nicotine content of Bonide®
Rabbit & Dog Chaser (EPA Reg. No. 4-465) must be substantiated by
preliminary analysis (830.1700) and certified limits (830.1750) product
chemistry data for the active ingredient, nicotine (alkaloid).

2.  Label should be amended to specify a maximum use rate in terms of lb
ai/A/application and seasonal limitations.

References:  TC \l1 "References: 

AGENCY MEMORANDA CITED IN THIS DOCUMENT

DP Barcode:	D276938

Subject:	Review of Nicotine Incident Reports.  Chemical #056702 and
#056703.

From:		J. Blondell and M. Spann

To:		B. Cropp-Kohlligian

Dated:		08/10/2001

MRIDs:	None

DP Barcode:	None

Subject:	Use Closure for Nicotine RED

From:		J. Bloom

To:		Nicotine RED team

Dated:		06/18/2007

MRIDs:	None

DP Barcode:	D341249

Subject:	Nicotine:  Occupational and Residential Exposure/Risk
Assessment for the Nicotine and derivatives Reregistration Eligibility
Decision (RED) Addressing the Fulex Nicotine Greenhouse Fumigator
Insecticide.

From:		B. Cropp-Kohlligian

To:		J. Bloom

Dated:		08/xx/2007

MRIDs:	46165801

DP Barcode:	D341897

Subject:	Nicotine (as a naturally occuring component of tobacco dust): 
Occupational and Residential Exposure/Risk Assessment for the Nicotine
and derivatives Reregistration Eligibility Decision (RED) Addressing the
Bonide Rabbit and Dog Repellent.

From:		B. Cropp-Kohlligian

To:		J. Bloom

Dated:		08/xx/2007

MRIDs:	None

MRID REFERENCES

46165801  Krake, A. (1997) Health Hazard Evaluation Report 96-0032-2649:
Fulex Nicotine Fumigator. Project Number: 96/0032/2649. Unpublished
study prepared by Cornell University. 20 p.

Appendix A:  Toxicology Assessment  TC \l1 "Appendix A:  Toxicology
Assessment 

A.1	Toxicology Data Requirements TC \l2 "A.1	Toxicology Data
Requirements 

The requirements (40 CFR 158.340) for non food for NICOTINE are in Table
1. Use of the new guideline numbers does not imply that the new (1998)
guideline protocols were used.

Test 

	Technical

	Required	Satisfied

870.1100    Acute Oral Toxicity	

870.1200    Acute Dermal Toxicity	

870.1300    Acute Inhalation Toxicity	

870.2400    Primary Eye Irritation	

870.2500    Primary Dermal Irritation	

870.2600    Dermal Sensitization		yes

yes

yes

yes

yes

yes	no

no

no

no

yes

yes

870.3100    Oral Subchronic (rodent)	

870.3150    Oral Subchronic (nonrodent)	

870.3200    21-Day Dermal	

870.3250    90-Day Dermal	

870.3465    90-Day Inhalation (21 – day)		NR

NR

Yes

NR

Yes	-

-

no

-

no

870.3700a  Developmental Toxicity (rodent)	

870.3700b  Developmental Toxicity (nonrodent)	

870.3800    Reproduction		Yes

NR

Yes	no

-

no

870.4100a  Chronic Toxicity (rodent)	

870.4100b  Chronic Toxicity (nonrodent)	

870.4200a  Oncogenicity (rat)	

870.4200b  Oncogenicity (mouse)	

870.4300    Chronic/Oncogenicity		NR

NR

NR

NR

NR	-

-

-

-

-

870.5100    Mutagenicity—Gene Mutation - bacterial	

870.5300    Mutagenicity—Gene Mutation - mammalian	

870.5xxx    Mutagenicity—Structural Chromosomal Aberrations	

870.5xxx    Mutagenicity—Other Genotoxic Effects		Yes

Yes

Yes

Yes	Yes$

Yes$

Yes$

Yes$

870.6100a  Acute Delayed Neurotox. (hen)	

870.6100b  90-Day Neurotoxicity (hen)	

870.6200a  Acute Neurotox. Screening Battery (rat)	

870.6200b  90-Day Neuro. Screening Battery (rat)	

870.6300    Develop. Neuro		NR

NR

NR

NR

NR	-

-

-

-

-

870.7485    General Metabolism	

870.7600    Dermal Penetration		NR

NR	-

-

Special Studies for Ocular Effects

Acute Oral (rat)	

Subchronic Oral (rat)	

Six-month Oral (dog)		

NR

NR

NR	

-

-

-

$  Published studies satisfy this requirement.

A.2	Toxicity Profiles TC \l2 "A.2	Toxicity Profiles 

  SEQ CHAPTER \h \r 1 TableA.2.1a.	Acute Toxicity Profile – Nicotine

  SEQ CHAPTER \h \r 1 Guideline No.	  SEQ CHAPTER \h \r 1 Study Type	 
SEQ CHAPTER \h \r 1 MRID No./Reference	  SEQ CHAPTER \h \r 1 Results	 
SEQ CHAPTER \h \r 1 Toxicity Category

Non-Guideline   SEQ CHAPTER \h \r 1 	Acute oral – rat

Acute oral - muse	IPCS INCHEM, 1991*	LD50 =  50 mg/kg

LD50 =  3 mg/kg	I

I

Non-Guideline	Acute oral – dog

	Matsushima et el, 1995	LD50 =  10-12 mg/kg

	I

Non-guideline	Acute i.p. - mice	Priestly and Plaa 1976	LD50 =  13.5
mg/kg

	I

  SEQ CHAPTER \h \r 1 870.1200	Acute dermal - rabbit	NA



  SEQ CHAPTER \h \r 1 870.1300	Acute inhalation - rat	NA



  SEQ CHAPTER \h \r 1 870.2400	Eye irritation - rabbit	NA



  SEQ CHAPTER \h \r 1 870.2500	Dermal irritation – rabbit

(40% nicotine formula)	41521101	Slightly  irritant	IV

  SEQ CHAPTER \h \r 1 870.2600	Skin sensitization

(40% nicotine formula)	41521102	Not a skin sensitizer

*   HYPERLINK
"http://www.inchem.org/documents/pims/chemical/nicotine.htm" 
http://www.inchem.org/documents/pims/chemical/nicotine.htm 

Table A.2.1b	Acute Toxicity Profile - F&B Rabbit and Dog Chaser (mixture
of tobacco, naphthalene and sterilized cattle blood)

Guideline No.	Study Type	MRID(s)	Results	Toxicity Category

870.1100	Acute oral: rat 	43523301	LD50 = > 5000 mg/kg	IV

870.1200	Acute dermal: rabbit	42631101	LD50 = greater than 2020 mg/kg	IV

870.1300	Acute inhalation: rat	42631102	LC50 = >5.39 mg/L	IV

870.2400	Acute eye irritation: rabbit	42631103	minimally irritating	IV

870.2500	Acute dermal irritation: rabbit 	42631104	Mildly irritating	IV

870.2600	Skin sensitization: guinea pig	42640001	Not sensitizer	NA



Table A.2.2	Subchronic, Chronic and Other Toxicity Profile of Nicotine

Guideline No. 	Study Type	MRID No. (year)/ Classification /Doses	Results

Non-guideline	Subacute

Pregnant and non-pregnant female rats	Yuen et al.  1995

Nicotine tartrate in drinking water for 10 days at 54 or 108 (mole/l
(8.76, 17.52 mg/l = 1.25 or 2.5 mg/kg/day)	Resulted in mild fatty
change, mild focal necrosis and mild dark cell change (containing
numerous prominent pore annuli in the nuclear membranes and the
mitochondria appeared decreased in size with a decrease in mitochondrial
granules and loss of aristae) in a dose proportional manner
statistically significant at 2.5 mg/kg/day. The NOAEL is 2.5 mg/kg/day
and the NOAEL is 1.25 mg/kg/day.

Non-guideline	Subchronic

Sprague Dawley Rats	Lau et al.  1990

adult male rats (185-225 g initial weight) were implanted with nicotine
pellets containing 0, 5, 15 or 50 mg nicotine (delivering 0, 9.9, 29.8,
and 99.2 ug/hr, respectively) for up to 12 weeks (equivalent to 0.0,
1.2, 3.6, 12.0 mg/kg/day)	Increased pancreatic enzyme biosynthesis and
accumulation of digestive enzymes within the pancreas were reported. 
Plasma nicotine at the 50 mg dose reached a steady state level of 76±19
ng/mL and cotinine exceeded 300 ng/mL.  Electron microscopy of pancreas
from rats treated with the 50 mg nicotine dose revealed intracytoplasmic
vacuoles appearing after 3 weeks of treatment, and persisting throughout
the remaining experimental period (evidence of morphological damage).  
Nicotine treatment and durations did not have an effect on body weight,
pancreatic weight, DNA, RNA or protein concentrations. A LOAEL of 12
mg/kg/day based on pancreatic morphologic change is suggested by the
study findings and a NOAEL is 3.6 mg/kg/day..

Non-guideline 	Subchronic

Sprague Dawley Rats 	(Dubick et al, 1988)

adult male rats (190-200 g) receiving nicotine via a time–release
pellet at a rate of 1.65 ug/min )equivalent to 12 mg/kg/day) for 3 weeks
Showed that increases in digestive pancreatic enzymes (amylase, trypsin
and chymotrypsin; 51%, 28%, 35% higher) were noted compared to controls.
Final body weight or pancreatic weight was not affected.

Non-guideline	Subchronic

CF-1 Swiss derived mice	(Priestly and Plaa 1976)

ess potent hepatotoxins chloroform or 1,1,1-trichloroethane nor the
cholestatic effect of α-naphthylisothiocyanate was modified.

Non-guideline	Subchronic

Sprague Dawley Male Rats	Kavitharaj and Vijayammal, 1999

Nicotine, subcutaneously at 0.0, 0.6 mg/kg/day for 21 days, or 0.6 mgk
nicotine for 21 days + mecamylamine at 0.8 mgk on Days 21, 23, and 25 (6
rats/group, 1.5 months old, 120-150 g weight) sacrificed on Day 35.
Administration of nicotine produced enhanced synthesis of cholesterol,
triglycerides, phospholipids and free fatty acids in the liver and
testes and lower serum testosterone and estradiol levels suggesting
gonadotoxic effects.  Body weight gain at sacrifice time in the nicotine
treated rats was less than the control rats (22.14 ± 0.86 g vs 34.28 ±
1.61 g in the controls). The activity of the lipogenic enzymes (NADPH
generating enzymes) was higher in the liver, but not altered in the
testes.  Mecamylamine (a known inhibitor of nicotine) counteracted the
effects seen in rats treated with nicotine only.

Non-guideline	Subchronic

Adult Female Rats	Fung et al, 1999

nicotine 3.0 or 4.5 mg/kg/day (9-10 rats/group) by subcutaneous osmotic
minipumps	had significantly lower levels of 25-hydroxyvitamin D than
controls. The high dose nicotine group had smaller vertebral areas and a
lower bone mineral content and significantly lower tibial endocortical
mineral apposition rate than the controls.  Nicotine serum levels at
sacrifice time were 60 ± 6 and 85 ± 5 ng/mL at the low and high dose,
respectively.  

Non-guideline	Subchronic

Male New Zealand white rabbits

	Booyse et al, 1981

Nicotine tartrate, administered orally (2.4 mg/kg/day) in drinking water
for 25 weeks to 10 rabbits	produced in vivo morphologic effect on
endothelial cells in the aortic arch.  Fasting serum levels of glucose,
triglycerides, total cholesterol, and LDL-cholesterol were significantly
(<0.001) elevated in nicotine-treated rabbits.   Endothelial cells from
nicotine-treated arched areas (Evans-blue-stained) showed extensive
changes such as increased cytoplasmic silver disposition, increased
formation of microvilli, and numerous focal areas of

 “ruffled” endothelium (projections on cell surfaces).

Non-guideline	Subchronic

Sprague Dawley Male Rats	Chowdhury et al, 1990

nicotine ingested for 16 weeks at 50 and 200 mg/l in drinking water
equivalent to 7  and 28 mg/kg/day	alters metabolic, endocrine, and
pathologic factors that may be responsible, at least in part, for the
development of gastrointestinal ulcers and pancreatitis.  
Endocrinological studies showed that the plasma levels of CCK were
significantly increased with nicotine but the amylase secretory response
of pancreatic acinar cells was inhibited in response to CCK-8 and
carbachol.  Prominent loss of gastric mucosal surface was found in
nicotine-treated animals with gross microscopic evidence of bleeding
ulcers.  All of the metabolic parameters except body weight gain were
reversed upon nicotine withdrawal. Histopathologic data showed a partial
but not a complete recovery of the pancreatic acinar cell morphology and
gastric mucosal surface following 4 weeks of nicotine withdrawal.

870.3200

	21/28-Day dermal toxicity (species)	NR

	870.3250

	90-Day dermal toxicity (species)	NR

	Non-Guideline

	Chronic, Inhalation

Female rats	Waldum et al, 1996

inhalation to 501±151 µg/m3 , 20 h/day, 5 days/week for 2 years
(equivalent to 0.3 ± 0.09 mg/kg/day)	resulted in slightly depressed
weight (Waldum et al, 1996).  Nicotine plasma concentration after 5 days
was 108.4 ± 55.1 ng/mL and remained fairly constant and after 24 months
it was 129.8±43.0 ng/mL.  All parameters measured were comparable to
the controls except for elevated adenomas of pituitary gland (4/59)
which was attributed to the neuroendocrine action of nicotine.  There
was no increase in mortality, in atherosclerosis or frequency of tumors
in these rats compared with controls. Particularly, there were no
microscopic or macroscopic lung tumors or any increase in pulmonary
neuroendocrine cells. Throughout the study, however, the body weight of
the nicotine exposed rats was reduced as compared with controls. 

Non-Guideline

	Developmental

Mouse	Saad et al, 1990, 1991

Pregnant CD-1 mice, exposed to ip injection of 0.1% nicotine sulfate at
a dose of 1.67 mg/kg body weight/day on gestational days 6-15	Depressed
maternal weight gain and fetal weight. Fetal crown-rump length fetal
head dimensions were significantly reduced.  Histological examination
revealed that 9.6% of fetuses of nicotine injected mothers presented
clefts of the palate (none in the control). Nicotine treatment also had
teratogenic effects on first molar odontogenesis in the mouse. The
mesiodistal diameter of molar tooth germs of nicotine treated fetuses
without cleft palate was significantly less than those of controls.

Non-Guideline	Developmental 

Rat	Chowdhury & Bromage 2000

experimental details of nicotine treatment were not presented	dental
asymmetries (calculated as a size difference between a tooth and its
antimere) were significantly increased while occlusal areas were
significantly decreased in nicotine-exposed rats compared to control
rats. Females tending to exhibit the deleterious effects of nicotine
more so than males

Non-Guideline	Developmental

Mouse	Nasrat et al, 1986

Nicotine administered subcutaneously to mice during gestation at ≥ 450
μg/kg/day dose	Nicotine increased the perinatal mortality.  The male to
female ratio was in favor of females in the nicotine treated mice during
the 2nd and 3rd trimester. When large doses of the drug were given
especially in the second and third trimesters of gestation, there was a
significant shortening of the gestation period.

Non-Guideline	Developmental

Mouse	Leblebicioglu-Bekcioglu et al, 1995

Nicotine (12 mkg or nicotine plus caffeine (125 mkg) by intubation
GD6-18. (7/group)	Nicotine had minimal ossification effects on the
fetuses as measured by staging and measuring craniofacial bones, and
counting ossification centra in sternbrae and in cervical and
sacrococcygeal vertebrae.  Caffeine had greater effect. 

Non-Guideline	Developmental 

Rat	Williams & Kanagasabai, 1984. 

2.46 ± 0.18 mg/kg/day in drinking water. GD 0-20

	Mean fetal body fat was significantly increased in fetuses of rats
administered nicotine during pregnancy.  Rate for maternal lypolysis
were higher in the nicotine treated animals.  Maternal body weights
gains were significantly lower (77.2% of controls, p < 0.001).

Non-Guideline

	Developmental

Rat	(Witschi et al, 1994)

Nicotine - delivering transdermal patches applied on the back of
pregnant female rats

3.5mg/day (2 rats GD 2-19; 8 rats GD 2-7; 3 rats GD 2-5)

1.75 mg/day patch (13 rats GD 2-19; 4 rats GD 2-7)	Nicotine –caused
100% pregnancy failure in animals treated GD2-19 with 3.5 mg/day and 50%
in animals treated GD 2-7, and 55% in animals exposed GD2-19 to 1.75
mg/day.  Litter size and pup weights were not affected. Nicotine and
cotinine not detected in animals exposed the first trimester of
pregnancy. In animals exposed the entire pregnancy at 1.75 mg/day
patches, 3 pregnant animals out of six had measurable nicotine levels
(43 ± 22 ng/mL) and all had cotinine levels (100 ± 48 ng/mL). The non
pregnant females of the 1.75 mg/day patches had 70 ± 57 ng/mL of plasma
nicotine and 231 ± 84 ng/mL of plasma cotinine. In the two animals
exposed to 3.5 mg/day patches and became nonpregnant, nicotine plasma
levels were 241 ± 51 ng/mL and cotinine levels of 302 ± 94 ng/mL.

Non-Guideline

	Developmental

Rat (Sprague Dawley	Slotkin et al, 1986

Subcutaneous injection of nicotine (3 mg/kg/day from gestation day 4
-20) to timed pregnant Sprague Dawley rats (41 control and 57 nicotine
treated)	significant reduced maternal weight gain and increased maternal
mortality (14%) and fetal resorptions (13%).  Litter size was not
affected. Biochemical changes in the fetal brain (elevation of fetal
ornithine decarboxylase activity, suppression of DNA synthesis (most
profound in cerebellum), desynchronization of the ontogenctic patterns
of DNA, RNA and proteins in every brain region)

Non-Guideline

	Developmental

Rat (Sprague Dawley)	Muneoka et al, 1999

Subcutaneous injections of nicotine (3 or 6 mg/kg/day, divided into two
doses) on GD 7-20 to pregnant rats (10/group)	Abnormal behavior such as
ataxic gate, decrease in spontaneous activity, creeping, or salivation
in pregnant rats (3 or 6 mg/kg/day) and convulsion and ptosis (6 mg/kg),
decreased maternal body weight at GD 14 (both groups) and reduced
offspring body weight.   Significant decreases in dihydroxyphenylacetic
acid (DOPAC) content in the neocortex and in both the neocortex and in
the midbrain plus pons medulla, respectively. 

Non-Guideline

	Developmental 

Rat	Hussein et al, 2007

nicotine dose of 6 mg/kg/day administered to Sprague Dawley time-mated
rats from gestation day 3 through 21 by osmotic mini pump	resulted in
nicotine plasma concentrations of 115-174 ng/mL.  Maternal hematocrit
was not affected by nicotine administration, or the number of pups per
litter, pup weight, placental weight or the weight of various pups'
organs. Maternal weight changes were comparable in the nicotine treated
and control groups. Maternal plasma coticosterone concentrations were
not affected by the nicotine infusion versus the control rats. 

Non-Guideline	Developmental 

Rat	Zahalka et al 1993

Nicotine's effects on ontogeny of postsynaptic muscarinic M1-receptors
in rat striatum and hippocampus were investigated after continuous
maternal infusions (15-21 rats/treatment) of 2 mg/kg/day or 6 mg/kg/day
from GD 4 through 20. 	Brain region weights were unaffected. Postnatal
development of striatal M1-receptor binding, as identified with
[3H]pirenzepine, was significantly impaired with either of the fetal
nicotine regimens. Treatment with 2 mkg also produced alterations in
striatal receptor affinity state, characterized by enhanced ability of
an agonist (oxotremorine-M) to displace [3H]pirenzepine; raising the
dose to 6  mkg masked the affinity shift by affecting G-protein
regulatory mechanisms, such that addition of the GTP analog, GppNHp,
produced a larger decrease in agonist affinity. In the hippocampus, no
such effects on receptor binding, affinity state, or G-protein
regulation were seen with either regimen.

870.3800

	Reproduction and fertility effects

(species)	NA

	870.4100a

	Chronic toxicity

(species)	NA

	870.4100b

	Chronic toxicity (dog)	NA

	870.4200

	Carcinogenicity

(rat)	NA

	870.4300

	Carcinogenicity

(mouse)	NA

	Gene Mutation

Non-Guideline

Tests by the R. J. Reynolds Tobacco Co. (Doolittle et al, 1995)	Nicotine
and its major metabolites: cotinine, nicotine-N'-oxide,
cotinine-N-oxide, and trans-3'-hydroxycotinine in the Salmonella
mutagenicity assay (strains TA98, TA100, TA1535, TA1537, and TA1538) at
0 to 1000 µg/plate and in the Chinese hamster ovary sister-chromatid
exchange (SCE) assay at 0 to 1000 µg /mL,  with and without S9
metabolic activation did not increase the frequency of mutations or the
frequency of SCEs. 

Cytogenetics 

Non-Guideline	Bacterial luminescence genotoxicity test	Yim & Hee 1995
Cotinine  was positive in the presence or absence of S9 at 1.25 - 2.5
mg/mL (9- 30 h incubation).   Nicotine was not positive to 20 mg/mL
concentrations for up to 40 hours of incubation. Nicotine/cotinine
mixtures were still positive at physiological concentrations, with
potentiation relative to cotinine alone with and without S9 

Other Effects 

Non-Guideline	Chromsomal aberration (CA) and sister chromatid exchange
(SCE)	Trivedi et al, 1990	Nicotine induced chromsomal aberration (CA)
and sister chromatid exchange (SCE) frequency in a dose and duration
dependent manner and at concentrations comparable to the saliva levels
of nicotine achieved during tobacco chewing.  Statistically significant
elevations in CA frequency were observed with nicotine concentrations >
375 (g/mL; SCE frequencies were increased significantly > 150 µg/mL.
Nicotine (150 µg/mL) and in combination with arecoline (an areca nut
alkaloid) incubated in Chinese hamster ovary (CHO) cell line (-S9)
increased the SCE frequency/cell.  Nicotine alone at 300 (g/mL did not
increase the frequency of CHE/cell, but in combination with arecoline it
did at ≥90 µg/mL.

Genotoxicity Non-Guideline 	Chromosomal aberrations	Bishun et al, 1972
Nicotine was cytotoxic at 1.5 - 2.0 µg/mL to human leucocytes in
culture in vitro without producing any chromosome damage.  However,
gross chromosomal aberrations including fuzzy chromosomes (stickiness),
aneuploidy and translocations were observed in the mice receiving low
tolerable doses of the drug (0.07 -0.09 µg/total body weight injected
to 5 weeks-4 month old mice in saline , two weekly injections for 3
weeks before sacrifice and preparing  the bone marrow from the femora.

Genotoxicity Non-Guideline	DNA damage	Kleinsasser et al, 2005

The genotoxicity of nicotine was tested with the alkaline single-cell
microgel electrophoresis (Comet) assay using human lymphocytes and
target cells from lymphatic tissue of the palatine tonsils from healthy
patients.	Nicotine exerted significant direct genotoxic effects in human
target cells in vitro.  One hour exposure to nicotine at 0.125, 0.25,
0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent
increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells
and lymphocytes, respectively. The minimum concentration eliciting
significant DNA damage was 0.5 mM nicotine. The genotoxic effect was
confirmed in a second series of experiments using nicotine of high
purity from two different suppliers.  Finally, DNA damage by nicotine
was compared in cells incubated in medium strictly adjusted to neutral
pH, with nonadjusted medium becoming alkaline with increasing nicotine
concentrations. Again no differences in DNA migration were observed. 
However, no differences in DNA damage were observed in cells from
smokers and nonsmokers incubated without nicotine. The lack of higher
DNA damage in smokers compared to nonsmokers could be a question of
nicotine dose, rapid DNA repair, or interactions with other smoke
constituents.

Genotoxicity Non-Guideline	Human nasal epithelia.	Sassen et al 2005
Nicotine produced genotoxic effects in human nasal epithelia.

Genotoxicity Non-Guideline	Human gingival fibroblasts (HGFs)	Argentin
and Cicchetti, 2004	Nicotine caused concomitant genotoxic and
antiapoptotic effect in human gingival fibroblasts (HGFs) in the
cytokinesis-block micronucleus (CBMN) test.   Treatment of HGFs with
nicotine, at a concentration of 1 uM, caused a statistically significant
increase of micronucleus (MN) frequency at the tested time intervals,
while no change was detected in cell growth under the same conditions. 
Preincubation of HGFs with 1 uM nicotine strongly attenuated
staurosporine (STP)-induced apoptosis. Cultures exposed to nicotine
showed an increase of reactive oxygen species, as determined by
increased levels of 2,7-dichlorofluorescein (DCF). When cells were
prelabeled with N-acetyl-cysteine (NAC), a substrate for glutathione
synthesis, and catalase (CAT), the oxygen free radical scavenger, a
significant reduction in cytogenetic damage was observed.

870.6200a

	Acute neurotoxicity screening battery	NA

	870.6200b

	Subchronic neurotoxicity screening battery	NA

	870.6300

Non-Guideline

	Developmental neurotoxicity	Temocin et al, 1993

subcutaneous injection of nicotine (0.125 - 0.375 mg/kg)	Decreased the
endurance time in swimming exercise significantly (10 minute after the
injection). At 0.125 mg/kg nicotine, the endurance time remained
unchanged, while at the doses of 0.25 and 0.375 mg/kg, it decreased
significantly (p < 0.05 and p < 0.01, respectively). This effect was
antagonized by pretreatment with hexamethonium 5 mg/kg s.c.



Non-Guideline

	Developmental neurotoxicity	Johns et al (1992)

0, 0.5, 1.5, or 2.5 mg/kg of nicotine hydrogen tartrate, injected 2X
daily throughout gestation period (15/dose).	Offspring exhibited
performance deficits in both learned and innate behavioral measures
throughout development and adulthood.  Offspring birth weight was not
affected by treatment nor the 32 day weight gain. Initial weight, weight
gain, gestation length, number of live or dead offspring, or mean food
consumption during pregnancy did not differ significantly in the treated
groups versus the controls. Dams receiving the nicotine injections
reacted with aversion.

Non-Guideline

	Developmental neurotoxicity	Navarro et al, 1989

2 mg/kg of nicotine infused per day from gestational days 4 through 20
Infusing pregnant rats with a nicotine dose that did not interfere with
maternal weight gain or resorption rate resulted in sufficient nicotine
penetrating the fetal brain to cause persistent alterations in [3H]
nicotine binding sites, abnormalities of cellular development [assessed
by measurements of ornithine decarboxylase (ODC) activity and
deoxyribonucleic acid (DNA)], and impairment of development of
peripheral noradrenergic projections (assessed by kidney norepinephrine
levels). 

Non-Guideline

	Developmental neurotoxicity	Levin et al, 1993

2 mg/kg of nicotine subcutaneous injection per day from gestational days
4 through 21	Prenatal nicotine exposure caused subtle alterations in
cognitive performance of the offspring which were magnified by
challenges of nicotinic and adrenergic systems.

Non-Guideline

	Developmental neurotoxicity	Fung, 1988

1.5 mg/kg/day, nicotine tartrate subcutaneous implanting of pregnant
rats) during gestation	14 – day old male and female offspring of rats
exposed to nicotinedemonstrated an increase in spontaneous locomotor
activity compared with controls.  The total number of pups born to the
treated group was significantly less than the controls.

Non-Guideline

	Developmental neurotoxicity	Peters et al, 1979 

60 - 80 days old offspring of rats treated with nicotine in drinking
water (6 mg/kg/day intake) four weeks before mating and during pregnancy
and throughout nursing and 6 weeks after weaning	Increased spontaneous
motor activity in the light which was not prevented by cross-fostering
to control dams at birth. The paternal rats had a marked reduction in
body weight gain (55% of controls for males and 63% of controls for
females after 4 month of nicotine treatment).  The dams were more active
during the day and exhibited a reduced plasma corticosterone response to
stress.  Male but not female offspring of nicotine treated rats were
significantly lighter at birth than control males.



Non-Guideline

	Developmental neurotoxicity	Peters & Ngan, 1982

Nicotine at 0, 1.5,  3 or 6.0 mg/kg per day; subcutaneously one week
prior to mating and throughout gestation	subtle neurological changes
which are manifested as behavioral alterations in the newborn (the
righting reflex, temperature  regulation, adherence to the inclined
screen, and in organ/body weight ratios for brain, heart,  lung, liver,
and kidney)  and adult offspring (prolonged time required and an
increase in number of mistakes made, during food maze testing and an
increased brain protein content). 

Non-Guideline

	Developmental neurotoxicity	Roy & Sabherwal, 1998

pregnant rats were injected ip with  nicotine (2.5 mg/kg/ day, given in
two divided doses) from GD 6 to term and pups were delivered  at term
normally	Reduction in cortical thickness and decreased cell size,
decreased dendritic branching and increased dendritic spine density,
irregular arrangement of cisternae of rough endoplasmic reticulum,
paucity of free ribosomes, and frequent cytoplasmic vacuoles of the
somatosensory cortex were observed in the pup up to postnatal day 40. 
Morphological changes in the hippocampus (significant reduction in the
neuronal area of nicotine-exposed brains in the dentate gyrus, CA3, and
CA1 regions) also resulted from the prental nicotine treatment, which
may contribute to the behavioral abnormalities.

Non-Guideline

	Developmental neurotoxicity	Ajarem & Ahmad, 1998

daily subcutaneous injections of nicotine  (0.5mg/kg) into the nape of
the neck during pregnancy	significant reduced postnatal body weight
gain, as well as significant delay in eye opening, in the appearance of
body hairs, and in sensory motor reflexes.  Motor activity was
significantly stimulated in early adulthood of mouse pups prenatally
exposed to nicotine, and had long-lasting hyperactive effects on mice.

Non-Guideline

	Developmental neurotoxicity	Marks et al, 1985

four inbred strains of mice (BALB, C57BL, DBA, C3H),  nicotine
administered ip at a single dose of 0, 0.5, 1.0 or 2.0 mg/kg 	Generally
nicotine administration increased the respiration rate, decreased the
body temperature and the heart rate, and a decline in the Y-maze crosses
and rears at different rates in the various strains. The startle
response was increased in the C3H strain only.

Non-Guideline

	Developmental neurotoxicity	McFarland et al, 1991

Nicotine given at an acute subcutaneous injection to neonatal rats as
nicotine dibitartrate (3 mg/kg of nicotine base) at 1, 3, 8, 10 or 15
days of age	Nicotine inhibited DNA synthesis in neonatal rat brain
regions as assessed by the incorporation of [3H]thymidine into DNA of
the brain tissues.  The inhibition potency correlated to the
concentration of nicotinic receptors: midbrain + brainstem (cerebral
cortex > cerebellum. The inhibitory effect of nicotine was also seen in
fetal brain on gestational day 20 after injection of nicotine (3 mg/kg)
to pregnant rats.  

Non-Guideline

	neurobehavioral teratology	Seidler et al, 1996

developing rats (1, 7, 14 and 21 days old) challenged acutely with
nicotine (0.3 mg/kg, i.p.) and the release of catecholamines was
evaluated in vivo in three brain regions that differ in nicotinic
receptor concentrations.	Nicotine did not stimulate catecholamine
release at birth, but developed the capacity to do so in parallel with
the ontogeny of nicotinic cholinergic receptors in the
midbrain+brainstem and in the forebrain. In the cerebellum, no response
was obtained at any age. Changes in sensitivity to nicotine were also
seen that corresponded to ontogenetic changes in endogenous cholinergic
tone, suggesting that receptor desensitization occurs normally during
developmental stages in which neuronal activity is high. The absence of
a catecholamine response to nicotine at birth in the rat indicates that
neurobehavioral teratology associated with fetal nicotine exposure does
not reflect secondary actions mediated through catecholamines. However,
because brain development in the neonatal rat corresponds to fetal
stages in man, the onset of these mechanisms may be relevant to human
fetal exposure.

Non-Guideline

	Endocrine effects

in vivo and in vitro  rat models	Blackburn et al, 1994

pregnant mare’s serum gonadotropin (PMSG) - primed and human chorionic
gonadotropin (hCG) - triggered rat ovaries exposed to nicotine  (ip 6.25
ng/g animal weight)	Nicotine inhibited ovulation, estradiol production,
and fertilization both in vivo and in vitro in rat models.  A dose
dependent reduction in oocytes within the fallopian tube was noted in
nicotine treated rats (p<0.001).  On the other hand cotinine did not
affect ovulation, estradiol production or fertilization in those tests.

 

Non-Guideline

	Endocrine effects

rats	Slotkin and Seidler, 1975

Nicotine at1 mg/kg or 10 mg/kg, subcutaneously twice daily for 1 or 2
weeks)	Nicotine produced alterations in catecholamines (CA) release,
tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and the
ability of isolated storage vesicles to incorporate 3H-epinephrine in
the adrenal glands and these alterations persisted when nicotine
administration was discontinued.

Non-Guideline

	Endocrine effects

Time-pregnant Sprague Dawley rats

	Lichtensteiger and Schlumpf 1985

Nicotine tartrate delivered in an osmotic minipump at a rate of 25
ug/100 g/ hr	Male fetuses of all control groups displayed the
characteristic rise in plasma testosterone at GD 18 (as compared to GD
17 and 19); this was abolished by nicotine. Adult offspring of untreated
or tartaric acid-treated dams exhibited a marked sexual dimorphism in
their preference for saccharin-containing drinking water at 0.06-0.25%.
No such sex difference was seen in offspring of nicotine-treated rats. 

Non-Guideline

	Endocrine effects

	Ehlers et al, 1997

Nicotine hydrogen-

tartrate (Sigma) dissolved in distilled water and added to the milk diet
(0 mg, 1 or 4mg/kg/day) administered to neonatal rat pups from PN4
through PN12 with an artificial rearing paradigm; Nicotine. All rats
were weaned at PN21 and housed in pairs.  Thirty one suckle controls (17
males, 14 females), 23 artificially reared rats (14 males, 9 females),
22 1 mg/kg/day rats (12 males, 10 females), and 23 4 mg/kg/day animals
(16 males, 7 females) were used in this study.	Nicotine exposure altered
responses of the P3 component of the event-related potential (ERP),
recorded in dorsal hippocampus, to changes in stimulus parameters. A
significant reduction in the response to the noise tone as compared with
the level of the infrequently presented tone also was seen in the P3B
component. No effects of drug exposure were found on the N1 component in
any lead, although artificial rearing produced specific effects on the
latency of the N1 component in cortex. No significant differences among
treatment groups were found on any of the EEG-dependent variables.
Female rats overall were found to have significantly higher
electroencephalography (EEG) amplitudes than the males. However, no
overall effects of gender were found on any ERP component. These studies
suggest that neonatal nicotine exposure specifically reduces the
electrophysiological response of the hippocampus to changes in auditory
stimuli

Non-Guideline

	Endocrine effects

Sprague Dawley rats

	Slawecki et al, 2000

Male rats were exposed to 6.0 mg/kg/day nicotine via gastric infusion
using an artificial rearing, “pup-in-the-cup,” technique for 6
consecutive days PND 4–9.  At adulthood, EEG and auditory ERPs were
recorded from the cortex and hippocampus	Examination of the hippocampal
EEG revealed significantly decreased power in the 1–2-Hz frequency
band of nicotine-treated rats. In addition, there was a significantly
attenuated P300 ERP response to a noise tone in the nicotine-treated
rats compared to controls. These data indicate that neonatal nicotine
exposure alters functional activity in the hippocampus of adult rats.
These effects are likely to be the result of synaptic disorganization in
the hippocampus, and indicate that neonatal nicotine exposure exerts
teratogenic effects on the developing central nervous system,
particularly the hippocampus, which persist into adulthood.

Non-Guideline

	Immune system	Maritz & Woolward, 1992

nicotine at1 mg/kg bw/d from GD 7 until weaning three weeks after birth)
resulted in a very low elastic tissue content in the lungs of 1- and
7-day old rat pups compared with those of the controls.  This may
interfere with normal lung development since elastic tissue is part of
the lung connective tissue structure and is involved in the formation of
alveoli.  Impaired elastic synthesis may thus make the rat pups more
susceptible to lung diseases such as emphysema

Non-Guideline

	Immune system	Geng et al, 1995

Nicotine at 1 mg/kg to young adult rats by intradermal implantation for
3-4 weeks	Nicotine inhibited both the T-dependent and T-independent AFC
responses and proliferation to anti-CD3.  Significantly fewer NT T cells
entered the S and G2/M phases than CON T cells, indicating an arrest in
the GO/G1 phase.  B and T cells were unable to elevate the intracellular
calcium levels normally in response to ligation of antigen receptors,
although Ca2+ responses of salivary gland cells to acetylcholine were
normal.  Serum cotinine levels of 219  ±  40 ng/mL were comparable to
average human smokers.

Non-Guideline

	Immune system	Basta et al, 2000

pregnant rats treated with nicotine (6 mg/kg/day) from GD 4-20
subcutaneously	Nicotine suppressed splenocyte responsiveness to
Concanavalin A or lipopolysaccharide in offspring and remained
subresponsive to stimulation well into adulthood.  Nicotine treatment
also resulted in reduced maternal weight gain during early gestation,
increased resorptions, increased offspring body weight and significantly
elevated offspring spleen weights.  In combination with ethanol, the
observed effects were more severe.

Non-Guideline

	Immune system

In vitro T-cells from female mice	Zhang & Petro, 1996

T-cells (splenic mononuclear cells) exposed to nicotine at 1-100 μg/100
mL)	Exposure of T-cells (splenic mononuclear cells from female mice) to
physiological concentration of nicotine (1-100 μg/100 mL) can alter T
cell expression of CD28 and CTLA-4 and the CD4 T cell cytokine
expression pattern

Non-Guideline

	Metabolism and pharmacokineti	Hukkanen et al, 2005 	Review article.
Rapid absorption of nicotine from the inhaled cigarette smoke and rapid
distribution via the blood stream to various tissues including the brain
within 10 to 20 seconds, faster than iv.  Poorly absorbed from stomach -
protonated in the acidic gastric fluid, but well absorbed in the small
intestine - more alkaline pH and a large surface area.  Oral ingestion
of nicotine capsules or nicotine in solution, peak concentrations in the
blood are reached in about 1 hour. Nicotine crosses the placental
barrier easily, and there is evidence for the accumulation of nicotine
in fetal serum and amnionic fluid in slightly higher concentrations than
in maternal serum. The plasma half-life of nicotine after iv infusion or
cigarette smoking averages about 2 h.  However, when half-life is
determined using the time course of urinary excretion of nicotine, which
is more sensitive in detecting lower levels of nicotine in the body, the
terminal half-life averages 11 h.

Nicotine is extensively metabolized – cotinine.

Non-Guideline	Dermal penetration

(species)	Zorin et al, 1999	In vitro tests of nicotine’s permeability
through human skin using Franz’ diffusion cells demonstrated the
dermal penetration of nicotine. Flux depended on the nicotine
concentration in a non-linear fashion, with the highest flux at 50% w/w
nicotine-water solution.  The lowest flux was in the acidic nicotine
solution and ethanol solution. Penetration continued after rinsing the
nicotine from the donor compartment, indicating that the skin acts as
reservoir for continued absorption. 

Non-Guideline

	Special studies:

Daily intake of nicotine from cigarette smoke	Benowitz znd Jacob III,
1984	Daily intake of nicotine in a group of 22 smokers averaged 37.6 (
17.7 mg (10.5 – 78.6 mg).  Nicotine intake per cigarette averaged
1.04( 0.36 mg.  Among several markers measured (carboxyhemoglobin level,
blood cotinine concentration), afternoon (4:00 pm) blood nicotine level
was the best marker for measuring nicotine intake in this group of
smokers.

A.3	Supplementary Toxicity Studies

	Subchronic Toxicity

Experimental data in male rats suggest that nicotine (free base)
ingested for 16 weeks at 50 or 200 mg/L in drinking water (3 and 14
mg/kg/day calculated on water intake) alters metabolic and  endocrine
factors that may be responsible, at least in part, for the development
of gastrointestinal ulcers and pancreatitis (Chowdhury et al, 1990).  
The plasma levels of CCK (cholecystokinin) were significantly increased
with nicotine intake but the amylase secretory response of pancreatic
acinar cells was inhibited in response to CCK-8
(cholecystokinin–octapeptide) and carbachol.  Prominent loss of
gastric mucosal surface was found in nicotine-treated animals with gross
microscopic evidence of bleeding ulcers.  Body weight gain in the
nicotine treated groups was significantly lower than in the controls at
every measurement point.  At the end of treatment period, body weight
gain in the low and high dose groups was approximately 74% and 53% of
the controls, respectively. Nicotine treatment was accompanied by
significantly lower food and water intake.  All of the metabolic
parameters except body weight gain were reversed upon nicotine
withdrawal. Histopathologic data showed a partial but not a complete
recovery of the pancreatic acinar cell morphology and gastric mucosal
surface following 4 weeks of nicotine withdrawal.

When adult male rats (185-225 g weight) were implanted subcutaneously
with nicotine pellets containing 0, 5, 15 or 50 mg nicotine (delivering
0, 9.9, 29.8, and 99.2 ug/hr, respectively; equivalent to 0.0, 1.2, 3.6,
12.0 mg/kg/day) for up to 12 weeks, increased pancreatic enzyme
biosynthesis and accumulation of digestive enzymes within the pancreas
were reported (Lau et al.  1990). Plasma nicotine concentrations at the
50 mg dose reached a steady state level of 76±19 ng/mL and cotinine
exceeded 300 ng/mL.  No effect on enzyme secretion was observed in rats
treated with 15 mg nicotine pellets, in rats treated with 15 mg pellets,
CCK-8-mediated amylase, trypsinogen, and chymotrypsinogen secretion was
maximally stimulated at 1.5 weeks and then decreased to near control
levels.  In rats treated with 50 mg nicotine pellets, enzyme release in
response to CCK-8 was also higher at 1.5 weeks, peaked at 3 weeks and
then decreased, such that at 12 weeks, enzyme release was at or below
control levels. Electron microscopy of pancreas from rats treated with
the 50 mg nicotine pellets revealed intracytoplasmic vacuoles appearing
after 3 weeks of treatment, and persisting throughout the remaining
experimental period (evidence of morphological damage).   Nicotine
treatment and durations did not have an effect on body weight,
pancreatic weight, DNA, RNA or protein concentrations.  From this study,
a LOAEL based on pancreatic morphogic damage is the 50 mg nicotine
pellet corresponding to a daily dose of 12 mg/kg and a NOAEL is 3.6
mg/kg/day.  Earlier work from the same laboratory (Dubick et al, 1988)
showed that adult male rats receiving nicotine via a time–release
pellet at a rate of 1.65 ug/min for 3 weeks, increases in digestive
pancreatic enzymes (amylase, trypsin and chymotrypsin) were noted
compared to controls. Final body weight or pancreatic weight was not
affected.

 hepatotoxin) or the less potent hepatotoxins chloroform or
1,1,1-trichloroethane nor the cholestatic effect of
α-naphthylisothiocyanate was modified.

Nicotine administration may adversely affect bone formation and decrease
body storage of vitamin D (Fung et al, 1999). Female rats (7-month old;
9-10/group) administered nicotine (nicotine hydrogen tartrate) at 0, 3.0
or 4.5 mg/kg/day dissolved in saline solution by subcutaneous osmotic
minipumps with a delivery rate of 0.25 ul/hr for three months had
significantly lower levels of 25-hydroxyvitamin D in the two nicotine
groups than controls. The high dose nicotine group had smaller vertebral
areas and a lower bone mineral content than the controls.  Tibial
endocortical mineral apposition rate was also significantly lower in the
high dose nicotine group than in the control group. Nicotine serum
levels at sacrifice time were 60 ± 6 ng/mL, and 85 ± 5 ng/mL at the
low and high dose, respectively. 

Nicotine tartrate, administered orally to 10 New Zealand white male
rabbits (2.4 mg/kg/day) in their drinking water for 25 weeks produced in
vivo morphologic effect on endothelial cells in the aortic arch (Booyse
et al, 1981).  It was estimated that each rabbit consumed about as much
as nicotine per day (2.4 mg/kg/day) as a person smoking 1- 2 packs of
cigarettes per day.  Fasting serum levels of glucose, triglycerides,
total cholesterol, and LDL-cholesterol were significantly (<0.001)
elevated in nicotine-treated rabbits.   Endothelial cells from
nicotine-treated arched areas (Evans-blue-stained) showed extensive
changes such as increased cytoplasmic silver disposition, increased
formation of microvilli, and numerous focal areas of  “ruffled”
endothelium (projections on cell surfaces).  Serum blood levels of
nicotine were not measured in this study.

	Chronic Toxicity

Dietary feeding of nicotine sulfate (0.0%, 0.00625%, 0.01255%, 0.025%,
0.05%, 0.1%, or 0.2% as nicotine base of the diet), nicotine tannate
(0.0%, 0.1%, 0.2%, or 0.4% of the diet) and nicotine bentonite (0.0%,
0.0625%, 0.1255%, 0.25%, 0.5%, 1.0%, or 2.0% of the diet) to rats
(initial weight 50-60 g) for 300 days resulted in retarded growth due to
nicotine toxicity and lower food consumption (Wilson & DeEds, 1936) with
a NOAEL of 0.006% nicotine base (4 mg/kg/day).  Retarded growth in rats
was reversed upon discontinuing the nicotine diets and rats resumed
normal growth.  Rats receiving the nicotine sulfate as 0.1% base and
above died within few days.  It was also demonstrated that “the
toxicity in terms of nicotine base was about the same for two nicotine
salts and the bentonite clay formulation” tested. Nicotine bentonite
for this study was prepared by mixing one part of nicotine base with
nine parts of finely powdered Wyoming bentonite and mixing thoroughly.
The bentonite nicotine complex was not merely an adsorption complex, but
rather some chemical bonding.  The nicotine was not removed by
extraction with alkali and only small amounts were removed by water, but
was readily extracted with dilute acid.

Developmental Toxicity

Pregnant CD-1 mice (N=19), exposed to ip injection of 0.1% nicotine
sulfate at a dose of 1.67 mg/kg body weight/day on gestational days 6-15
had significantly depressed maternal weight gain (37% decrease on GD18)
and fetal weight (64% decrease) compared to the controls. Fetal
crown-rump length fetal head dimensions (width, height and
circumference) were significantly reduced.  Histological examination
revealed that 9.6% of fetuses of nicotine injected mothers presented
clefts of the palate, whereas none of the control fetuses had that
anomaly. Nicotine treatment also had teratogenic effects on first molar
odontogenesis in the mouse. The mesiodistal diameter of molar tooth
germs of nicotine treated fetuses without cleft palate was significantly
less than those of controls (Saad et al, 1990 & 1991).  It was suggested
that nicotine, or its metabolic byproducts, interfere with normal
interaction between the epithelial and mesenchymal components of the
developing tooth.

Subcutaneous injection of nicotine to timed pregnant Sprague Dawley rats
(41 control and 57 nicotine treated) (3 mg/kg/day from gestation day 4
-20) resulted in significant reduced maternal weight gain and increased
maternal mortality (14%) and fetal resorptions (13%).  Litter size was
not affected. Nicotine exposure produced biochemical changes in the
fetal brain (elevation of fetal ornithine decarboxylase activity,
suppression of DNA synthesis (most profound in cerebellum),
desynchronization of the ontogenctic patterns of DNA, RNA and proteins
in every brain region) (Slotkin et al, 1986).

Subcutaneous injections of nicotine (3 or 6 mg/kg/day, divided into two
doses) on GD 7-20 to pregnant rats (10/group) resulted in abnormal
behavior such as ataxic gate, decrease in spontaneous activity,
creeping, or salivation in pregnant rats (3 or 6 mg/kg/day) and
convulsion and ptosis (6 mg/kg), decreased maternal body weight at GD 14
(both groups) and reduced offspring body weight. These nicotine
exposures also resulted in significant decreases in
dihydroxyphenylacetic acid (DOPAC) content in the neocortex and in both
the neocortex and in the midbrain plus pons medulla, respectively,
without any effects on the other brain regions such as the hypothalamus
or striatum.  Norepinephrine, serotonin, or 5-hydroxy-3-indolacetic acid
levels were not affected.  These data demonstrated that prenatal
nicotine exposure induced disturbances in the dopaminergic system in the
young adult period.  Furthermore, the region-specific reductions in the
DOPAC content suggests that the exposure to a high dose of nicotine in
utero might cause a predisposition to diseases related to a dopaminergic
dysfunction in the frontal cortex (Muneoka et al, 1999).

A recent study questioned developmental effects of nicotine reported in
the scientific literature (Hussein et al, 2007).  They found that a
nicotine dose of 6 mg/kg/day administered to Sprague Dawley time-mated
rats from gestation day 3 through 21 by osmotic mini pump resulted in
nicotine plasma concentrations of 115-174 ng/mL.  This is 3-10X higher
than published values observed in heavy smokers (10-50 ng/mL) and
pregnant mothers (2-10 ng/mL).  The total nicotine dose of 6 mg/kg/day
in the rat was considered to be equivalent to smoking 480 and 560
cigarettes per day (1 mg NIC per cigarette) by pregnant women weighing
60 and 70 kg, respectively, which is unrealistic. Maternal hematocrit
was not affected by nicotine administration, or the number of pups per
litter, pup weight, placental weight or the weight of various pups'
organs. Maternal weight changes were comparable in the nicotine treated
and control groups. Maternal plasma coticosterone concentrations were
not affected by the nicotine infusion versus the control rats.
Measurements of plasma nicotine concentration in this study demonstrated
that total nicotine clearance increases towards the second trimester but
remains unchanged thereafter, whereas the plasma concentrations
decrease. Thus plasma nicotine concentrations are maintained for at
least two thirds of the gestation period and only a marginal decrease is
observed during the third trimester. In earlier studies in CD-1 mice by
Paulson et al (1989), fetal retardation was observed at plasma nicotine
concentrations of 218-1284 ng/mL corresponding to 60 mg/kg/day, but not
at lower doses of 12 or 36 mg/kg/day corresponding to 100 and 400 ng/mL.

Genotoxicity

g/mL, whereas, SCE frequencies were increased significantly (150
g/mL concentrations (Trivedi et al, 1990). Nicotine (150 µg/mL) and
in combination with arecoline (an areca nut alkaloid) incubated in
Chinese hamster ovary (CHO) cell line (without S9 metabolic activation)
increased the SCE frequency/cell.  Nicotine alone at 300 µg/mL did not
increase the frequency of chromosomal aberrations/cell, but in
combination with arecoline it did at ≥90 µg/mL (Trivedi et al, 1993).

Nicotine was cytotoxic at 1.5 - 2.0 g/mL to human leucocytes in
culture in vitro without producing any chromosome damage.  However,
gross chromosomal aberrations including fuzzy chromosomes (stickiness),
aneuploidy and translocations were observed in mice receiving low
tolerable doses of the drug ( 0.07 -0.09 g/total body weight injected
to 5 weeks-4 month old mice in saline, two weekly injections for 3 weeks
before sacrifice and preparing  the bone marrow from the femora (Bishun
et al, 1972).

Nicotine has been shown to cause concomitant genotoxic and antiapoptotic
effect in human gingival fibroblasts (HGFs) in the cytokinesis-block
micronucleus (CBMN) test (Argentin and Cicchetti, 2004).   Treatment of
HGFs with nicotine, at a concentration of 1 uM, caused a statistically
significant increase of micronucleus (MN) frequency at the tested time
intervals, while no change was detected in cell growth under the same
conditions.  Preincubation of HGFs with 1 uM nicotine strongly
attenuated staurosporine (STP)-induced apoptosis. Cultures exposed to
nicotine showed an increase of reactive oxygen species, as determined by
increased levels of 2,7-dichlorofluorescein (DCF). When cells were
prelabeled with N-acetyl-cysteine (NAC), a substrate for glutathione
synthesis, and catalase (CAT), the oxygen free radical scavenger, a
significant reduction in cytogenetic damage was observed. 

Cotinine (a metabolite of nicotine and a biological monitoring marker of
nicotine absorption in humans) was positive in the presence or absence
of S9 in the bacterial luminescence genotoxicity test at 1.25 - 2.5
mg/mL (9- 30 h incubation).   In contrast, nicotine was not positive to
20 mg/mL concentrations for up to 40 hours of incubation.
Nicotine/cotinine mixtures were still positive at physiological
concentrations, with potentiation relative to cotinine alone with and
without S9 (Yim & Hee 1995). 

Tests by the R. J. Reynolds Tobacco Co. (Doolittle et al, 1995) on
nicotine and four of its major metabolites: cotinine, nicotine-N'-oxide,
cotinine-N-oxide, and trans-3'-hydroxycotinine in the Salmonella
mutagenicity assay (strains TA98, TA100, TA1535, TA1537, and TA1538) at
concentrations ranging from 0 to 1000 micrograms/plate and in the
Chinese hamster ovary sister-chromatid exchange (SCE) assay at
concentrations ranging from 0 to 1000 micrograms/mL,  with and without
S9 metabolic activation showed that none of the five compounds increased
the frequency of mutations or the frequency of SCEs. 

Recent data indicate that nicotine exerts significant direct genotoxic
effects in human target cells in vitro (Kleinsasser et al, 2005).  The
genotoxicity of nicotine was tested with the alkaline single-cell
microgel electrophoresis (Comet) assay using human lymphocytes and
target cells from lymphatic tissue of the palatine tonsils from healthy
patients.  One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and
4 mM induced a statistically significant dose-dependent increase of DNA
migration up to 3.8-fold and 3.2-fold in tonsillar cells and
lymphocytes, respectively. The minimum concentration eliciting
significant DNA damage was 0.5 mM nicotine. The genotoxic effect was
confirmed in a second series of experiments using nicotine of high
purity from two different suppliers.  Finally, DNA damage by nicotine
was compared in cells incubated in medium strictly adjusted to neutral
pH, with nonadjusted medium becoming alkaline with increasing nicotine
concentrations. Again no differences in DNA migration were observed. 
However, no differences in DNA damage were observed in cells from
smokers and nonsmokers incubated without nicotine. The lack of higher
DNA damage in smokers compared to nonsmokers could be a question of
nicotine dose, rapid DNA repair, or interactions with other smoke
constituents.  Further work by Sassen et al (2005) confirmed the
genotoxic effects of nicotine on human nasal epithelia. 

Many studies have demonstrated that nicotine penetrates the fetal brain
to cause various biochemical changes with behavioral consequences on the
offspring.   These studies are listed in the tox profile in Appendix 
A.2.  In all of these studies, nicotine was administered subcutaneously
or intraperitonealy.  Performance deficits in both learned and innate
behavioral measures throughout development and adulthood in offspring of
animals exposed to nicotine during gestation have been reported.  Doses
as low as 0.25 mg/kg/day produced behavioral changes.            

Immune System Toxicity

There is considerable evidence suggesting immune toxicity by nicotine. 
Few of these studies are discussed below. Other studies are listed in
the tox profile in Appendix A.2.

Maternal nicotine exposure (1 mg/kg body mass/d from GD 7 until weaning
three weeks after birth) resulted in a very low elastic tissue content
in the lungs of 1- and 7-day old rat pups compared with those of the
controls.  This may interfere with normal lung development since elastic
tissue is part of the lung connective tissue structure and is involved
in the formation of alveoli.  Impaired elastic synthesis may thus make
the rat pups more susceptible to lung diseases such as emphysema (Maritz
& Woolward, 1992).

	

Nicotine at 1 mg/kg (administered to young adult rats by intradermal
implantation for 3-4 weeks), inhibited both the T-dependent and
T-independent AFC responses and proliferation to anti-CD3. 
Significantly fewer NT T cells entered the S and G2/M phases than CON T
cells, indicating an arrest in the GO/G1 phase.  B and T cells were
unable to elevate the intracellular calcium levels normally in response
to ligation of antigen receptors, although Ca2+ responses of salivary
gland cells to acetylcholine were normal.  Serum cotinine levels of 219
± 40 ng/mL were comparable to average human smokers (Geng et al, 1995).

Prenatal nicotine exposure can cause long-term suppression of the
proliferative response of offspring immune cells (Basta et al, 2000). 
Thus in offspring of pregnant rats treated with nicotine (6 mg/kg/day)
from GD 4-20 subcutaneously, nicotine suppressed splenocyte
responsiveness to Concanavalin A or lipopolysaccharide and remained
subresponsive to stimulation well into adulthood.  Nicotine treatment
also resulted in reduced maternal weight gain during early gestation,
increased resorptions, increased offspring body weight and significantly
elevated offspring spleen weights.  In combination with ethanol, the
observed effects were more severe.

Exposure of T-cells (splenic mononuclear cells) from female mice to
physiological concentration of nicotine (1-100 μg/100 mL) can alter T
cell expression of CD28 and CTLA-4 and the CD4 T cell cytokine
expression pattern (Zhang & Petro, 1996). 

A.4	References

Registrant References:

MRID 41521101. Naas, D. (1990) Primary Dermal Irritation Study in Albino
Rabbits with Nicotine, Black Leaf 40: Lab Project Number: WIL-149014. 
Unpublished study prepared by WIL Research Laboratories, Inc. April 2,
1990. 19 p.

MRID 41521102.  Naas, D. (1990) Skin Sensitization Study in Albino
Guinea Pigs with               Nicotine, Black Leaf 40: Lab Project
Number: WIL-149015, unpublished study prepared by WIL Research
Laboratories, Inc., April 2, 1990..   38 p.   30 pages.

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 TC \l2 "A.4	References 

DRAFT

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