**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. \[Note: This document includes minor editorial
modifications made after August 22, 2007.\]

![](media/image1.png){width="0.8486111111111111in"
height="0.9243055555555556in"}

**UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**

WASHINGTON, D.C. 20460

OFFICE OF

PREVENTION, PESTICIDES

AND TOXIC SUBSTANCES

[August xx, 2007]{.mark}

**[MEMORANDUM]{.underline}**

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]{.underline}**

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 LD~50~ 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/m^3^) 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
(<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.

A subchronic oral rat toxicity study (Yuen *et al*. 1995) conducted with
nicotine hydrogen tartrate was selected as a basis for estimating the
incidental oral, dermal, and inhalation toxicity endpoints. Nicotine
hydrogen tartrate was administered to pregnant and non-pregnant female
rats in the drinking water for 10 days at 54 or 108 μmole/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)]{.underline}**

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 ft^3^. Each 6 ounce canister contains 0.05 lbs a.i. and treats
10,000 ft^3^.

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/m^3^ 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/m^3^ 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/m^3^ 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]{.underline}

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]{.underline}

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]{.underline} 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]{.underline}

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]{.underline} 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]{.underline} 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]{.underline} 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]{.underline} 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)]{.underline}**

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]{.underline}

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]{.underline} 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]{.underline} 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]{.underline}

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]{.underline} 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**

**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 ft^3^. Each 6 ounce canister contains 0.05 lbs a.i. and treats
10,000 ft^3^. 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  | Use      | Ap        | Timing of       |
|                  | of      | Sites    | plication | Application and |
|                  | Appl    |          | Rates     | Restrictions    |
|                  | ication |          |           |                 |
+------------------+---------+----------+-----------+-----------------+
| Fulex Nicotine   | Smoke   | Gre      | 1         | At fumigation,  |
| Fumigator        | fu      | enhouses | canister  | greenhouse      |
|                  | migator | on       | (12 oz)/  | temperature     |
| Insecticide      |         | or       |           | between 70-90   |
| (13.4% ai)       |         | namental | 20,000    | F. Foliage and  |
|                  |         | **       | ft^3^     | blossoms must   |
| EPA Reg. No.     |         | plants** |           | be dry. Avoid   |
|                  |         | (to      | or        | rainy or windy  |
| 1327-41          |         | control  |           | days. Close all |
|                  |         | aphids   | 1         | vents prior to  |
| Note: Available  |         | and      | canister  | use. Repeat     |
| in ready-to-use  |         | thrips)  | (6 oz)/   | treatment in    |
| 6- and 12-oz.    |         |          |           | one week if     |
| screw-capped     |         |          | 10,000    | necessary.      |
| steel canisters  |         |          | ft^3^     |                 |
| with wire        |         |          |           | **Note:         |
| igniters which   |         |          | Note:     | According to    |
| look and act     |         |          | Both      | information     |
| like sparklers.  |         |          | canister  | provided by the |
|                  |         |          | sizes     | registrant (Use |
|                  |         |          | provide   | Closure for     |
|                  |         |          | the same  | Nicotine RED,   |
|                  |         |          | ap        | J. Bloom,       |
|                  |         |          | plication | 06/18/2007), in |
|                  |         |          | rate.     | 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
ft^2^. 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         |          |          |                                 |
| F           |          |          |                                 |
| ormulations |          |          |                                 |
| for the     |          |          |                                 |
| B           |          |          |                                 |
| onideRabbit |          |          |                                 |
| and Dog     |          |          |                                 |
| R           |          |          |                                 |
| epellant.** |          |          |                                 |
+-------------+----------+----------+---------------------------------+
| Formulation | Method   | Use      | Application Rates, Timing of    |
|             | of       | Sites    | Application, and Restrictions   |
|             | App      |          |                                 |
|             | lication |          |                                 |
+-------------+----------+----------+---------------------------------+
| Bonide®     | Rabbit   | B        | According to the label, a       |
| Rabbit &    | and dog  | arrier/p | 3-pound package of product will |
| Dog Chaser  | r        | erimeter | produce a band of product 1     |
| EPA Reg.    | epellant | tr       | inch wide and 85 feet long and  |
| No. 4-465   | applied  | eatments | a 1-pound package of product    |
|             | by hand. | for use  | will produce a band of product  |
| (The        | The      | by       | 1 inch wide and 28 feet long.   |
| product     | product  | ho       | The product is applied in 2 or  |
| contains    | is       | meowners | more inch-wide bands around     |
| 0.35%       | s        | around   | areas to be protected.          |
| nicotine    | prinkled | res      |                                 |
| and is      | onto     | idential | **The reviewer estimates that a |
| present in  | treated  | lawns,   | 3-pound package of product will |
| this        | area     | flower   | provide a barrier/perimeter     |
| product as  | directly | gardens, | treatment around ≤144 ft^2^     |
| a naturally | from the | v        | area as follows:**              |
| occurring   | package. | egetable |                                 |
| component   |          | gardens, | A 3-pound package of product    |
| of tobacco  |          | orna     | will provide 42.5 linear feet   |
| dust;       |          | mentals, | of 2 inch-wide bands of         |
| hence, the  |          | trees    | barrier/perimeter treatment. If |
| formulation |          | and      | this product is applied in a    |
| is          |          | shrubs.  | closed circle it will encompass |
| considered  |          |          | at most a 144 ft^2^ of area     |
| a dust for  |          |          | (Area=πr ^2^ ) with a           |
| this risk   |          |          | circumference (Circumference =  |
| a           |          |          | 2πr) of 42.5 ft.                |
| ssessment.) |          |          |                                 |
|             |          |          | According to the label, before  |
| Note:       |          |          | treatment, try to remove all    |
| Marketed in |          |          | traces of animal droppings and  |
| r           |          |          | urine from the areas to be      |
| eady-to-use |          |          | protected. Be careful not to    |
| in 1 and    |          |          | apply product directly to       |
| 3-pound     |          |          | foliage or stems. Repeat        |
| poly bags.  |          |          | 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**

+---------------+------------------------------------------------------+
| **Table 2.2.  |                                                      |
| Test Compound |                                                      |
| No            |                                                      |
| menclature.** |                                                      |
+===============+======================================================+
| Compound      | ![](media/image2.wmf)                                |
+---------------+------------------------------------------------------+
| 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  | 54-11-5                                              |
| number        |                                                      |
+---------------+------------------------------------------------------+
| End-use       | Fulex Nicotine Fumigator (13.4% nicotine): EPA Reg.  |
| products (EP) | 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**

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       Merck Index
                          (partial decomposition) 

  pH                                              

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

  Water solubility                                

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

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

  Dissociation constant,                          
  pK~a~                                           

  Octanol/water partition                         
  coefficient, Log(K~OW~)                         

  UV/visible absorption                           
  spectrum                                        
  ----------------------- ----------------------- -----------------------

**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 LD~50~ 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.

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/m^3^) 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
(<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**

**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)**

**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 LD~50~ 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 LD~50~ 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 μmole/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 10^th^ 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 μmole/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 μmole/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 μmole/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
μmole/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**

Chronic exposure of Sprague Dawley female rats (initial weight 240 g)
through inhalation to 0.0 (34 rats) or 501±151 μg/ m^3^ of \>99% pure
nicotine/m^3^ (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 μg/m^3^ is equivalent to 0.34 ± 0.1
mg/kg/day (based on inhalation rate of 0.29 m^3^/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**

The genotoxic potential of nicotine and its metabolites has been
evaluated in numerous published studies. In some tests nicotine induced
chromosomal aberration (CA) and sister chromatid exchange (SCE)
frequencies in Chinese hamster ovary (CHO) cells at concentrations as
low as 375 µg/mL for CA and 150 µg/mL for SCE (Trivedi *et al*, 1990).
Whereas, other tests indicated that nicotine and its major metabolites
including cotinine were negative in the Salmonella mutagenicity assay
and the CHO SCE assay at concentrations ranging from 0 to 1000 µg/mL,
with and without S9 metabolic activation (Doolittle *et al*, 1995).
However, gross chromosomal aberrations including fuzzy chromosomes
(stickiness), aneuploidy and translocations were observed in mice
receiving low tolerable doses (0.07-0.09 μg/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."

(<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**

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

**3.2 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)**

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**

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)**

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**

+-----------------------+-----------+-----------------+--------------+
| **Table 3.2.6 Summary |           |                 |              |
| of Levels of Concern  |           |                 |              |
| for Risk Assessment   |           |                 |              |
| of Nicotine.**        |           |                 |              |
+-----------------------+-----------+-----------------+--------------+
| Route                 | S         | In              | Long-Term    |
|                       | hort-Term | termediate-Term |              |
|                       |           |                 | (\> 6        |
|                       | (1 - 30   | (1 - 6 Months)  | Months)      |
|                       | Days)     |                 |              |
+-----------------------+-----------+-----------------+--------------+
| **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**

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
(<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**

+---------+---------+---------+-----------+--------------------------+
| **Table |         |         |           |                          |
| 3.2.8.  |         |         |           |                          |
| Summary |         |         |           |                          |
| of      |         |         |           |                          |
| Toxico  |         |         |           |                          |
| logical |         |         |           |                          |
| Doses   |         |         |           |                          |
| and     |         |         |           |                          |
| En      |         |         |           |                          |
| dpoints |         |         |           |                          |
| for     |         |         |           |                          |
| N       |         |         |           |                          |
| icotine |         |         |           |                          |
| for Use |         |         |           |                          |
| in      |         |         |           |                          |
| Occup   |         |         |           |                          |
| ational |         |         |           |                          |
| and     |         |         |           |                          |
| Resi    |         |         |           |                          |
| dential |         |         |           |                          |
| Human   |         |         |           |                          |
| Health  |         |         |           |                          |
| Risk    |         |         |           |                          |
| Assess  |         |         |           |                          |
| ments** |         |         |           |                          |
+=========+=========+=========+===========+==========================+
| Ex      | Point   | Unce    | Level of  | Study and Toxicological  |
| posure/ | of      | rtainty | Concern   | Effects                  |
|         | De      | Factors | for Risk  |                          |
| S       | parture |         | A         |                          |
| cenario |         |         | ssessment |                          |
+---------+---------+---------+-----------+--------------------------+
| Inc     | NOA     | UF      | Re        | Yuen *et al*. 1995: oral |
| idental | EL=1.25 | ~A~=10x | sidential | study in drinking water  |
| Oral,   | mg      |         | LOC for   |                          |
| sho     | n       | UF      | MOE =     | LOAEL = 2.5 mg nicotine  |
| rt-term | icotine | ~H~=10x | 1000      | alkaloid/kg/day based on |
| e       | a       |         |           | hepatotoxicity: mild     |
| xposure | lkaloid | UF~     |           | fatty change, mild focal |
| (1-30   | /kg/day | DB~=10x |           | necrosis and mild dark   |
| days)   |         |         |           | 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, | NOA     | UF      | Occ       | Yuen *et al*. 1995: oral |
| Sho     | EL=1.25 | ~A~=10x | upational | study in drinking water  |
| rt-Term | mg      |         | and       |                          |
| (1-30   | n       | UF      | Re        |                          |
| days)   | icotine | ~H~=10x | sidential |                          |
| and     | a       |         | LOC for   |                          |
| Int     | lkaloid | UF~     | MOE =     |                          |
| ermedia | /kg/day | DB~=10x | 1000      |                          |
| te-Term |         |         |           |                          |
| (1-6    |         |         |           |                          |
| months) |         |         |           |                          |
+---------+---------+---------+-----------+--------------------------+
| Inha    | NOA     | UF      | Occ       | Yuen *et al*. 1995: oral |
| lation, | EL=1.25 | ~A~=10x | upational | study in drinking water  |
| Sho     | mg      |         | and       |                          |
| rt-Term | n       | UF      | Re        |                          |
| (1-30   | icotine | ~H~=10x | sidential |                          |
| days)   | a       |         | LOC for   |                          |
| and     | lkaloid | UF~     | MOE =     |                          |
| Int     | /kg/day | DB~=10x | 1000      |                          |
| ermedia |         |         |           |                          |
| te-Term |         |         |           |                          |
| (1-6    |         |         |           |                          |
| months) |         |         |           |                          |
+---------+---------+---------+-----------+--------------------------+
| Cancer  | C       |         |           |                          |
| (oral,  | lassifi |         |           |                          |
| dermal, | cation: |         |           |                          |
| inha    | N       |         |           |                          |
| lation) | icotine |         |           |                          |
|         | has not |         |           |                          |
|         | been    |         |           |                          |
|         | ev      |         |           |                          |
|         | aluated |         |           |                          |
|         | by the  |         |           |                          |
|         | NCI     |         |           |                          |
|         | N       |         |           |                          |
|         | ational |         |           |                          |
|         | Tox     |         |           |                          |
|         | icology |         |           |                          |
|         | P       |         |           |                          |
|         | rogram, |         |           |                          |
|         | the     |         |           |                          |
|         | Intern  |         |           |                          |
|         | ational |         |           |                          |
|         | Agency  |         |           |                          |
|         | for     |         |           |                          |
|         | R       |         |           |                          |
|         | esearch |         |           |                          |
|         | on      |         |           |                          |
|         | Cancer  |         |           |                          |
|         | and the |         |           |                          |
|         | EPA     |         |           |                          |
|         | Int     |         |           |                          |
|         | egrated |         |           |                          |
|         | Risk    |         |           |                          |
|         | Info    |         |           |                          |
|         | rmation |         |           |                          |
|         | System  |         |           |                          |
|         | for its |         |           |                          |
|         | po      |         |           |                          |
|         | tential |         |           |                          |
|         | ca      |         |           |                          |
|         | rcinoge |         |           |                          |
|         | nicity. |         |           |                          |
|         | It has  |         |           |                          |
|         | not     |         |           |                          |
|         | been    |         |           |                          |
|         | tested  |         |           |                          |
|         | in      |         |           |                          |
|         | rodents |         |           |                          |
|         | exempt  |         |           |                          |
|         | for the |         |           |                          |
|         | Waldum  |         |           |                          |
|         | *et     |         |           |                          |
|         | al*,    |         |           |                          |
|         | 1996    |         |           |                          |
|         | inh     |         |           |                          |
|         | alation |         |           |                          |
|         | study   |         |           |                          |
|         | which   |         |           |                          |
|         | tested  |         |           |                          |
|         | one     |         |           |                          |
|         | dose    |         |           |                          |
|         | only.   |         |           |                          |
|         | Ac      |         |           |                          |
|         | cording |         |           |                          |
|         | to the  |         |           |                          |
|         | Intern  |         |           |                          |
|         | ational |         |           |                          |
|         | Program |         |           |                          |
|         | on      |         |           |                          |
|         | C       |         |           |                          |
|         | hemical |         |           |                          |
|         | Safety  |         |           |                          |
|         | (IPCS)  |         |           |                          |
|         | review, |         |           |                          |
|         | lit     |         |           |                          |
|         | erature |         |           |                          |
|         | reports |         |           |                          |
|         | i       |         |           |                          |
|         | ndicate |         |           |                          |
|         | that    |         |           |                          |
|         | n       |         |           |                          |
|         | icotine |         |           |                          |
|         | is      |         |           |                          |
|         | neither |         |           |                          |
|         | an      |         |           |                          |
|         | in      |         |           |                          |
|         | itiator |         |           |                          |
|         | nor a   |         |           |                          |
|         | p       |         |           |                          |
|         | romoter |         |           |                          |
|         | of      |         |           |                          |
|         | tumors  |         |           |                          |
|         | in      |         |           |                          |
|         | mice.   |         |           |                          |
|         | There   |         |           |                          |
|         | is      |         |           |                          |
|         | incon   |         |           |                          |
|         | clusive |         |           |                          |
|         | e       |         |           |                          |
|         | vidence |         |           |                          |
|         | to      |         |           |                          |
|         | suggest |         |           |                          |
|         | that    |         |           |                          |
|         | co      |         |           |                          |
|         | tinine, |         |           |                          |
|         | an      |         |           |                          |
|         | o       |         |           |                          |
|         | xidized |         |           |                          |
|         | met     |         |           |                          |
|         | abolite |         |           |                          |
|         | of      |         |           |                          |
|         | ni      |         |           |                          |
|         | cotine, |         |           |                          |
|         | may be  |         |           |                          |
|         | carci   |         |           |                          |
|         | nogenic |         |           |                          |
|         | 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.
UF~A~ = extrapolation from animal to human (interspecies). UF~H~ =
potential variation in sensitivity among members of the human population
(intraspecies). UF~L~ = use of a LOAEL to extrapolate a NOAEL. UF~S\ =~
use of a short-term study for long-term risk assessment. UF~DB~ = 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**

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.

Nicotine administration (1 mg/kg or 10 mg/kg, subcutaneously twice daily
to rats for 1 or 2 weeks) 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 (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**

**4.1 Incident Reports**

1.  **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 81^st^ 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 178^th^ 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**

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**

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.

**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**

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 ft^2^ area at most.

Residential MOEs equal to or greater than 1000 are [not]{.underline} 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]{.underline} 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.*

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:]{.underline}

-   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.

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```
-   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.

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```
-   The application rate is based on the currently registered Bonide®
    Rabbit & Dog Chaser (EPA Reg. No. 4-465; label date 12/28/2006)
    end-use product label. **The label does not specify a maximum use
    rate in terms of lb ai/A. The reviewer has estimated that a 3-pound
    package of product, containing 0.35% nicotine (as the alkaloid),
    will provide a barrier/perimeter treatment around ≤144 ft^2^ area.**

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```
-   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:]{.underline}

-   **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.

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```
-   **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)\_\_\_\_]{.underline}**

    **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) ]{.underline}**

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

<table>
<colgroup>
<col style="width: 9%" />
<col style="width: 7%" />
<col style="width: 8%" />
<col style="width: 7%" />
<col style="width: 9%" />
<col style="width: 18%" />
<col style="width: 8%" />
<col style="width: 6%" />
<col style="width: 9%" />
<col style="width: 8%" />
<col style="width: 6%" />
</colgroup>
<tbody>
<tr class="odd">
<td colspan="11"><strong>Table 6.2.1. Residential Handler (Applicator
Only) Short-term Exposure and Risk for Nicotine (as a naturally
occurring component of tobacco dust).</strong></td>
</tr>
<tr class="even">
<td><strong>Scenario</strong></td>
<td><strong>Use Site</strong></td>
<td><p><strong>Dermal</strong></p>
<p><strong>Unit</strong></p>
<p><strong>Exposure (mg/lb) <sup>1</sup></strong></p></td>
<td><p><strong>Inhalation</strong></p>
<p><strong>Unit</strong></p>
<p><strong>Exposure (mg/lb) <sup>1</sup></strong></p></td>
<td><p><strong>Application</strong></p>
<p><strong>Rate <sup>2</sup></strong></p>
<p><strong>(lb ai/site/day)</strong></p></td>
<td><p><strong>Area</strong></p>
<p><strong>Treated</strong></p></td>
<td><p><strong>Dermal</strong></p>
<p><strong>Dose <sup>3</sup></strong></p>
<p><strong>(mg/kg/day)</strong></p></td>
<td><p><strong>Dermal</strong></p>
<p><strong>MOE <sup>5</sup></strong></p></td>
<td><p><strong>Inhalation</strong></p>
<p><strong>Dose <sup>4</sup> (mg/kg/day)</strong></p></td>
<td><p><strong>Inhalation</strong></p>
<p><strong>MOE <sup>6</sup></strong></p></td>
<td><p><strong>Total</strong></p>
<p><strong>MOE <sup>7</sup></strong></p></td>
</tr>
<tr class="odd">
<td colspan="11"><strong>Applicator</strong></td>
</tr>
<tr class="even">
<td rowspan="3"><p>Nicotine (0.35% a.i) <strong>dust</strong></p>
<p>Reg.# 4-465</p></td>
<td rowspan="3">In and around homes as a barrier/perimeter treatment to
repel rabbits and dogs</td>
<td rowspan="3">4.4</td>
<td rowspan="3">0.0434</td>
<td><p>0.0105</p>
<p>(1 3-pound package)</p></td>
<td><p>42.5 linear feet; could provide a barrier/perimeter around a 144
ft<sup>2</sup> area (or 0.0033 acres)</p>
<p>42.5 ft/2π = 6.77 ft</p>
<p>area = π(6.77 ft)<sup>2</sup> = 144 ft<sup>2</sup></p></td>
<td>6.6E-4</td>
<td>1894</td>
<td>6.5E-6</td>
<td>1.9E5</td>
<td>1875</td>
</tr>
<tr class="odd">
<td><p>0.0210</p>
<p>(2 3-pound packages)</p></td>
<td>288 ft<sup>2</sup> area (or 0.0066 acres)</td>
<td>1.32E-3</td>
<td>947</td>
<td>1.3E-5</td>
<td>9.6E4</td>
<td>938</td>
</tr>
<tr class="even">
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>

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**

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]{.underline} 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/ft^3^. 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/ft^3^ x 454 g/lb x 1ft^3^/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]{.underline}:

-   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.

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-   Average weight of toddler is 15 kg

[Equations and Calculations]{.underline}:

-   **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**              | *    | **F** | *    | **Dose  | **MOE   |
|                           | *IgR |       | *CF1 | ^1^     | ^2^**   |
|                           | (    |       | (mg/ | (mg/kg  |         |
|                           | g/da |       | g)** | /day)** |         |
|                           | y)** |       |      |         |         |
+---------------------------+------+-------+------+---------+---------+
| Episodic Ingestion        | 5    | 0.35% | 1000 | 1.17    | 1       |
+---------------------------+------+-------+------+---------+---------+
|                           | 0    |       |      | 0.00117 | *       |
|                           | .005 |       |      |         | *1000** |
|                           |      |       |      |         |         |
|                           | (0   |       |      |         |         |
|                           | .002 |       |      |         |         |
|                           | tsp) |       |      |         |         |
+---------------------------+------+-------+------+---------+---------+

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.)**

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**

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**

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
[[http://www.epa.gov/pesticides/cumulative/.]{.underline}](http://www.epa.gov/pesticides/cumulative/.)

**9.0 Occupational Exposure/Risk Pathway**

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.

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                             |
|                                    |                                 |
| ![](media/image3.wmf)              | ![](media/image4.wmf)           |
+------------------------------------+---------------------------------+

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)**

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/m^3^ 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/m^3^ 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/m^3^ 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**

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]{.underline} 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 ft^3^ or one 6
ounce canister per 10,000 ft^3^. 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:]{.underline}

-   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.

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```
-   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 ft^3^)(1 lb/16 ounces)(0.134 ai)
= 0.10 lb ai/20,000 ft^3^**

-   The air concentration was calculated at the maximum application
    rate. The application rate was adjusted to an air concentration of
    80.1 mg ai/m^3^ (milligrams of active ingredient per cubic meters).
    The air concentration was calculated using the following equation:

> **(0.10 lb ai/20,000 ft^3^)(35.3 ft^3^/m^3^)(454,000 mg/lb) = 80.1 mg
> ai/m^3^**

-   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.

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```
-   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.

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```
-   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.

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```
-   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.

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```
-   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.

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```
-   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.

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```
-   Air Concentration Equations:

**Inhalation Dose (mg/kg/day) = (C~a~ x BR x ET)/BW**

Where,

C~a~ = air concentration at time of application (time = 0)

BR = adult breathing rate (1 m^3^/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    | C~a~  | BR  | ET       | BW | Inha   | Inhal |
|                     |       |     |          |    | lation | ation |
|                     | (mg   | (m^ | (hr/day) | (k | Dose   | MOE   |
|                     | ai/   | 3^/ |          | g) | ^1^    | ^2^   |
|                     | m^3^) | hr) |          |    | (mg/k  |       |
|                     |       |     |          |    | g/day) |       |
+---------------------+-------+-----+----------+----+--------+-------+
| Baseline            | 80.1  | 1   | 0.5      | 70 | 0.57   | 2     |
|                     |       |     |          |    |        |       |
| No Respirator       |       |     |          |    |        |       |
+---------------------+-------+-----+----------+----+--------+-------+
|                     |       |     | 1.0      |    | 1.14   | 1     |
+---------------------+-------+-----+----------+----+--------+-------+
| PF10 Respirator     | 8.01  | 1   | 0.5      | 70 | 0.057  | 22    |
|                     |       |     |          |    |        |       |
| Half-face           |       |     |          |    |        |       |
| org                 |       |     |          |    |        |       |
| anic-vapor-removing |       |     |          |    |        |       |
| respirator          |       |     |          |    |        |       |
| providing 90%       |       |     |          |    |        |       |
| protection          |       |     |          |    |        |       |
+---------------------+-------+-----+----------+----+--------+-------+
|                     |       |     | 1.0      |    | 0.114  | 11    |
+---------------------+-------+-----+----------+----+--------+-------+
|                     | 8.01  | 1   | 0.011    | 70 | 0      | 1000  |
|                     |       |     |          |    | .00125 |       |
|                     |       |     | (\<1     |    |        |       |
|                     |       |     | minutes) |    |        |       |
+---------------------+-------+-----+----------+----+--------+-------+
| PF50 Full-face      | 1.6   | 1   | 0.5      | 70 | 0.01   | 125   |
| org                 |       |     |          |    |        |       |
| anic-vapor-removing |       |     |          |    |        |       |
| respirator          |       |     |          |    |        |       |
| providing 98%       |       |     |          |    |        |       |
| protection          |       |     |          |    |        |       |
+---------------------+-------+-----+----------+----+--------+-------+
|                     |       |     | 1.0      |    | 0.02   | 63    |
+---------------------+-------+-----+----------+----+--------+-------+
|                     | 1.6   | 1   | 0.055    | 70 | 0      | 1000  |
|                     |       |     |          |    | .00125 |       |
|                     |       |     | (\~3     |    |        |       |
|                     |       |     | minutes) |    |        |       |
+---------------------+-------+-----+----------+----+--------+-------+
| PF10,000            | 0.008 | 1   | 1.0      | 70 | 0.0001 | 12500 |
| Self-contained      |       |     |          |    |        |       |
| breathing apparatus |       |     |          |    |        |       |
| (SCBA) providing    |       |     |          |    |        |       |
| 99.99% protection   |       |     |          |    |        |       |
+---------------------+-------+-----+----------+----+--------+-------+

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) = \[C~a~ (mg ai/m^3^) x BR (m^3^/hr) x ET
(hr/day)\] / BW (70 kg)

Where,

C~a~ = air concentration at time of application (time = 0)

BR = adult breathing rate (1 m^3^/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**

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]{.underline} 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]{.underline} 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

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```
-   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 ft^3^)(43,560 ft^2^/acre)(10
ft ceiling) = 2.178 lb ai/acre**

-   Exposure Duration = 8 hours per day

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```
-   Body Weight = 70 kg

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```
-   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      **Activities**      **Reference**
                    Coefficients                        
                    (cm^2^/hr)**                        

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

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

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

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

**[Equations/Calculations:]{.underline}**

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

-   **Disoldgeable Foliar Residue (DFR)**

> **DFR~t~ (ug/cm^2^) = Application Rate (lb ai/acre) x F x (1-D)^t^ x
> 4.54E8 µg/lb x 2.47E-8 acre/cm^2^**
>
> Where:
>
> DFR~t~ = dislodgeable foliage residue on day \"t\" (ug/cm^2^)
>
> F = fraction of ai retained on foliage (0.2 unitless)
>
> D = fraction of residue that dissipates daily (0.1 unitless)

-   **Dermal Dose ~t~ = [DFR~t~ (µg/cm^2^) x 1E-3 mg/µg x Tc (cm^2^/hr)
    x ET (hrs)]{.underline} BW (kg)**

> Where,
>
> t = number of days after application day (days)
>
> DFR~t~ = dislodgeable foliage residue on day \"t\" (ug/cm^2^)
>
> Tc = transfer coefficient (cm^2^/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]{.underline}
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:

**C~AC~ = C~i~ x 0.5 ^(#\ ACH/0.693)^**

Where,

C~AC~ = air concentration after number of air changes of interest (mg
ai/m^3^)

C~i~ = initial air concentration at time of application (80.1 mg
ai/m^3^)

\# ACH = number of air changes (10)

**Inhalation Dose (mg/kg/day) = (C~AC~ x BR x ET)/BW**

Where,

C~AC~= air concentration after number of air changes of interest

BR = adult breathing rate (1 m^3^/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  |          |       |        |          |       |           |
| P       |          |       |        |          |       |           |
| ostappl |          |       |        |          |       |           |
| ication |          |       |        |          |       |           |
| E       |          |       |        |          |       |           |
| xposure |          |       |        |          |       |           |
| and     |          |       |        |          |       |           |
| Risk    |          |       |        |          |       |           |
| for     |          |       |        |          |       |           |
| N       |          |       |        |          |       |           |
| icotine |          |       |        |          |       |           |
| Used in |          |       |        |          |       |           |
| Greenho |          |       |        |          |       |           |
| uses.** |          |       |        |          |       |           |
+---------+----------+-------+--------+----------+-------+-----------+
| Crops   | DAT^1^   | D     | Dermal |          | D     |           |
|         |          | FR^2^ | D      |          | ermal |           |
|         |          | (ug/c | ose^3^ |          | M     |           |
|         |          | m^2^) | (mg/k  |          | OE^4^ |           |
|         |          |       | g/day) |          |       |           |
+---------+----------+-------+--------+----------+-------+-----------+
|         |          |       | Low    | High     | Low   | High      |
|         |          |       |        |          |       |           |
|         |          |       | (T     | (Tc=     |       |           |
|         |          |       | c=175) | 5100)    |       |           |
+---------+----------+-------+--------+----------+-------+-----------+
| Ornam   | 0        | 4.88  | 0.0976 | 2.844    | 13    | \<1       |
| entals, |          |       |        |          |       |           |
| B       |          |       |        |          |       |           |
| aseline |          |       |        |          |       |           |
+---------+----------+-------+--------+----------+-------+-----------+
|         | 3        | 3.56  | 0.0712 | 2.075    | 18    | \<1       |
|         | (RTI^5^  |       |        |          |       |           |
|         | )        |       |        |          |       |           |
+---------+----------+-------+--------+----------+-------+-----------+
|         | 40       | 0     | 0      | 0.042    | 868   | 30        |
|         |          | .0721 | .00144 |          |       |           |
+---------+----------+-------+--------+----------+-------+-----------+
|         |          |       |        |          |       |           |
+---------+----------+-------+--------+----------+-------+-----------+

1 DAT = Days after treatment

2 DFR = Dislodgeable Foliar Residue

3 Dermal Dose = \[DFR (ug/cm^2^) x Tc (cm^2^/hr) x 0.001 mg/ug x 8
hrs/day\] ÷ 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>
<colgroup>
<col style="width: 14%" />
<col style="width: 10%" />
<col style="width: 0%" />
<col style="width: 7%" />
<col style="width: 11%" />
<col style="width: 9%" />
<col style="width: 9%" />
<col style="width: 7%" />
<col style="width: 1%" />
<col style="width: 10%" />
<col style="width: 0%" />
<col style="width: 16%" />
</colgroup>
<thead>
<tr class="header">
<th colspan="12"><strong>Table 9.1.3.2. Non-cancer Inhalation
Postapplication Exposure and Risk for Nicotine Used in
Greenhouses.</strong></th>
</tr>
<tr class="odd">
<th>Exposure Scenario (Scenario #)</th>
<th><p>C<sub>i</sub></p>
<p>(mg/ai/m<sup>3</sup>)</p></th>
<th colspan="2">#ACH</th>
<th><p>C<sub>AC</sub> <sup>1</sup></p>
<p>(mg ai/m<sup>3</sup>)</p></th>
<th><p>BR</p>
<p>(m<sup>3</sup>/hr)</p></th>
<th><p>ET</p>
<p>(hr/day)</p></th>
<th><p>BW</p>
<p>(kg)</p></th>
<th colspan="2">Inhalation Dose <sup>2</sup> (mg/kg/day)</th>
<th colspan="2">Inhalation MOE <sup>3</sup></th>
</tr>
</thead>
<tbody>
<tr class="odd">
<td colspan="12"></td>
</tr>
<tr class="even">
<td>Greenhouse Re-entry</td>
<td colspan="2">80.1</td>
<td>10</td>
<td>0.0036</td>
<td>1</td>
<td>8</td>
<td colspan="2">70</td>
<td colspan="2">0.00041</td>
<td>3049</td>
</tr>
</tbody>
</table>

1 C~AC~ = C~i~ x 0.5 ^(#\ ACH/0.693)^

> Where,
>
> C~AC~ = air concentration after number of air changes of interest (mg
> ai/m^3^)
>
> C~i~ = initial air concentration at time of application (80.1 mg
> ai/m^3^)
>
> \# ACH = number of air changes (10)

2 Inhalation Dose (mg/kg/day) = \[C~AC~ (mg ai/m^3^) x BR (m^3^/hr) x ET
(hr/day)\] / BW

> Where,
>
> C~a~= air concentration at time of application (time = 0)
>
> BR= adult breathing rate (1 m^3^/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.

1.  **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**

**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**

**[Fulex Nicotine Fumigator (EPA Reg. No. 1327-41)]{.underline}**

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]{.underline} 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]{.underline} 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)]{.underline}**

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:**

**[AGENCY MEMORANDA CITED IN THIS DOCUMENT]{.underline}**

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]{.underline}**

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**

**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            | yes         | no          |
|                                         |             |             |
| 870.1200 Acute Dermal Toxicity          | yes         | no          |
|                                         |             |             |
| 870.1300 Acute Inhalation Toxicity      | yes         | no          |
|                                         |             |             |
| 870.2400 Primary Eye Irritation         | yes         | no          |
|                                         |             |             |
| 870.2500 Primary Dermal Irritation      | yes         | yes         |
|                                         |             |             |
| 870.2600 Dermal Sensitization           | yes         | yes         |
+-----------------------------------------+-------------+-------------+
| 870.3100 Oral Subchronic (rodent)       | NR          | \-          |
|                                         |             |             |
| 870.3150 Oral Subchronic (nonrodent)    | NR          | \-          |
|                                         |             |             |
| 870.3200 21-Day Dermal                  | Yes         | no          |
|                                         |             |             |
| 870.3250 90-Day Dermal                  | NR          | \-          |
|                                         |             |             |
| 870.3465 90-Day Inhalation (21 -- day)  | Yes         | no          |
+-----------------------------------------+-------------+-------------+
| 870.3700a Developmental Toxicity        | Yes         | no          |
| (rodent)                                |             |             |
|                                         | NR          | \-          |
| 870.3700b Developmental Toxicity        |             |             |
| (nonrodent)                             | Yes         | no          |
|                                         |             |             |
| 870.3800 Reproduction                   |             |             |
+-----------------------------------------+-------------+-------------+
| 870.4100a Chronic Toxicity (rodent)     | NR          | \-          |
|                                         |             |             |
| 870.4100b Chronic Toxicity (nonrodent)  | NR          | \-          |
|                                         |             |             |
| 870.4200a Oncogenicity (rat)            | NR          | \-          |
|                                         |             |             |
| 870.4200b Oncogenicity (mouse)          | NR          | \-          |
|                                         |             |             |
| 870.4300 Chronic/Oncogenicity           | NR          | \-          |
+-----------------------------------------+-------------+-------------+
| 870.5100 Mutagenicity---Gene Mutation - | Yes         | Yes^\$^     |
| bacterial                               |             |             |
|                                         | Yes         | Yes^\$^     |
| 870.5300 Mutagenicity---Gene Mutation - |             |             |
| mammalian                               | Yes         | Yes^\$^     |
|                                         |             |             |
| 870.5xxx Mutagenicity---Structural      | Yes         | Yes^\$^     |
| Chromosomal Aberrations                 |             |             |
|                                         |             |             |
| 870.5xxx Mutagenicity---Other Genotoxic |             |             |
| Effects                                 |             |             |
+-----------------------------------------+-------------+-------------+
| 870.6100a Acute Delayed Neurotox. (hen) | NR          | \-          |
|                                         |             |             |
| 870.6100b 90-Day Neurotoxicity (hen)    | NR          | \-          |
|                                         |             |             |
| 870.6200a Acute Neurotox. Screening     | NR          | \-          |
| Battery (rat)                           |             |             |
|                                         | NR          | \-          |
| 870.6200b 90-Day Neuro. Screening       |             |             |
| Battery (rat)                           | NR          | \-          |
|                                         |             |             |
| 870.6300 Develop. Neuro                 |             |             |
+-----------------------------------------+-------------+-------------+
| 870.7485 General Metabolism             | NR          | \-          |
|                                         |             |             |
| 870.7600 Dermal Penetration             | NR          | \-          |
+-----------------------------------------+-------------+-------------+
| Special Studies for Ocular Effects      | NR          | \-          |
|                                         |             |             |
| > Acute Oral (rat)                      | NR          | \-          |
| >                                       |             |             |
| > Subchronic Oral (rat)                 | NR          | \-          |
| >                                       |             |             |
| > Six-month Oral (dog)                  |             |             |
+-----------------------------------------+-------------+-------------+

^\$^ Published studies satisfy this requirement.

**A.2 Toxicity Profiles**

+----------+----------------+-------------+-------------------+-------+
| Number   |                |             |                   |       |
| range    |                |             |                   |       |
| CHAPT    |                |             |                   |       |
| ER**Tabl |                |             |                   |       |
| eA.2.1a. |                |             |                   |       |
| Acute    |                |             |                   |       |
| Toxicity |                |             |                   |       |
| Profile  |                |             |                   |       |
| --       |                |             |                   |       |
| Ni       |                |             |                   |       |
| cotine** |                |             |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Number   | Number range   | Number      | Number range      | N     |
| range    | CHAPTERStudy   | range       | CHAPTERResults    | umber |
| CHAPTERG | Type           | CHAPTERMRID |                   | range |
| uideline |                | No          |                   | CHAPT |
| No.      |                | ./Reference |                   | ERTox |
|          |                |             |                   | icity |
|          |                |             |                   | Cat   |
|          |                |             |                   | egory |
+----------+----------------+-------------+-------------------+-------+
| Non-G    | Acute oral --  | IPCS        | LD~50\ =~ 50      | I     |
| uideline | rat            | INCHEM,     | mg/kg             |       |
| Number   |                | 1991\*      |                   | I     |
| range    | Acute oral -   |             | LD~50\ =~ 3 mg/kg |       |
| CHAPTER  | muse           |             |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Non-G    | Acute oral --  | Matsushima  | LD~50\ =~ 10-12   | I     |
| uideline | dog            | *et el*,    | mg/kg             |       |
|          |                | 1995        |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Non-g    | Acute i.p. -   | Priestly    | LD~50\ =~ 13.5    | I     |
| uideline | mice           | and Plaa    | mg/kg             |       |
|          |                | 1976        |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Number   | Acute dermal - | NA          |                   |       |
| range    | rabbit         |             |                   |       |
| CHAPTER  |                |             |                   |       |
| 870.1200 |                |             |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Number   | Acute          | NA          |                   |       |
| range    | inhalation -   |             |                   |       |
| CHAPTER  | rat            |             |                   |       |
| 870.1300 |                |             |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Number   | Eye            | NA          |                   |       |
| range    | irritation -   |             |                   |       |
| CHAPTER  | rabbit         |             |                   |       |
| 870.2400 |                |             |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Number   | Dermal         | 41521101    | Slightly irritant | IV    |
| range    | irritation --  |             |                   |       |
| CHAPTER  | rabbit         |             |                   |       |
| 870.2500 |                |             |                   |       |
|          | (40% nicotine  |             |                   |       |
|          | formula)       |             |                   |       |
+----------+----------------+-------------+-------------------+-------+
| Number   | Skin           | 41521102    | Not a skin        |       |
| range    | sensitization  |             | sensitizer        |       |
| CHAPTER  |                |             |                   |       |
| 870.2600 | (40% nicotine  |             |                   |       |
|          | formula)       |             |                   |       |
+----------+----------------+-------------+-------------------+-------+

\* <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    LD~50~ = \>     IV
                                                      5000 mg/kg      

  870.1200      Acute dermal: rabbit      42631101    LD~50~ =        IV
                                                      greater than    
                                                      2020 mg/kg      

  870.1300      Acute inhalation: rat     42631102    LC~50~ = \>5.39 IV
                                                      mg/L            

  870.2400      Acute eye irritation:     42631103    minimally       IV
                rabbit                                irritating      

  870.2500      Acute dermal irritation:  42631104    Mildly          IV
                rabbit                                irritating      

  870.2600      Skin sensitization:       42640001    Not sensitizer  NA
                guinea pig                                            
  ------------- ------------------------- ----------- --------------- ----------

+----------+----------+-----------------+------------------------------+
| **Table  |          |                 |                              |
| A.2.2    |          |                 |                              |
| Sub      |          |                 |                              |
| chronic, |          |                 |                              |
| Chronic  |          |                 |                              |
| and      |          |                 |                              |
| Other    |          |                 |                              |
| Toxicity |          |                 |                              |
| Profile  |          |                 |                              |
| of       |          |                 |                              |
| Ni       |          |                 |                              |
| cotine** |          |                 |                              |
+==========+==========+=================+==============================+
| **G      | **Study  | **MRID No.      | **Results**                  |
| uideline | Type**   | (year)/         |                              |
| No.**    |          | Classification  |                              |
|          |          | /Doses**        |                              |
+----------+----------+-----------------+------------------------------+
| Non-g    | Subacute | Yuen *et al*.   | Resulted in mild fatty       |
| uideline |          | 1995            | change, mild focal necrosis  |
|          | Pregnant |                 | and mild dark cell change    |
|          | and      | Nicotine        | (containing numerous         |
|          | non-     | tartrate in     | prominent pore annuli in the |
|          | pregnant | drinking water  | nuclear membranes and the    |
|          | female   | for 10 days at  | mitochondria appeared        |
|          | rats     | 54 or 108       | decreased in size with a     |
|          |          | μmole/l (8.76,  | decrease in mitochondrial    |
|          |          | 17.52 mg/l =    | granules and loss of         |
|          |          | 1.25 or 2.5     | aristae) in a dose           |
|          |          | mg/kg/day)      | 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-g    | Su       | Lau *et al*.    | Increased pancreatic enzyme  |
| uideline | bchronic | 1990            | biosynthesis and             |
|          |          |                 | accumulation of digestive    |
|          | Sprague  | adult male rats | enzymes within the pancreas  |
|          | Dawley   | (185-225 g      | were reported. Plasma        |
|          | Rats     | initial weight) | nicotine at the 50 mg dose   |
|          |          | were implanted  | reached a steady state level |
|          |          | with nicotine   | of 76±19 ng/mL and cotinine  |
|          |          | pellets         | exceeded 300 ng/mL. Electron |
|          |          | containing 0,   | microscopy of pancreas from  |
|          |          | 5, 15 or 50 mg  | rats treated with the 50 mg  |
|          |          | nicotine        | nicotine dose revealed       |
|          |          | (delivering 0,  | intracytoplasmic vacuoles    |
|          |          | 9.9, 29.8, and  | appearing after 3 weeks of   |
|          |          | 99.2 ug/hr,     | treatment, and persisting    |
|          |          | respectively)   | throughout the remaining     |
|          |          | for up to 12    | experimental period          |
|          |          | weeks           | (evidence of morphological   |
|          |          | (equivalent to  | damage). Nicotine treatment  |
|          |          | 0.0, 1.2, 3.6,  | and durations did not have   |
|          |          | 12.0 mg/kg/day) | 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-g    | Su       | (Dubick *et     | Showed that increases in     |
| uideline | bchronic | al*, 1988)      | digestive pancreatic enzymes |
|          |          |                 | (amylase, trypsin and        |
|          | Sprague  | adult male rats | chymotrypsin; 51%, 28%, 35%  |
|          | Dawley   | (190-200 g)     | higher) were noted compared  |
|          | Rats     | receiving       | to controls. Final body      |
|          |          | nicotine via a  | weight or pancreatic weight  |
|          |          | time--release   | was not affected.            |
|          |          | pellet at a     |                              |
|          |          | rate of 1.65    |                              |
|          |          | ug/min          |                              |
|          |          | )equivalent to  |                              |
|          |          | 12 mg/kg/day)   |                              |
|          |          | for 3 weeks     |                              |
+----------+----------+-----------------+------------------------------+
| Non-g    | Su       | (Priestly and   | no evidence of               |
| uideline | bchronic | Plaa 1976)      | hepatotoxicity as measured   |
|          |          |                 | by serum glutamic-pyruvic    |
|          | CF-1     | Male mice,      | transaminase or serum        |
|          | Swiss    | 20-25 g initial | alkaline phosphatase         |
|          | derived  | weight)         | activities. The LD~50~ of    |
|          | mice     | consuming       | nicotine HCl administered    |
|          |          | nicotine HCl in | i.p. in mice was 13.5 mg/kg. |
|          |          | their drinking  | Nicotine did not modify the  |
|          |          | water (25 mg/L; | hepatotoxic response of CCl4 |
|          |          | equivalent to 7 | or the less potent           |
|          |          | mg/kg/day)      | hepatotoxins chloroform or   |
|          |          | daily for 2-3   | 1,1,1-trichloroethane nor    |
|          |          | months or       | the cholestatic effect of    |
|          |          | injected i.p.   | α-naphthylisothiocyanate was |
|          |          | with 5 mkg once | modified.                    |
|          |          | or for 3 weeks  |                              |
+----------+----------+-----------------+------------------------------+
| Non-g    | Su       | Kavitharaj and  | Administration of nicotine   |
| uideline | bchronic | Vijayammal,     | produced enhanced synthesis  |
|          |          | 1999            | of cholesterol,              |
|          | Sprague  |                 | triglycerides, phospholipids |
|          | Dawley   | Nicotine,       | and free fatty acids in the  |
|          | Male     | subcutaneously  | liver and testes and lower   |
|          | Rats     | at 0.0, 0.6     | serum testosterone and       |
|          |          | mg/kg/day for   | estradiol levels suggesting  |
|          |          | 21 days, or 0.6 | gonadotoxic effects. Body    |
|          |          | mgk nicotine    | weight gain at sacrifice     |
|          |          | for 21 days +   | time in the nicotine treated |
|          |          | mecamylamine at | rats was less than the       |
|          |          | 0.8 mgk on Days | control rats (22.14 ± 0.86 g |
|          |          | 21, 23, and 25  | vs 34.28 ± 1.61 g in the     |
|          |          | (6 rats/group,  | controls). The activity of   |
|          |          | 1.5 months old, | the lipogenic enzymes (NADPH |
|          |          | 120-150 g       | generating enzymes) was      |
|          |          | weight)         | higher in the liver, but not |
|          |          | sacrificed on   | altered in the testes.       |
|          |          | Day 35.         | Mecamylamine (a known        |
|          |          |                 | inhibitor of nicotine)       |
|          |          |                 | counteracted the effects     |
|          |          |                 | seen in rats treated with    |
|          |          |                 | nicotine only.               |
+----------+----------+-----------------+------------------------------+
| Non-g    | Su       | Fung *et al*,   | had significantly lower      |
| uideline | bchronic | 1999            | levels of 25-hydroxyvitamin  |
|          |          |                 | D than controls. The high    |
|          | Adult    | nicotine 3.0 or | dose nicotine group had      |
|          | Female   | 4.5 mg/kg/day   | smaller vertebral areas and  |
|          | Rats     | (9-10           | a lower bone mineral content |
|          |          | rats/group) by  | and significantly lower      |
|          |          | subcutaneous    | tibial endocortical mineral  |
|          |          | osmotic         | apposition rate than the     |
|          |          | minipumps       | controls. Nicotine serum     |
|          |          |                 | levels at sacrifice time     |
|          |          |                 | were 60 ± 6 and 85 ± 5 ng/mL |
|          |          |                 | at the low and high dose,    |
|          |          |                 | respectively.                |
+----------+----------+-----------------+------------------------------+
| Non-g    | Su       | Booyse *et al*, | produced *in vivo*           |
| uideline | bchronic | 1981            | morphologic effect on        |
|          |          |                 | endothelial cells in the     |
|          | Male New | Nicotine        | aortic arch. Fasting serum   |
|          | Zealand  | tartrate,       | levels of glucose,           |
|          | white    | administered    | triglycerides, total         |
|          | rabbits  | orally (2.4     | cholesterol, and             |
|          |          | mg/kg/day) in   | LDL-cholesterol were         |
|          |          | drinking water  | significantly (\<0.001)      |
|          |          | for 25 weeks to | elevated in nicotine-treated |
|          |          | 10 rabbits      | 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-g    | Su       | Chowdhury *et   | alters metabolic, endocrine, |
| uideline | bchronic | al*, 1990       | and pathologic factors that  |
|          |          |                 | may be responsible, at least |
|          | Sprague  | nicotine        | in part, for the development |
|          | Dawley   | ingested for 16 | of gastrointestinal ulcers   |
|          | Male     | weeks at 50 and | and pancreatitis.            |
|          | Rats     | 200 mg/l in     | Endocrinological studies     |
|          |          | drinking water  | showed that the plasma       |
|          |          | equivalent to 7 | levels of CCK were           |
|          |          | and 28          | significantly increased with |
|          |          | mg/kg/day       | 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 | 2        | NR              |                              |
|          | 1/28-Day |                 |                              |
|          | dermal   |                 |                              |
|          | toxicity |                 |                              |
|          | (        |                 |                              |
|          | species) |                 |                              |
+----------+----------+-----------------+------------------------------+
| 870.3250 | 90-Day   | NR              |                              |
|          | dermal   |                 |                              |
|          | toxicity |                 |                              |
|          | (        |                 |                              |
|          | species) |                 |                              |
+----------+----------+-----------------+------------------------------+
| Non-G    | Chronic, | Waldum *et al*, | resulted in slightly         |
| uideline | In       | 1996            | depressed weight (Waldum *et |
|          | halation |                 | al*, 1996). Nicotine plasma  |
|          |          | inhalation to   | concentration after 5 days   |
|          | Female   | 501±151 µg/m^3^ | was 108.4 ± 55.1 ng/mL and   |
|          | rats     | , 20 h/day, 5   | remained fairly constant and |
|          |          | days/week for 2 | after 24 months it was       |
|          |          | years           | 129.8±43.0 ng/mL. All        |
|          |          | (equivalent to  | parameters measured were     |
|          |          | 0.3 ± 0.09      | comparable to the controls   |
|          |          | mg/kg/day)      | 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-G    | Devel    | Saad *et al*,   | Depressed maternal weight    |
| uideline | opmental | 1990, 1991      | gain and fetal weight. Fetal |
|          |          |                 | crown-rump length fetal head |
|          | Mouse    | Pregnant CD-1   | dimensions were              |
|          |          | mice, exposed   | significantly reduced.       |
|          |          | to ip injection | Histological examination     |
|          |          | of 0.1%         | revealed that 9.6% of        |
|          |          | nicotine        | fetuses of nicotine injected |
|          |          | sulfate at a    | mothers presented clefts of  |
|          |          | dose of 1.67    | the palate (none in the      |
|          |          | mg/kg body      | control). Nicotine treatment |
|          |          | weight/day on   | also had teratogenic effects |
|          |          | gestational     | on first molar odontogenesis |
|          |          | days 6-15       | 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-G    | Devel    | Chowdhury &     | dental asymmetries           |
| uideline | opmental | Bromage 2000    | (calculated as a size        |
|          |          |                 | difference between a tooth   |
|          | Rat      | experimental    | and its antimere) were       |
|          |          | details of      | significantly increased      |
|          |          | nicotine        | while occlusal areas were    |
|          |          | treatment were  | significantly decreased in   |
|          |          | not presented   | nicotine-exposed rats        |
|          |          |                 | compared to control rats.    |
|          |          |                 | Females tending to exhibit   |
|          |          |                 | the deleterious effects of   |
|          |          |                 | nicotine more so than males  |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Nasrat *et al*, | Nicotine increased the       |
| uideline | opmental | 1986            | perinatal mortality. The     |
|          |          |                 | male to female ratio was in  |
|          | Mouse    | Nicotine        | favor of females in the      |
|          |          | administered    | nicotine treated mice during |
|          |          | subcutaneously  | the 2^nd^ and 3^rd^          |
|          |          | to mice during  | trimester. When large doses  |
|          |          | gestation at ≥  | of the drug were given       |
|          |          | 450 μg/kg/day   | especially in the second and |
|          |          | dose            | third trimesters of          |
|          |          |                 | gestation, there was a       |
|          |          |                 | significant shortening of    |
|          |          |                 | the gestation period.        |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Leblebic        | Nicotine had minimal         |
| uideline | opmental | ioglu-Bekcioglu | ossification effects on the  |
|          |          | *et al*, 1995   | fetuses as measured by       |
|          | Mouse    |                 | staging and measuring        |
|          |          | Nicotine (12    | craniofacial bones, and      |
|          |          | mkg or nicotine | counting ossification centra |
|          |          | plus caffeine   | in sternbrae and in cervical |
|          |          | (125 mkg) by    | and sacrococcygeal           |
|          |          | intubation      | vertebrae. Caffeine had      |
|          |          | GD6-18.         | greater effect.              |
|          |          | (7/group)       |                              |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Williams &      | Mean fetal body fat was      |
| uideline | opmental | Kanagasabai,    | significantly increased in   |
|          |          | 1984.           | fetuses of rats administered |
|          | Rat      |                 | nicotine during pregnancy.   |
|          |          | 2.46 ± 0.18     | Rate for maternal lypolysis  |
|          |          | mg/kg/day in    | were higher in the nicotine  |
|          |          | drinking water. | treated animals. Maternal    |
|          |          | GD 0-20         | body weights gains were      |
|          |          |                 | significantly lower (77.2%   |
|          |          |                 | of controls, p \< 0.001).    |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | (Witschi *et    | Nicotine --caused 100%       |
| uideline | opmental | al*, 1994)      | pregnancy failure in animals |
|          |          |                 | treated GD2-19 with 3.5      |
|          | Rat      | Nicotine -      | mg/day and 50% in animals    |
|          |          | delivering      | treated GD 2-7, and 55% in   |
|          |          | transdermal     | animals exposed GD2-19 to    |
|          |          | patches applied | 1.75 mg/day. Litter size and |
|          |          | on the back of  | pup weights were not         |
|          |          | pregnant female | affected. Nicotine and       |
|          |          | rats            | cotinine not detected in     |
|          |          |                 | animals exposed the first    |
|          |          | 3.5mg/day (2    | trimester of pregnancy. In   |
|          |          | rats GD 2-19; 8 | animals exposed the entire   |
|          |          | rats GD 2-7; 3  | pregnancy at 1.75 mg/day     |
|          |          | rats GD 2-5)    | patches, 3 pregnant animals  |
|          |          |                 | out of six had measurable    |
|          |          | 1.75 mg/day     | nicotine levels (43 ± 22     |
|          |          | patch (13 rats  | ng/mL) and all had cotinine  |
|          |          | GD 2-19; 4 rats | levels (100 ± 48 ng/mL). The |
|          |          | GD 2-7)         | 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-G    | Devel    | Slotkin *et     | significant reduced maternal |
| uideline | opmental | al*, 1986       | weight gain and increased    |
|          |          |                 | maternal mortality (14%) and |
|          | Rat      | Subcutaneous    | fetal resorptions (13%).     |
|          | (Sprague | injection of    | Litter size was not          |
|          | Dawley   | nicotine (3     | affected. Biochemical        |
|          |          | mg/kg/day from  | changes in the fetal brain   |
|          |          | gestation day 4 | (elevation of fetal          |
|          |          | -20) to timed   | ornithine decarboxylase      |
|          |          | pregnant        | activity, suppression of DNA |
|          |          | Sprague Dawley  | synthesis (most profound in  |
|          |          | rats (41        | cerebellum),                 |
|          |          | control and 57  | desynchronization of the     |
|          |          | nicotine        | ontogenctic patterns of DNA, |
|          |          | treated)        | RNA and proteins in every    |
|          |          |                 | brain region)                |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Muneoka *et     | Abnormal behavior such as    |
| uideline | opmental | al*, 1999       | ataxic gate, decrease in     |
|          |          |                 | spontaneous activity,        |
|          | Rat      | Subcutaneous    | creeping, or salivation in   |
|          | (Sprague | injections of   | pregnant rats (3 or 6        |
|          | Dawley)  | nicotine (3 or  | mg/kg/day) and convulsion    |
|          |          | 6 mg/kg/day,    | and ptosis (6 mg/kg),        |
|          |          | divided into    | decreased maternal body      |
|          |          | two doses) on   | weight at GD 14 (both        |
|          |          | GD 7-20 to      | groups) and reduced          |
|          |          | pregnant rats   | offspring body weight.       |
|          |          | (10/group)      | Significant decreases in     |
|          |          |                 | dihydroxyphenylacetic acid   |
|          |          |                 | (DOPAC) content in the       |
|          |          |                 | neocortex and in both the    |
|          |          |                 | neocortex and in the         |
|          |          |                 | midbrain plus pons medulla,  |
|          |          |                 | respectively.                |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Hussein *et     | resulted in nicotine plasma  |
| uideline | opmental | al*, 2007       | concentrations of 115-174    |
|          |          |                 | ng/mL. Maternal hematocrit   |
|          | Rat      | nicotine dose   | was not affected by nicotine |
|          |          | of 6 mg/kg/day  | administration, or the       |
|          |          | administered to | number of pups per litter,   |
|          |          | Sprague Dawley  | pup weight, placental weight |
|          |          | time-mated rats | or the weight of various     |
|          |          | from gestation  | pups\' organs. Maternal      |
|          |          | day 3 through   | weight changes were          |
|          |          | 21 by osmotic   | comparable in the nicotine   |
|          |          | mini pump       | treated and control groups.  |
|          |          |                 | Maternal plasma              |
|          |          |                 | coticosterone concentrations |
|          |          |                 | were not affected by the     |
|          |          |                 | nicotine infusion versus the |
|          |          |                 | control rats.                |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Zahalka *et al* | Brain region weights were    |
| uideline | opmental | 1993            | unaffected. Postnatal        |
|          |          |                 | development of striatal      |
|          | Rat      | Nicotine\'s     | M1-receptor binding, as      |
|          |          | effects on      | identified with              |
|          |          | ontogeny of     | \[^3^H\]pirenzepine, was     |
|          |          | postsynaptic    | significantly impaired with  |
|          |          | muscarinic      | either of the fetal nicotine |
|          |          | M1-receptors in | regimens. Treatment with 2   |
|          |          | rat striatum    | mkg also produced            |
|          |          | and hippocampus | alterations in striatal      |
|          |          | were            | receptor affinity state,     |
|          |          | investigated    | characterized by enhanced    |
|          |          | after           | ability of an agonist        |
|          |          | continuous      | (oxotremorine-M) to displace |
|          |          | maternal        | \[^3^H\]pirenzepine; raising |
|          |          | infusions       | the dose to 6 mkg masked the |
|          |          | (15-21          | affinity shift by affecting  |
|          |          | rats/treatment) | G-protein regulatory         |
|          |          | of 2 mg/kg/day  | mechanisms, such that        |
|          |          | or 6 mg/kg/day  | addition of the GTP analog,  |
|          |          | from GD 4       | GppNHp, produced a larger    |
|          |          | through 20.     | 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 | Repr     | NA              |                              |
|          | oduction |                 |                              |
|          | and      |                 |                              |
|          | f        |                 |                              |
|          | ertility |                 |                              |
|          | effects  |                 |                              |
|          |          |                 |                              |
|          | (        |                 |                              |
|          | species) |                 |                              |
+----------+----------+-----------------+------------------------------+
| 8        | Chronic  | NA              |                              |
| 70.4100a | toxicity |                 |                              |
|          |          |                 |                              |
|          | (        |                 |                              |
|          | species) |                 |                              |
+----------+----------+-----------------+------------------------------+
| 8        | Chronic  | NA              |                              |
| 70.4100b | toxicity |                 |                              |
|          | (dog)    |                 |                              |
+----------+----------+-----------------+------------------------------+
| 870.4200 | Carcino  | NA              |                              |
|          | genicity |                 |                              |
|          |          |                 |                              |
|          | (rat)    |                 |                              |
+----------+----------+-----------------+------------------------------+
| 870.4300 | Carcino  | NA              |                              |
|          | genicity |                 |                              |
|          |          |                 |                              |
|          | (mouse)  |                 |                              |
+----------+----------+-----------------+------------------------------+
| Gene     |          | Tests by the R. | Nicotine and its major       |
| Mutation |          | J. Reynolds     | metabolites: cotinine,       |
|          |          | Tobacco Co.     | nicotine-N\'-oxide,          |
| Non-G    |          | (Doolittle *et  | cotinine-N-oxide, and        |
| uideline |          | al*, 1995)      | 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.                     |
+----------+----------+-----------------+------------------------------+
| Cyto     | B        | Yim & Hee 1995  | Cotinine was positive in the |
| genetics | acterial |                 | presence or absence of S9 at |
|          | lumi     |                 | 1.25 - 2.5 mg/mL (9- 30 h    |
| Non-G    | nescence |                 | incubation). Nicotine was    |
| uideline | geno     |                 | not positive to 20 mg/mL     |
|          | toxicity |                 | concentrations for up to 40  |
|          | test     |                 | hours of incubation.         |
|          |          |                 | Nicotine/cotinine mixtures   |
|          |          |                 | were still positive at       |
|          |          |                 | physiological                |
|          |          |                 | concentrations, with         |
|          |          |                 | potentiation relative to     |
|          |          |                 | cotinine alone with and      |
|          |          |                 | without S9                   |
+----------+----------+-----------------+------------------------------+
| Other    | Ch       | Trivedi *et     | Nicotine induced chromsomal  |
| Effects  | romsomal | al*, 1990       | aberration (CA) and sister   |
|          | ab       |                 | chromatid exchange (SCE)     |
| Non-G    | erration |                 | frequency in a dose and      |
| uideline | (CA) and |                 | duration dependent manner    |
|          | sister   |                 | and at concentrations        |
|          | c        |                 | comparable to the saliva     |
|          | hromatid |                 | levels of nicotine achieved  |
|          | exchange |                 | during tobacco chewing.      |
|          | (SCE)    |                 | Statistically significant    |
|          |          |                 | elevations in CA frequency   |
|          |          |                 | were observed with nicotine  |
|          |          |                 | concentrations \> 375 μg/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 μg/mL  |
|          |          |                 | did not increase the         |
|          |          |                 | frequency of CHE/cell, but   |
|          |          |                 | in combination with          |
|          |          |                 | arecoline it did at ≥90      |
|          |          |                 | µg/mL.                       |
+----------+----------+-----------------+------------------------------+
| Geno     | Chr      | Bishun *et al*, | Nicotine was cytotoxic at    |
| toxicity | omosomal | 1972            | 1.5 - 2.0 µg/mL to human     |
| Non-G    | abe      |                 | leucocytes in culture *in    |
| uideline | rrations |                 | 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.             |
+----------+----------+-----------------+------------------------------+
| Geno     | DNA      | Kleinsasser *et | Nicotine exerted significant |
| toxicity | damage   | al*, 2005       | direct genotoxic effects in  |
| Non-G    |          |                 | human target cells *in       |
| uideline |          | The             | vitro*. One hour exposure to |
|          |          | genotoxicity of | nicotine at 0.125, 0.25,     |
|          |          | nicotine was    | 0.5, 1, 2, and 4 mM induced  |
|          |          | tested with the | a statistically significant  |
|          |          | alkaline        | dose-dependent increase of   |
|          |          | single-cell     | DNA migration up to 3.8-fold |
|          |          | microgel        | and 3.2-fold in tonsillar    |
|          |          | electrophoresis | cells and lymphocytes,       |
|          |          | (Comet) assay   | respectively. The minimum    |
|          |          | using human     | concentration eliciting      |
|          |          | lymphocytes and | significant DNA damage was   |
|          |          | target cells    | 0.5 mM nicotine. The         |
|          |          | from lymphatic  | genotoxic effect was         |
|          |          | tissue of the   | confirmed in a second series |
|          |          | palatine        | of experiments using         |
|          |          | tonsils from    | nicotine of high purity from |
|          |          | healthy         | two different suppliers.     |
|          |          | patients.       | 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.          |
+----------+----------+-----------------+------------------------------+
| Geno     | Human    | Sassen *et al*  | Nicotine produced genotoxic  |
| toxicity | nasal    | 2005            | effects in human nasal       |
| Non-G    | ep       |                 | epithelia.                   |
| uideline | ithelia. |                 |                              |
+----------+----------+-----------------+------------------------------+
| Geno     | Human    | Argentin and    | Nicotine caused concomitant  |
| toxicity | gingival | Cicchetti, 2004 | genotoxic and antiapoptotic  |
| Non-G    | fib      |                 | effect in human gingival     |
| uideline | roblasts |                 | fibroblasts (HGFs) in the    |
|          | (HGFs)   |                 | 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.                    |
+----------+----------+-----------------+------------------------------+
| 8        | Acute    | NA              |                              |
| 70.6200a | neuro    |                 |                              |
|          | toxicity |                 |                              |
|          | s        |                 |                              |
|          | creening |                 |                              |
|          | battery  |                 |                              |
+----------+----------+-----------------+------------------------------+
| 8        | Su       | NA              |                              |
| 70.6200b | bchronic |                 |                              |
|          | neuro    |                 |                              |
|          | toxicity |                 |                              |
|          | s        |                 |                              |
|          | creening |                 |                              |
|          | battery  |                 |                              |
+----------+----------+-----------------+------------------------------+
| 870.6300 | Devel    | Temocin *et     | Decreased the endurance time |
|          | opmental | al*, 1993       | in swimming exercise         |
| Non-G    | neuro    |                 | significantly (10 minute     |
| uideline | toxicity | subcutaneous    | after the injection). At     |
|          |          | injection of    | 0.125 mg/kg nicotine, the    |
|          |          | nicotine        | endurance time remained      |
|          |          | (0.125 - 0.375  | unchanged, while at the      |
|          |          | mg/kg)          | 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-G    | Devel    | Johns *et al*   | Offspring exhibited          |
| uideline | opmental | (1992)          | performance deficits in both |
|          | neuro    |                 | learned and innate           |
|          | toxicity | 0, 0.5, 1.5, or | behavioral measures          |
|          |          | 2.5 mg/kg of    | throughout development and   |
|          |          | nicotine        | adulthood. Offspring birth   |
|          |          | hydrogen        | weight was not affected by   |
|          |          | tartrate,       | treatment nor the 32 day     |
|          |          | injected 2X     | weight gain. Initial weight, |
|          |          | daily           | weight gain, gestation       |
|          |          | throughout      | length, number of live or    |
|          |          | gestation       | dead offspring, or mean food |
|          |          | period          | consumption during pregnancy |
|          |          | (15/dose).      | did not differ significantly |
|          |          |                 | in the treated groups versus |
|          |          |                 | the controls. Dams receiving |
|          |          |                 | the nicotine injections      |
|          |          |                 | reacted with aversion.       |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Navarro *et     | Infusing pregnant rats with  |
| uideline | opmental | al*, 1989       | a nicotine dose that did not |
|          | neuro    |                 | interfere with maternal      |
|          | toxicity | 2 mg/kg of      | weight gain or resorption    |
|          |          | nicotine        | rate resulted in sufficient  |
|          |          | infused per day | nicotine penetrating the     |
|          |          | from            | fetal brain to cause         |
|          |          | gestational     | persistent alterations in    |
|          |          | days 4 through  | \[^3^H\] nicotine binding    |
|          |          | 20              | 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-G    | Devel    | Levin *et al*,  | Prenatal nicotine exposure   |
| uideline | opmental | 1993            | caused subtle alterations in |
|          | neuro    |                 | cognitive performance of the |
|          | toxicity | 2 mg/kg of      | offspring which were         |
|          |          | nicotine        | magnified by challenges of   |
|          |          | subcutaneous    | nicotinic and adrenergic     |
|          |          | injection per   | systems.                     |
|          |          | day from        |                              |
|          |          | gestational     |                              |
|          |          | days 4 through  |                              |
|          |          | 21              |                              |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Fung, 1988      | 14 -- day old male and       |
| uideline | opmental |                 | female offspring of rats     |
|          | neuro    | 1.5 mg/kg/day,  | exposed to                   |
|          | toxicity | nicotine        | nicotinedemonstrated an      |
|          |          | tartrate        | increase in spontaneous      |
|          |          | subcutaneous    | locomotor activity compared  |
|          |          | implanting of   | with controls. The total     |
|          |          | pregnant rats)  | number of pups born to the   |
|          |          | during          | treated group was            |
|          |          | gestation       | significantly less than the  |
|          |          |                 | controls.                    |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Peters *et al*, | Increased spontaneous motor  |
| uideline | opmental | 1979            | activity in the light which  |
|          | neuro    |                 | was not prevented by         |
|          | toxicity | 60 - 80 days    | cross-fostering to control   |
|          |          | old offspring   | dams at birth. The paternal  |
|          |          | of rats treated | rats had a marked reduction  |
|          |          | with nicotine   | in body weight gain (55% of  |
|          |          | in drinking     | controls for males and 63%   |
|          |          | water (6        | of controls for females      |
|          |          | mg/kg/day       | after 4 month of nicotine    |
|          |          | intake) four    | treatment). The dams were    |
|          |          | weeks before    | more active during the day   |
|          |          | mating and      | and exhibited a reduced      |
|          |          | during          | plasma corticosterone        |
|          |          | pregnancy and   | response to stress. Male but |
|          |          | throughout      | not female offspring of      |
|          |          | nursing and 6   | nicotine treated rats were   |
|          |          | weeks after     | significantly lighter at     |
|          |          | weaning         | birth than control males.    |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Peters & Ngan,  | subtle neurological changes  |
| uideline | opmental | 1982            | which are manifested as      |
|          | neuro    |                 | behavioral alterations in    |
|          | toxicity | Nicotine at 0,  | the newborn (the righting    |
|          |          | 1.5, 3 or 6.0   | reflex, temperature          |
|          |          | mg/kg per day;  | regulation, adherence to the |
|          |          | subcutaneously  | inclined screen, and in      |
|          |          | one week prior  | organ/body weight ratios for |
|          |          | to mating and   | brain, heart, lung, liver,   |
|          |          | throughout      | and kidney) and adult        |
|          |          | gestation       | offspring (prolonged time    |
|          |          |                 | required and an increase in  |
|          |          |                 | number of mistakes made,     |
|          |          |                 | during food maze testing and |
|          |          |                 | an increased brain protein   |
|          |          |                 | content).                    |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Roy &           | Reduction in cortical        |
| uideline | opmental | Sabherwal, 1998 | thickness and decreased cell |
|          | neuro    |                 | size, decreased dendritic    |
|          | toxicity | pregnant rats   | branching and increased      |
|          |          | were injected   | dendritic spine density,     |
|          |          | ip with         | irregular arrangement of     |
|          |          | nicotine (2.5   | cisternae of rough           |
|          |          | mg/kg/ day,     | endoplasmic reticulum,       |
|          |          | given in two    | paucity of free ribosomes,   |
|          |          | divided doses)  | and frequent cytoplasmic     |
|          |          | from GD 6 to    | vacuoles of the              |
|          |          | term and pups   | somatosensory cortex were    |
|          |          | were delivered  | observed in the pup up to    |
|          |          | at term         | postnatal day 40.            |
|          |          | normally        | 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-G    | Devel    | Ajarem & Ahmad, | significant reduced          |
| uideline | opmental | 1998            | postnatal body weight gain,  |
|          | neuro    |                 | as well as significant delay |
|          | toxicity | daily           | in eye opening, in the       |
|          |          | subcutaneous    | appearance of body hairs,    |
|          |          | injections of   | and in sensory motor         |
|          |          | nicotine        | reflexes. Motor activity was |
|          |          | (0.5mg/kg) into | significantly stimulated in  |
|          |          | the nape of the | early adulthood of mouse     |
|          |          | neck during     | pups prenatally exposed to   |
|          |          | pregnancy       | nicotine, and had            |
|          |          |                 | long-lasting hyperactive     |
|          |          |                 | effects on mice.             |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | Marks *et al*,  | Generally nicotine           |
| uideline | opmental | 1985            | administration increased the |
|          | neuro    |                 | respiration rate, decreased  |
|          | toxicity | four inbred     | the body temperature and the |
|          |          | strains of mice | heart rate, and a decline in |
|          |          | (BALB, C57BL,   | the Y-maze crosses and rears |
|          |          | DBA, C3H),      | at different rates in the    |
|          |          | nicotine        | various strains. The startle |
|          |          | administered ip | response was increased in    |
|          |          | at a single     | the C3H strain only.         |
|          |          | dose of 0, 0.5, |                              |
|          |          | 1.0 or 2.0      |                              |
|          |          | mg/kg           |                              |
+----------+----------+-----------------+------------------------------+
| Non-G    | Devel    | McFarland *et   | Nicotine inhibited DNA       |
| uideline | opmental | al*, 1991       | synthesis in neonatal rat    |
|          | neuro    |                 | brain regions as assessed by |
|          | toxicity | Nicotine given  | the incorporation of         |
|          |          | at an acute     | \[3H\]thymidine into DNA of  |
|          |          | subcutaneous    | the brain tissues. The       |
|          |          | injection to    | inhibition potency           |
|          |          | neonatal rats   | correlated to the            |
|          |          | as nicotine     | concentration of nicotinic   |
|          |          | dibitartrate (3 | receptors: midbrain +        |
|          |          | mg/kg of        | brainstem ≥cerebral cortex   |
|          |          | nicotine base)  | \> cerebellum. The           |
|          |          | at 1, 3, 8, 10  | inhibitory effect of         |
|          |          | or 15 days of   | nicotine was also seen in    |
|          |          | age             | fetal brain on gestational   |
|          |          |                 | day 20 after injection of    |
|          |          |                 | nicotine (3 mg/kg) to        |
|          |          |                 | pregnant rats.               |
+----------+----------+-----------------+------------------------------+
| Non-G    | neurobe  | Seidler *et     | Nicotine did not stimulate   |
| uideline | havioral | al*, 1996       | catecholamine release at     |
|          | te       |                 | birth, but developed the     |
|          | ratology | developing rats | capacity to do so in         |
|          |          | (1, 7, 14 and   | parallel with the ontogeny   |
|          |          | 21 days old)    | of nicotinic cholinergic     |
|          |          | challenged      | receptors in the             |
|          |          | acutely with    | midbrain+brainstem and in    |
|          |          | nicotine (0.3   | the forebrain. In the        |
|          |          | mg/kg, i.p.)    | cerebellum, no response was  |
|          |          | and the release | obtained at any age. Changes |
|          |          | of              | in sensitivity to nicotine   |
|          |          | catecholamines  | were also seen that          |
|          |          | was evaluated   | corresponded to ontogenetic  |
|          |          | *in vivo* in    | changes in endogenous        |
|          |          | three brain     | cholinergic tone, suggesting |
|          |          | regions that    | that receptor                |
|          |          | differ in       | desensitization occurs       |
|          |          | nicotinic       | normally during              |
|          |          | receptor        | developmental stages in      |
|          |          | concentrations. | 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-G    | E        | Blackburn *et   | Nicotine inhibited           |
| uideline | ndocrine | al*, 1994       | ovulation, estradiol         |
|          | effects  |                 | production, and              |
|          |          | pregnant mare's | fertilization both *in vivo* |
|          | *in      | serum           | and *in vitro* in rat        |
|          | vivo*    | gonadotropin    | models. A dose dependent     |
|          | and *in  | (PMSG) - primed | reduction in oocytes within  |
|          | vitro*   | and human       | the fallopian tube was noted |
|          | rat      | chorionic       | in nicotine treated rats     |
|          | models   | gonadotropin    | (p\<0.001). On the other     |
|          |          | (hCG) -         | hand cotinine did not affect |
|          |          | triggered rat   | ovulation, estradiol         |
|          |          | ovaries exposed | production or fertilization  |
|          |          | to nicotine (ip | in those tests.              |
|          |          | 6.25 ng/g       |                              |
|          |          | animal weight)  |                              |
+----------+----------+-----------------+------------------------------+
| Non-G    | E        | Slotkin and     | Nicotine produced            |
| uideline | ndocrine | Seidler, 1975   | alterations in               |
|          | effects  |                 | catecholamines (CA) release, |
|          |          | Nicotine at1    | tyrosine hydroxylase (TH),   |
|          | rats     | mg/kg or 10     | dopamine β-hydroxylase       |
|          |          | mg/kg,          | (DBH), and the ability of    |
|          |          | subcutaneously  | isolated storage vesicles to |
|          |          | twice daily for | incorporate 3H-epinephrine   |
|          |          | 1 or 2 weeks)   | in the adrenal glands and    |
|          |          |                 | these alterations persisted  |
|          |          |                 | when nicotine administration |
|          |          |                 | was discontinued.            |
+----------+----------+-----------------+------------------------------+
| Non-G    | E        | Lichtensteiger  | Male fetuses of all control  |
| uideline | ndocrine | and Schlumpf    | groups displayed the         |
|          | effects  | 1985            | characteristic rise in       |
|          |          |                 | plasma testosterone at GD 18 |
|          | Time-    | Nicotine        | (as compared to GD 17 and    |
|          | pregnant | tartrate        | 19); this was abolished by   |
|          | Sprague  | delivered in an | nicotine. Adult offspring of |
|          | Dawley   | osmotic         | untreated or tartaric        |
|          | rats     | minipump at a   | acid-treated dams exhibited  |
|          |          | rate of 25      | a marked sexual dimorphism   |
|          |          | ug/100 g/ hr    | 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-G    | E        | Ehlers *et al*, | Nicotine exposure altered    |
| uideline | ndocrine | 1997            | responses of the P3          |
|          | effects  |                 | component of the             |
|          |          | Nicotine        | event-related potential      |
|          |          | hydrogen-       | (ERP), recorded in dorsal    |
|          |          |                 | hippocampus, to changes in   |
|          |          | tartrate        | stimulus parameters. A       |
|          |          | (Sigma)         | significant reduction in the |
|          |          | dissolved in    | response to the noise tone   |
|          |          | distilled water | as compared with the level   |
|          |          | and added to    | of the infrequently          |
|          |          | the milk diet   | presented tone also was seen |
|          |          | (0 mg, 1 or     | in the P3B component. No     |
|          |          | 4mg/kg/day)     | effects of drug exposure     |
|          |          | administered to | were found on the N1         |
|          |          | neonatal rat    | component in any lead,       |
|          |          | pups from PN4   | although artificial rearing  |
|          |          | through PN12    | produced specific effects on |
|          |          | with an         | the latency of the N1        |
|          |          | artificial      | component in cortex. No      |
|          |          | rearing         | significant differences      |
|          |          | paradigm;       | among treatment groups were  |
|          |          | Nicotine. All   | found on any of the          |
|          |          | rats were       | EEG-dependent variables.     |
|          |          | weaned at PN21  | Female rats overall were     |
|          |          | and housed in   | found to have significantly  |
|          |          | pairs. Thirty   | higher                       |
|          |          | one suckle      | electroencephalography (EEG) |
|          |          | controls (17    | amplitudes than the males.   |
|          |          | males, 14       | However, no overall effects  |
|          |          | females), 23    | of gender were found on any  |
|          |          | artificially    | ERP component. These studies |
|          |          | reared rats (14 | suggest that neonatal        |
|          |          | males, 9        | nicotine exposure            |
|          |          | females), 22 1  | specifically reduces the     |
|          |          | mg/kg/day rats  | electrophysiological         |
|          |          | (12 males, 10   | response of the hippocampus  |
|          |          | females), and   | to changes in auditory       |
|          |          | 23 4 mg/kg/day  | stimuli                      |
|          |          | animals (16     |                              |
|          |          | males, 7        |                              |
|          |          | females) were   |                              |
|          |          | used in this    |                              |
|          |          | study.          |                              |
+----------+----------+-----------------+------------------------------+
| Non-G    | E        | Slawecki *et    | Examination of the           |
| uideline | ndocrine | al*, 2000       | hippocampal EEG revealed     |
|          | effects  |                 | significantly decreased      |
|          |          | Male rats were  | power in the 1--2-Hz         |
|          | Sprague  | exposed to 6.0  | frequency band of            |
|          | Dawley   | mg/kg/day       | nicotine-treated rats. In    |
|          | rats     | nicotine via    | addition, there was a        |
|          |          | gastric         | significantly attenuated     |
|          |          | infusion using  | P300 ERP response to a noise |
|          |          | an artificial   | tone in the nicotine-treated |
|          |          | rearing,        | rats compared to controls.   |
|          |          | "p              | These data indicate that     |
|          |          | up-in-the-cup," | neonatal nicotine exposure   |
|          |          | technique for 6 | alters functional activity   |
|          |          | consecutive     | in the hippocampus of adult  |
|          |          | days PND 4--9.  | rats. These effects are      |
|          |          | At adulthood,   | likely to be the result of   |
|          |          | EEG and         | synaptic disorganization in  |
|          |          | auditory ERPs   | the hippocampus, and         |
|          |          | were recorded   | indicate that neonatal       |
|          |          | from the cortex | nicotine exposure exerts     |
|          |          | and hippocampus | teratogenic effects on the   |
|          |          |                 | developing central nervous   |
|          |          |                 | system, particularly the     |
|          |          |                 | hippocampus, which persist   |
|          |          |                 | into adulthood.              |
+----------+----------+-----------------+------------------------------+
| Non-G    | Immune   | Maritz &        | resulted in a very low       |
| uideline | system   | Woolward, 1992  | elastic tissue content in    |
|          |          |                 | the lungs of 1- and 7-day    |
|          |          | nicotine at1    | old rat pups compared with   |
|          |          | mg/kg bw/d from | those of the controls. This  |
|          |          | GD 7 until      | may interfere with normal    |
|          |          | weaning three   | lung development since       |
|          |          | weeks after     | elastic tissue is part of    |
|          |          | birth)          | 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-G    | Immune   | Geng *et al*,   | Nicotine inhibited both the  |
| uideline | system   | 1995            | T-dependent and              |
|          |          |                 | T-independent AFC responses  |
|          |          | Nicotine at 1   | and proliferation to         |
|          |          | mg/kg to young  | anti-CD3. Significantly      |
|          |          | adult rats by   | fewer NT T cells entered the |
|          |          | intradermal     | S and G2/M phases than CON T |
|          |          | implantation    | cells, indicating an arrest  |
|          |          | for 3-4 weeks   | 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 Ca^2+^   |
|          |          |                 | responses of salivary gland  |
|          |          |                 | cells to acetylcholine were  |
|          |          |                 | normal. Serum cotinine       |
|          |          |                 | levels of 219 ± 40 ng/mL     |
|          |          |                 | were comparable to average   |
|          |          |                 | human smokers.               |
+----------+----------+-----------------+------------------------------+
| Non-G    | Immune   | Basta *et al*,  | Nicotine suppressed          |
| uideline | system   | 2000            | splenocyte responsiveness to |
|          |          |                 | Concanavalin A or            |
|          |          | pregnant rats   | lipopolysaccharide in        |
|          |          | treated with    | offspring and remained       |
|          |          | nicotine (6     | subresponsive to stimulation |
|          |          | mg/kg/day) from | well into adulthood.         |
|          |          | GD 4-20         | Nicotine treatment also      |
|          |          | subcutaneously  | 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-G    | Immune   | Zhang & Petro,  | Exposure of T-cells (splenic |
| uideline | system   | 1996            | mononuclear cells from       |
|          |          |                 | female mice) to              |
|          | *In      | T-cells         | physiological concentration  |
|          | vitro*   | (splenic        | of nicotine (1-100 μg/100    |
|          | T-cells  | mononuclear     | mL) can alter T cell         |
|          | from     | cells) exposed  | expression of CD28 and       |
|          | female   | to nicotine at  | CTLA-4 and the CD4 T cell    |
|          | mice     | 1-100 μg/100    | cytokine expression pattern  |
|          |          | mL)             |                              |
+----------+----------+-----------------+------------------------------+
| Non-G    | Me       | Hukkanen *et    | Review article. Rapid        |
| uideline | tabolism | al*, 2005       | absorption of nicotine from  |
|          | and      |                 | the inhaled cigarette smoke  |
|          | pharma   |                 | and rapid distribution via   |
|          | cokineti |                 | 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-G    | Dermal   | Zorin *et al*,  | *In vitro* tests of          |
| uideline | pen      | 1999            | nicotine's permeability      |
|          | etration |                 | through human skin using     |
|          |          |                 | Franz' diffusion cells       |
|          | (        |                 | demonstrated the dermal      |
|          | species) |                 | 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-G    | Special  | Benowitz znd    | Daily intake of nicotine in  |
| uideline | studies: | Jacob III, 1984 | a group of 22 smokers        |
|          |          |                 | averaged 37.6 ± 17.7 mg      |
|          | Daily    |                 | (10.5 -- 78.6 mg). Nicotine  |
|          | intake   |                 | intake per cigarette         |
|          | of       |                 | averaged 1.04± 0.36 mg.      |
|          | nicotine |                 | Among several markers        |
|          | from     |                 | measured (carboxyhemoglobin  |
|          | c        |                 | level, blood cotinine        |
|          | igarette |                 | concentration), afternoon    |
|          | smoke    |                 | (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.

Male mice (CF-1 Swiss derived, 20-25 g initial weight) consuming
nicotine hydrochloride in their drinking water (25 mg/l; equivalent to 7
mg/kg/day) daily for 2-3 months or injected i.p. with 5 mg/kg/day
acutely or for 3 weeks showed no evidence of hepatotoxicity as measured
by serum glutamic-pyruvic transaminase or serum alkaline phosphatase
activities (Priestly and Plaa 1976). The LD~50~ of nicotine HCl
administered i.p. in mice was 13.5 mg/kg. Acute or subchhronic
administration of nicotine did not modify the hepatotoxic response of
carbon tetrachloride (potent 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**

Nicotine was found to induce chromsomal aberration (CA) and sister
chromatid exchange (SCE) frequency in Chinese hamster ovary cells (CHO)
in a dose and duration dependent manner and at concentrations comparable
to the saliva levels of nicotine achieved during tobacco chewing. In one
experiment CHO cells were incubated with free base nicotine (analytical
grade from Sigma Chem. Co.) at 625 µg/mL or 1000 µg/mL for 2 or 4 hours.
In a second experiment, CHO cells were incubated with free base nicotine
(analytical grade from Sigma Chem. Co.) at 150 µg/mL, 250 µg/mL, 375
µg/mL, 500 µg/mL or 625 µg/mL for 24 and 48 hours for CA and SCE
analysis, respectively. Statistically significant elevations in CA
frequency were observed with nicotine concentrations ≥375 μg/mL,
whereas, SCE frequencies were increased significantly ≥150 μg/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 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 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 (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 Ca^2+^ 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.

**MRID 42631101.** Kuhn, J. (1992) Acute Dermal Toxicity Study in
Rabbits: F&B Rabbit & Dog Chaser: Lab Project Number: 9643-92.
Stillmeadow, Inc. Unpublished. 11 p.

**MRID 42631102.** Holbert, M. (1992) Acute Inhalation Toxicity Study in
Rats: F&B Rabbit & Dog Chaser: Lab Project Number: 9644-92. Stillmeadow,
Inc. Unpublished. 17 pages.

**MRID 42631103.** Kuhn, J. (1992) Primary Eye Irritation Study in
Rabbits: F&B Rabbit & Dog Chaser: Lab Project Number: 9645-92.
Stillmeadow, Inc. Unpublished. 17 p.

**MRID 42631104.** Kuhn, J. (1992) Primary Dermal Irritation Study in
Rabbits: F&B Rabbit & Dog Chaser: Lab Project Number: 9646-92.
Stillmeadow, Inc. Report no. 9646-92. Unpublished. 12 pages.

**MRID 42640001.** Kuhn, J. (1993) Dermal Sensitization Study in Guinea
Pigs: F&B Rabbit & Dog Chaser: Lab Project Number: 9647-92. Stillmeadow,
Inc. Unpublished. 17p.

**MRID 43375601.** Kuhn, J. (1994) Dermal Sensitization study in Guinea
Pigs: F&B Rabbit & Dog Chaser: Amendment: Lab Project Number: 9647-92.
Stillmeadow, Inc. Unpublished. 10 p.

**MRID 43523301.** Kuhn, J. (1994) Acute Oral Toxicity Study in Rats: (F
& B Rabbit and Dog Chaser): Final Report: Lab Project Number:1607-94.
Stillmeadow, Inc. **MRID 43523301.** Unpublished. 11 pages.

**MRID 43874001.** Bennick, J. (1995) F & B Rabbit and Dog Chaser: Acute
Inhalation Toxicity Study in Rats: Final Report: Lab Project Number:
2376-95: S9-FF81-3. Stillmeadow, Inc. Unpublished. 10 P.

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