UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C.  20460

OFFICE OF

PREVENTION, PESTICIDES

AND TOXIC SUBSTANCES

Date: September 30, 2007

MEMORANDUM

SUBJECT:	Sodium Fluoride Toxicology Chapter  for the Reregistration
Eligibility Decision (RED) Document.  PC Code: 075202  (active). Case
No.  3132

FROM:	Timothy F. McMahon, Ph.D.

		Senior Toxicologist

		Antimicrobials Division (AD) (7510P)

		And

		Jonathan Chen, Ph.D., Toxicologist

		Timothy Leighton, Exposure/Risk Assessor

		Richard Petrie, Agronomist

		A. Najm Shamim, Ph.D. Chemist

	            Antimicrobials Division (AD) (7510P)

TO:		Sanyvette Williams, D.V.M, Chemical Review Manager

		And

		Mark Hartman, Branch Chief

		Regulatory Management Branch II

		Antimicrobials Division (7510P)

Attached is the Toxicology chapter for the Sodium Fluoride RED document.

Table of Contents

1.0	Hazard
Characterization........................................................
...............................3

2.0	Toxicology
Data....................................................................
................................8

3.0	Data
Gaps....................................................................
.........................................9

4.0	Hazard
Assessment..............................................................
................................9

	4.1	Acute
Toxicity................................................................
...........................9

	4.2	Subchronic
Toxicity................................................................
.................10

	4.3	Prenatal Developmental
Toxicity...........................................................15

	4.4	Reproductive
Toxicity................................................................
.............18

	4.5	Chronic
Toxicity................................................................
......................20

	4.6
Carcinogenicity.........................................................
...............................20

	4.7
Mutagenicity............................................................
................................24

4.8
Neurotoxicity...........................................................
.................................42

4.9	Metabolism and
Pharmacokinetics........................................................
44

5.0	Toxicity End Point
Selection...............................................................
................44

6.0	FQPA
Considerations..........................................................
................................44

7.0	Summary of toxicological doses and
endpoints.................................................44

8.0	Toxicity Profile
Tables..................................................................
......................46

9.0
References..............................................................
..............................................73

1.0	HAZARD CHARACTERIZATION

Sodium fluoride is registered for commercial use only as a wood
preservative for utility poles and railroad ties.  Sodium fluoride
products are used as supplemental wood treatments and are not intended
for primary wood preservative or pressure treated wood preservation.

Sodium Fluoride is an inorganic substance which does not undergo
hydrolysis typically like an organic compound. Sodium fluoride is water
soluble and dissociates in water.

The acute toxicity database for sodium fluoride is considered complete.
Sodium fluoride has a high order of toxicity via the oral route of
exposure (Toxicity Category II) and a moderate order of toxicity via the
dermal and inhalation routes of exposure (Toxicity Category III).
Primary eye irritation studies classify sodium fluoride as corrosive
(Toxicity Category I) whereas dermal irritation studies classify sodium
fluoride as a mild or slight irritant (Toxicity Category IV). Sodium
fluoride is not a dermal sensitizer.

For subchronic toxicity two National Toxicology Program (NTP) studies
and five open literature studies (Bohatyrewicz, A. (1999); Paul et al.
(1998); Pillai et al. (1988); Chinoy and Patel. (2001) and Shahshi et
al. (1994)) were evaluated and considered acceptable. In one NTP study
10 F344/N rats/sex/dose were administered sodium fluoride in drinking
water at doses of 0, 10, 30, 100, or 300 ppm for 6 months. The NOAEL was
30 ppm and the LOAEL was 100 ppm, based on the presence of hyperplasia
in the glandular stomach. There were no treatment-related effects on
mortality. In another NTP study, sodium fluoride was administered to
8-12 B6C3F1 mice/sex/dose in deionized water at doses of 0, 10, 50, 100,
200, 300 or 600 ppm for 6 months. The NOAEL was determined to be 50 ppm
in female mice, and could not be determined in males based on the
observation of increased osteoid of the tibia in 5/10 males dosed at 50
ppm.   The LOAEL was established to be 50 ppm in male mice and 100 ppm
in female mice, based on histopathology observed in bone. In one
non-guideline study (Bohatyrewicz, A. 1999) 10 female 6-week-old Wistar
rats/group were administered NaF at levels of 0, 8, 30, and 60 mg of
fluoride/L in drinking water for 6 weeks. High fluoride intake (30 and
60 mg/L) was found to significantly decrease bone quality of the femoral
shaft and neck of young rats while lower concentrations (8 mg/L)
significantly increased the strength of the femoral neck from the
control. In another study (Paul et al. 1998) 10 colony-bred adult Wistar
female rats were treated orally (intubation) with daily, non-lethal
doses of sodium fluoride at concentrations of 20 or 40 mg/kg for 60
days. The subchronic toxicity NOAEL was less than 20 mg/kg/day (lowest
dose tested). The LOAEL was ≤ 20 mg/kg/day, based on significant
reductions in body weight gain and suppressed spontaneous motor
activity. A non-guideline toxicity study (Pillai et al. 1988)
administered sodium fluoride daily to 5 Swiss albino mice (Haffkine
strain) at a concentration of 5.2 mg/kg/day for 35 days. The subchronic
toxicity NOAEL was < 5.2 mg/kg/day (lowest dose tested). The LOAEL was
≤ 5.2 mg/kg/day, based on significant decreases in body weight gain,
and food and water consumption. In another study (Chinoy and Patel.
2001) 20 Adult female albino mice were administered 10 mg/kg/day sodium
fluoride (in 0.2 mL water) for 30 days. A significant decline of ovarian
protein, 3-beta-and 17-beta-hydroxysteroid dehydrogenase activities was
observed. This could be related to increased cholesterol levels in the
ovary suggesting altered steroidogenesis.

The database for developmental toxicity is considered complete with five
studies, four in the rat and one in the rabbit. In one study (Bates et
al. 1994), sodium fluoride (purity >99%) was administered ad libitum in
drinking water to groups of 26 Sprague-Dawley rats/dose at dose levels
of 0, 7, 18, or 27 mg NaF/kg/day (0, 50, 150, and 300 ppm, respectively)
from gestation days (GD) 6 to 15. The Maternal NOAEL was determined to
be 18 mg/kg/day and maternal LOAEL 27 mg/kg/day, based on reduced
maternal body weight. There were no treatment-related clinical signs,
increases in mortality or decreases in body weight in rats dosed with
sodium fluoride. There were no treatment-related effects on mean live
fetal body weight /litter, and the number of live fetuses. The
Developmental toxicity NOAEL is greater than or equal to 27 mg/kg/day
(highest dose tested).  The Developmental toxicity LOAEL is greater than
27 mg/kg/day (not established).

A developmental toxicity NOAEL of ≥ 250 ppm (25.1 mg/kg/day; highest
dose tested) and LOAEL > 250 ppm (25.1 mg/kg/day; not established) were
determined. There were no treatment-related effects in fetal body
weight, litter sizes, or viable fetuses.

In a developmental toxicity study in the rat (Heindel et al 1996),
Sprague-Dawley rats (26/group) were administered sodium fluoride ad
libitum in deionized/filtered drinking water on gestation days 6-15 at
levels 0, 50, 150, or 300 ppm (0, 6.6, 18.3, or 27.1 mg/kg/day,
respectively). The feed contained 15.6 ppm of sodium fluoride (purity
>99%). There were no treatment-related clinical signs, increases in
mortality (100% survival), or decreases in body weights in rabbits dosed
with sodium fluoride at the low- and mid-dose. A maternal toxicity NOAEL
was 18.3 mg/kg/day and LOAEL was 27.1 mg/kg/day, based on reduced
maternal body weight gain. The developmental NOAEL was ≥ 27.1
mg/kg/day (highest dose tested) and LOAEL > 27.1 mg/kg/day (not
established). There were no treatment-related effects on mean live fetal
body weight/litter, live fetal number, and prevalence of malformations.

In a study in rabbits (Heindel et al. 1996), New Zealand White rabbits
(26/group) were administered sodium fluoride feed (15.6 ppm, purity
>99%) ad libitum in deionized/filtered drinking water on gestation days
(6-19 at levels of 0, 50, 150, or 300 ppm. The maternal toxicity NOAEL
was NOAEL 18 mg/kg/day and LOAEL was 29 mg/kg/day, based on reduced
maternal body weight gain. There were no treatment-related clinical
signs, increases in mortality (100% survival), or decreases in body
weights in rabbits dosed with sodium fluoride at the low- and mid-dose.
The developmental toxicity NOAEL was established to be ≥ 29 mg/kg/day
(highest dose tested) and LOAEL at > 29 mg/kg/day (not established).
There were no treatment-related effects in mean live fetal body
weight/litter, live fetal number, and prevalence of malformations.

≥ 250 ppm and LOAEL > 250 ppm were determined for maternal toxicity.
There were no treatment-related effects on maternal mortality. A
significant decrease from control in fluid consumption (30%) was
observed at the 250 ppm dose level. The reproductive toxicity NOAEL and
LOAEL were determined to be ≥ 250 ppm (highest dose tested) and > 250
ppm, respectively. There were no treatment-related effects in the mean
number of corpora lutea, mean number of implantation sites, implantation
efficiency, mean number of viable fetuses, and average percentage of
early and late deaths per litter of dams. The developmental toxicity
NOAEL was 175 ppm and the LOAEL was 250 ppm, based on decreased
ossification of the hyoid bone. Fetal body weight was not affected by
treatment with sodium fluoride. There was no evidence of toxicity in
fetuses or pups of the F1 generation. The F2 generation fetuses and pups
were unaffected by treatment with sodium fluoride with the exception of
decreased ossification of the hyoid bone in the F2 fetuses at the 175
(not significant) and 250 ppm (significant) dose groups.

L is ≥ 250 ppm (highest dose tested) and the maternal toxicity LOAEL >
250 ppm (not established). The reproductive toxicity NOAEL was ≥ 250
ppm and the corresponding LOAEL > 250 ppm (not established). There were
no dose-related clinical effects observed and no significant differences
were observed in F0 female food consumption while there was a 5%
decrease (significant) reduction in F0 males at 250 ppm (in the first 7
weeks, and week 9 of the 10 week growth period). Fluid consumption was
significantly reduced from control levels in the 175 and 250 ppm dose
groups with decreases of 11 and 20% for F0 females, 9 and 20% for F0
males, 19 and 29% for F1 females, and 15 and 25% for F1 males,
respectively. F1 males in the 100 ppm dose group drank significantly
less (9%) than control animals. There were no significant or
dose-related effects observed in implantation and reproductive
parameters of any generation.  In a third study (Messer et al), 0, 50,
100, and 200 ppm sodium fluoride was administered via drinking water to
58, 55, 50, and 50 weaning female albino mice, respectively. The females
mated and litters were normalized to 6 pups. Second generation mice from
control and 50 ppm groups (38 and 44 animals, respectively) were mated
and followed the same parameters as the parental group. Retardation of
growth was observed in the 100 and 200 ppm F1 groups, with death in 50%
of animals in the 200 ppm groups by 8 weeks of age. No litter production
was seen at the 200 ppm group and only 9 litters at the 100 ppm were
observed over a ten-week period.

The database for chronic toxicity is considered incomplete. In one study
(Varner et al. 1998), adult male Long-Evans rats (7/group) received
double deionized water (ddw) and 0.5 ppm aluminum fluoride, or ddw and
2.1 ppm sodium fluoride for 52 weeks. No differences were found between
the body weights of rats in the different treatment groups although more
rats died in the aluminum fluoride (5) ad the sodium fluoride group (3)
than the control group (1). All levels in samples of brain and kidney
were higher in both the aluminum fluoride and sodium fluoride groups
relative to controls. The effects of the two treatments on
cerebrovascular and neuronal integrity were qualitatively and
quantitatively different. These alterations were greater in animals in
the aluminum fluoride group than in the sodium fluoride group and
greater in the sodium fluoride group than in controls.

The database for carcinogenicity consists of three studies; one open
literature study in the rat and two National Toxicology Program studies.
In one study (Maurer et al), Sprague-Dawley rats (70/group) were fed a
diet containing 0, 4, 10, or 25 mg/kg/day sodium fluoride added to a
low-fluoride diet for up to 99 weeks. There was no evidence of
treatment-related incidence of carcinogenicity in Sprague-Dawley rats
administered dietary sodium fluoride in concentrations up to 25
mg/kg/day for 2 years. All bone neoplasms observed were considered to be
incidental and spontaneous and not related to sodium fluoride treatment,
because of their low incidence and random distribution.

In one NTP study, 100, 70, 70, or 100 B6C3F1 mice/sex were administered
sodium fluoride (purity >99%) in the drinking water at doses of 0, 25,
100, or 175 ppm (mice/sex) for 103 weeks (Male: 0, 2.4, 9.6, or 16.7
mg/kg/day; female: 0, 2.8, 11.3, or 18.8 mg/kg/day). There were no
compound-related effects on mortality, body weight, food consumption,
water consumption, hematology, or organ weights.  Treatment-related
clinical findings included a dose-dependent increase in white
discoloration of the teeth (27%, 39%, 80%, and 100% in males and 19%,
43%, 84%, and 100% in females, from control to high dose, respectively)
which occurred as early as Day 74 in the high-dose animals compared to
Day 508 in the control animals. Serum alkaline phosphatase was
significantly increased in high-dose females at 24 (29%) and 66 weeks
(88%) and in high dose-males at 66 weeks (11%).  Serum phosphorus levels
were significantly decreased (13%) in high-dose males at 66 weeks.
There was a significant increase in incisor dentine dysplasia in
high-dose males (78% in controls versus 91% at the high dose).  There
was an increase in the incidence of myelofibrosis (femoral, humerus,
maxilla, and thoracic) in female mice at all doses. The NOAEL in male
mice was 9.6 mg/kg/day and the corresponding  LOAEL 16.7 mg/kg/day,
based on the clinical chemistry changes in alkaline phosphatase and
serum phosphorus (males) at 66 weeks and bone lesions (dentine
dysplasia). The NOAEL for female mice was 11.3 mg/kg/day and the LOAEL
was18.8 mg/kg/day, based on the clinical chemistry changes in alkaline
phosphatase and bone lesions (myelofibrosis).

In a combined chronic toxicity/oncogenicity study (NTP), 100, 70, 70, or
100 F344/N rats/sex were administered sodium fluoride (purity >99%) in
the drinking water at doses of 0, 25, 100, or 175 ppm (mice/sex) for 103
weeks (male: 0, 1.3, 5.2, or 8.6 mg/kg/day; female: 0, 1.3, 5.5, or 9.5
mg/kg/day). The NOAEL was determined to be < 1.3 mg/kg/day (lowest dose
tested) and the  LOAEL was determined to be  1.3 mg/kg/day, based on
dentine dysplasia in males and females, and ameloblast degeneration in
males. Mortality, body weight, body weight gain, food consumption, water
consumption, hematology, and organ weights were not affected by exposure
to sodium fluoride. Histopathology of the incisors noted dentine
dysplasia (all dosed animals), degeneration of the ameloblasts (mid- and
high-dose animals), and, to a lesser extent, degeneration of the
odontoblasts (principally dosed males).  Increases in the incidence and
severity of osteosclerosis of the long bones were noted in the high-dose
females (6/80 control; 18/81 high- dose, P=0.04).  Three bone
osteosarcomas were noted in high-dose males and one in a mid-dose male,
with none in controls.  A fourth osteosarcoma, not originating in the
bone, was observed in an additional high-dose male

The database for mutagenicity is considered complete with five National
Toxicology Program studies and numerous open literature studies. The
results can be summarized as follows: (1) all studies conducting the
bacterial gene mutation test concluded that sodium fluoride was not
mutagenic to any of the 5 Salmonella typhimurium bacterial strains in
the presence or absence of metabolic activation; (2) of the four studies
that performed the in vitro mammalian cell gene mutation test, two
concluded that sodium fluoride was not mutagenic at the HGPRT locus of
Chinese hamster ovary cells and the rat epithelial cells (ARL 1),
respectively. Two studies (Caspary et al., 1987; NTP) observed positive
results in L5179Y and L5178Y mouse lymphoma cells in the absence and
presence of metabolic activation; (3) six studies performed the in vitro
mammalian chromosome aberration test and observed a dose- and
time-dependent increase in chromosomal aberrations following
administration of sodium fluoride. Hence, all of them found sodium
fluoride to be positively mutagenic (4) three studies conducted the
mammalian spermatogonial chromosomal aberration test, two of which
reported positive results for mutagenicity; (5) of  four studies that
performed the mammalian bone marrow chromosomal  aberration test, two
reported positive results; (6) of  four studies that conducted the
mammalian erythrocyte micronucleus test, three reported sodium fluoride
to be non-mutagenic; (7) only one study (Tong et al. 1988) conducted the
bacterial DNA damage or repair test with negative results reported (8)
two studies reported sodium fluoride to positively induce unscheduled
DNA synthesis in mammalian cell culture (9) six studies conducted the in
vitro sister chromatid exchange assay, only two of which reported sodium
fluoride positively inducing sister chromatid exchanges (10) there was
only one study that conducted an in vivo sister chromatid exchange assay
(Li Y, et al. 1987). In this study, Male Chinese hamsters were
administered sodium fluoride at concentrations of 0.1, 1, 10, 60 or 130
mg/kg. Sodium fluoride did not induce a SCE increase in CHBM cells;
there was no evidence of mutagenicity.  Death occurred in three out of
the eight hamsters in the 130 mg/kg/day group. Although toxic effects
were seen in the high dose group, there were no treatment-related
increases in SCE.

In 1996, the EPA’s Office of Prevention, Pesticides, and Toxic
Substances classified sodium aluminofluoride (cryolite) as a “Group
D” carcinogen (not classifiable as to carcinogenicity), citing the
National Toxicology Program’s carcinogenicity study of sodium fluoride
(NTP, 1990). More recently, the National Acedemy of Sciences (NAS, 2006)
at the request of the EPA, conducted a review of the toxicologic,
epidemiologic, and clinical data on fluoride since the 1993 NAS report.
With respect to carcinogenicity, the 2006 NAS report concluded that “
on the basis of the committee’s collective consideration of data from
humans, genotoxicity assays, and studies of mechanism of action in cell
systems…the evidence on the potential of fluoride to initiate or
promote cancers, particularly of the bone, is tentative and mixed.”
This recent conclusion is consistent with the past conclusion of OPPTS
regarding carcinogenic potential of fluoride.

The database for metabolism consists of one study from the open
literature. In a study by Hall et al. 1977, 6 adult male New Zealand
rabbits were administered sodium fluoride in the diet (15 ppm), water (1
ppm), and  in a single oral dose injected (0.5 mg/kg ) directly into
stomach through nasal catheter. Urine excretion following oral
administration of sodium fluoride was 5 and 13% for 60 and 600 minutes,
respectively.  Under steady state conditions approximately 15% of
fluoride ingested in food and water was absorbed by the animals. 15% was
excreted in urine and 85% of ingested fluoride was removed via fecal
excretion.

2.0	TOXICOLOGY DATA

The available toxicology data for Sodium Fluoride is listed below.

Table 1. Toxicology data for sodium fluoride

Test

	MRID	Acceptable

870.1100	Acute Oral Toxicity

870.1200	Acute Dermal Toxicity

870.1300	Acute Inhalation Toxicity

870.2400	Primary Eye Irritation

870.2500	Primary Dermal Irritation

870.2600    Dermal
Sensitization...................................................

162945, 40928201,

43778501, 40932003

43778502, 40928202, 162946, 40932002

43778503

162948, 40928204,

40932001, 41204001

43778504

43778505, 162947,

40928203, 40932004

43778506, 40866801

40866901

Yes

Yes

Yes

Yes

Yes

Yes

870.3100	Oral Subchronic (rodent)

NTP, 1990

yes

870.3700a	Developmental Toxicity (rodent)

870.3700b	Developmental Toxicity (nonrodent)

870.3800
Reproduction............................................................
...

Open literature

open literature

open literature

yes

yes

yes

870.4200a	Oncogenicity (rat)

870.4200b	Oncogenicity (mouse)

870.4300
Chronic/Oncogenicity.................................................

             NTP study

 NTP study

NTP study

yes

yes

yes

870.5100	Mutagenicity-Gene Mutation-bacterial

870.5300	Mutagenicity-Gene Mutation mammalian

870.5375    Mutagenicity-In Vitro mammalian chromosome aberration
test..............................................................

870.5380	Mutagenicity-Spermatogonial chromosomal test

870.5385    Mutagenicity-Mammalian bone marrow

                   chromosome aberration
test........................................

870.5395    Mutagenicity-Mammalian erythrocyte micronucleus

Test....................................................................
..........

870.5550    Mutagenicity-UDS in mammalian cells in culture......

870.5900    Mutagenicity-In Vitro sister chromatid exchange

assay...................................................................
........

870.5915    Mutagencity-In Vivo sister chromatid exchange

Assay...................................................................
.......

.

Open literature

Open literature

Open literature

Open literature

Open literature

Open literature

Open literature

Open literature

Open literature

yes

yes

yes

yes

yes

yes

yes

Yes

yes

870.7485	General Metabolism

870.7600    Dermal
Penetration......................................................

Hall, 1977

           No study

no

--

* Open Literature studies

3.0	DATA GAPS

Intermediate- and long-term dermal risks that are of concern from
occupational exposures involving pre-drilled hole spray applications
using the mechanical pressure pumps and ground-line brush-on treatments
could be refined if a dermal absorption study is conducted to determine
actual dermal absorption of sodium fluoride and the result shows a low
potential for dermal absorption, or from conduct of  a repeated dose
dermal toxicity study (90-day) to determine the route-specific NOAEL.

4.0	HAZARD ASSESSMENT

	4.1	Acute Toxicity

Adequacy of database for Acute Toxicity: The acute toxicity database for
sodium fluoride is considered complete. For the technical grade active
ingredient, sodium fluoride has a high order of toxicity via the oral
route of exposure (Toxicity Category II) and a moderate order of
toxicity via the dermal and inhalation routes of exposure (Toxicity
Category III). Primary eye irritation studies classify sodium fluoride
as corrosive (Toxicity Category I) whereas dermal irritation studies
classify sodium fluoride as a mild or slight irritant (Toxicity Category
IV). Sodium fluoride is not a dermal sensitizer.

The acute toxicity data for sodium fluoride is summarized below in Table
2.

Table 2. Acute toxicity data for sodium fluoride technical a.i.

Guideline Number	Study Type/Test substance (% a.i.)	MRID Number/

Citation	Results	Toxicity Category

870.1100

(§81-1)

Acute Oral – Rat

Purity 95.6% - Sodium Fluoride

43778501

LD50 (combined) = 105 (93-119 CL)

Male LD50 = 120 mg/kg

Female LD50 = 89 mg/kg

II

870.1200

(§81-2)

Acute Dermal – Rat

Purity 95.6% - Sodium Fluoride

43778502

LD50 > 2000 mg/kg

III

870.1300

(§81-3)

Acute Inhalation - Rat

Purity 95.6% - Sodium Fluoride

43778503

LC50 = 1.00 mg/L

III

870.2400

(§81-4)

Primary Eye Irritation - Rabbit

Purity 95.6% - Sodium Fluoride

43778504

Severely irritating to unwashed eyes

II

870.2500

(§81-5)

Primary Dermal Irritation- Rabbit

purity 95.6% – Sodium Fluoride

43778505

Slightly Irritating

IV

870.2600

(§81-6)

	Dermal Sensitization - Guinea pig

purity 95.6 % - Sodium Fluoride

43778506

Buehler: Not a skin sensitizer

No

870.2600

(§81-6)

	Dermal Sensitization - Guinea pig

purity not reported

40866801

Not a dermal sensitizer

No

4.2	Subchronic Toxicity

Adequacy of database for Subchronic Toxicity: Two National Toxicology
Program studies and five open literature studies were evaluated and
considered acceptable.

870.3100	90-Day Oral Toxicity study in rodents – Rats

A 6-month oral toxicity study (NIH Publication No. 91/2848, 1990) was
designed to determine the subchronic oral toxicity of sodium fluoride
(>99% purity) administered to 10 F344/N rats/sex/dose in deionized
drinking water at dose levels of 0, 10, 30, 100, or 300 ppm
(approximately  0, 0.05, 1.5, 5.0, and 15.0 mg/kg/day).

There were no treatment-related effects on mortality.  Food and water
consumption was decreased compared to the control at the 300 ppm dose
level with reductions of 13 and 8% for males and 18 and 19% for females,
respectively.  There were significant decreases in mean body weight in
both males (84% of controls) and females (90% of controls) of the 300
ppm dose group.  Mean body weight changes were also decreased in the 300
ppm-treated rats; however, this reduction was only significant in the
males (21%).  Additional treatment-related effects were noted in 300-ppm
treated rats including clinical observations of dental fluorosis (chalky
white appearance of teeth from week 6 to 26, overgrowth of upper
incisors from week 6 to 17, unusual wear pattern of incisors, occlusal
surface of the lower incisor worn to the gumline) and rough hair coats
observed from week 18 to 26.

The principle pathological effects associated with the administration of
sodium fluoride were observed in the incisor teeth and stomach.  Five
male rats (of 6 total) treated with 300 ppm sodium fluoride effects had
focal or multifocal degeneration of the enamel organ (degeneration was
termed dysplasia for this lesion, small aggregation of enamel-like
material trapped within cell layers collectively termed dysplasia),
columnar ameloblasts flattened or lost (atrophy), disorganized cells of
the stratum intermedium that contained less cytoplasm and fewer
secretory vacuoles.  Effects of the glandular stomach were observed with
sodium fluoride treatment.  Acute inflammation (7/10 rats), hyperplasia
(10/10 rats), necrosis (10/10 rats), and inflammatory, infiltrate,
lymphocytic effects (7/10 rats) were observed at the 300 ppm dose level.
 Inflammatory, infiltrate, lymphocytic effects were also observed at 30
ppm in 2 or 10 male rats while hyperplasia was found in 5 or 10 male
rats at 100 ppm.  Mucosa of glandular stomach of most male rats in the
300 ppm dose groups appeared thickened and focal or multifocal punctate
hemorrhages were observed in 4/10 males.  Multiple, small non-perforated
ulcers were observed in 1 male rat of the 300 ppm dose group.

Glandular stomach inflammatory, infiltrate, lymphocytic effects were
observed in 1 and 4 of 10 female rats at 30 and 300 ppm, respectively.
Additional effects were observed at 100 ppm (2/10, hyperplasia) and 300
ppm (9/10, hyperplasia; 9/10, necrosis).  Focal or multifocal punctate
hemorrhages of the glandular stomach were observed in 1/10 females.
Perforated ulcers of the glandular stomach were found in the 300
ppm-treated females.

Measurement of fluoride content revealed a dose-dependent increase in
fluoride concentration in bone and urine, while elevated levels of
fluoride in plasma were only observed in 300-ppm treated rats.

Histologically, the hyperplasia of the mucosal epithelium of the
glandular stomach, which ranged from subtly focal to diffuse, was found
in 100 and 90% of the males and females, respectively, at 300 ppm.  This
effect was accompanied by minimal individual cell necrosis (apoptosis)
in the pyloric region as evidenced by acute inflammation in several
males at 300 ppm.  Compared to the control, mucous cells in the
epithelium was slightly decreased and the number of mitotic figures at
the base of the gastric pits was increased.  Columnar cells were stained
more basophilic and epithelium lining the gastric pits contained 1 or
several cells with pyknotic nuclei, fragments of nuclear debris, or
residual bodies.  Focal basal cell hyperplasia of the stratified
squamous epithelium was located adjacent to the lining ridge (junction
of the glandular stomach and forestomach) in nearly all 300-ppm treated
rats.  Hyperplasia of the mucosal epithelium of the glandular stomach
was also observed in males (5/10) and females (2/10) of the 100 ppm dose
group, but individual necrosis was not.  Microscopic evidence of the
effects of the test article on the incisors included focal or multifocal
degeneration of the enamel organ in 300-ppm males (5/10), localized in
the maturation zone near the apical end of the tooth.

The Subchronic Toxicity NOAEL is 100 ppm.  The Subchronic Toxicity LOAEL
is 300 ppm, based on decreased body weight and food and water
consumption, increased body weight changes, and macroscopic effects
(dental fluorosis and glandular stomach effects including acute
inflammation, apoptosis in the pyloric region, focal or multifocal
punctate hemorrhages, perforated ulcer of the glandular stomach, and
multiple, small, nonperforated ulcers).

This 6-month oral (drinking) toxicity study in F344/N rats is
ACCEPTABLE-NONGUIDELINE as a range-finding study for the 2-year chronic
toxicity in rats, and does not satisfy the guideline requirement for a
subchronic oral toxicity study (OPPTS 870.3100; OECD 408) in rats.  The
study is classified as nonguideline based on the omission of hematology
and clinical chemistry analyses, urinalysis, organ weights,
ophthalmoscopic examinations, and various organs/tissues for microscopic
examination.  In addition, without test article intake data, it is
difficult to draw definitive conclusions regarding the dose level and
treatment-related effects.

870.3100	90-Day Oral Toxicity study in rodents – Mouse

A 6-month oral toxicity study (NIH Publication No. 91/2848, 1990) was
designed to examine the subchronic toxic effects of sodium fluoride
(>99% purity) administered to 8-12 B6C3F1 mice/sex/dose in deionized
water at dose levels of 0, 10, 50, 100, 200, 300 or 600 ppm
(approximately 0, 1.5, 7.5, 15.0, 30.0, 45.0, and 90 mg/kg bw/day)

There were premature deaths, including sacrifice due to moribundity,
observed at 300 ppm (male, 12.5%) and 600 ppm (males, 44%; females, 82%)
dose levels.  Clinical signs of thin appearance, hunched posture and
weakness were observed in several of the decedents prior to premature
sacrifice.  Clinical signs in surviving animals included chalky white
incisors (≥100 ppm) and chipped teeth (≥300 ppm).  The effects on
the incisors correlated with microscopic findings, which included focal
or multifocal degeneration of the enamel organ.

Food consumption in 600 ppm males was approximately 77% of controls.
Food consumption in the other treatment groups, and water consumption in
all treatment groups were within 20% of control values.  Mean body
weight was significantly decreased in 600 ppm treated males (20%) and in
200 and 300 ppm treated females (16 and 13%, respectively).  Mean body
weight gain was significantly decreased form control in 200, 300, and
600 ppm males (9, 5, and 20%, respectively) and in 200 and 300 ppm
females (16 and 13%, respectively).  These parameters were also
decreased in the 600 ppm females, but did not reach statistical
significance, likely due to the reduced number of animals in this group
as a result of premature deaths.

There was a dose-dependent increase in fluoride content in bone and
urine.  Due to the pooling of plasma samples for sufficient volume for
analysis, meaningful statistical analyses in this fluid could not be
performed.  The data indicate that there was generally a dose-dependent
increase in fluoride concentration in the plasma.

s of ≥50 ppm for males and ≥100 ppm for females.  This
histopathology is indicative of altered rates of bone deposition and
remodeling as a result of treatment.  Further, treatment with sodium
fluoride induced degeneration of the incisors as evidenced by
degeneration (dysplasia) in ≥300-ppm treated mice.

The Subchronic Toxicity NOAEL is 10 ppm in male mice and 50 ppm in
female mice.  The LOAEL is 50 ppm for males and 100 ppm for females,
based on histopathology observed in bone with degeneration in tibias and
femurs of animals.

This 6-month oral toxicity study in the B6C3F1 mice is
ACCEPTABLE-NONGUIDELINE as a range-finding study for the 2-year chronic
toxicity in mice, and does not satisfy the guideline requirement for a
subchronic oral toxicity study (OPPTS 870.3100; OECD 408) in mice.  The
study is classified as nonguideline based on the omission of hematology
and clinical chemistry analyses, urinalysis, organ weights,
ophthalmoscopic examination, and various organs/tissues for microscopic
examination.  In addition, without test article intake data, it is
difficult to draw definitive conclusions regarding the dose level and
treatment-related effects.

Non-guideline Oral Subchronic Toxicity - Rat

A non-guideline toxicity study (Paul, Ekambaram, et al. 1998) was
designed to determine spontaneous motor activity and motor coordination
in 10 colony-bred adult Wistar female rats that were treated orally
(intubation) with daily, non-lethal doses of sodium fluoride at
concentrations of 20 or 40 mg/kg/day for 60 days.

There were significant dose-dependent decreases of 17 and 30% for food
intake and 14 and 37% for body weight gain at the 20 and 40 mg/kg/day
dose levels, respectively.   Total protein concentrations in serum
(low-dose, 13%; high-dose, 38%), liver (low-dose, 22%; high-dose, 42%),
and skeletal muscle (low-dose, 15%; high-dose, 31%) were also
significantly reduced in a dose-related manner in animals treated with
sodium fluoride.

Spontaneous motor activity was suppressed in a dose-dependent manner
with decreases of 15 and 29% at the 20 and 40 mg/kg/day dose levels,
respectively.  However, motor coordination was not altered in treated
animals. Total blood cholinesterase activity was reduced at the low- and
high-dose, although there was no evidence of change in
acetyl-cholinesterase activity of the cerebral cortex, brain stem, or
cerebellum.

Food intake reductions may account for the decrease in protein
concentration or a direct deleterious action of fluoride on protein
metabolism can also play a role in depleting protein in sensitive
tissues.  Thus, a decreased food intake together with a depletion of
protein in soft tissues accounted for an inhibition of body growth in
sodium fluoride-treated animals.  Sodium fluoride deprived skeletal
muscle of total protein and suppressed blood cholinesterase activity;
although, these effects are unlikely to have a deteriorating action on
neuromuscular function.  However, similar sodium fluoride doses can
produce neurobehavioral deficit resulting in an inhibition of
spontaneously occurring locomotor activity.

The Subchronic Toxicity NOAEL is less than 20 mg/kg/day (lowest dose
tested).  The LOAEL is less than or equal to 20 mg/kg/day, based on
significant reductions in body weight gain and suppressed spontaneous
motor activity.

Non-guideline Oral Subchronic Toxicity - Mouse

A non-guideline subchronic toxicity study (Pillai, Mathai, et al. 1988)
was designed to assess the toxicity effects of sodium fluoride (purity
not reported) on the male mouse.  Sodium fluoride was administered daily
to 5 Swiss albino mice of a laboratory colony Haffkine strain at a
concentration of 5.2 mg/kg/day for 35 days.  These mice were also
provided with a low-fluoride diet and water supply (< 1ppm fluoride).

There were significant changes in hematological analyses with decreases
in red blood cells, lymphocytes, hemoglobin, albumin, total protein,
cholesterol, glucose, and alkaline phosphatase.  Statistically
significant increases were observed in white blood cells, monocytes,
basophils, and eosinophils.  Food and water consumption was
significantly decreased in treated animals compared to controls.  There
were significant, treatment-related decreases from control in body
weight gain of sodium fluoride-treated mice after day 19 of the
treatment period.  A significant relationship between food and water
consumption and the body weight was observed in the controls, but not in
the treated animals.

Significant increases in fluoride content were measured in the kidneys,
stomach, brain, liver, and intestines of the sodium fluoride-treated
animals when compared to the controls.  The increases were 3.5- and
1.5-fold greater than control in the kidneys and stomach, respectively,
while the brain, intestines, and liver exhibited 2-fold increases over
control.  There was no evidence of sperm abnormalities following
treatment with sodium fluoride.

The Subchronic toxicity NOAEL is less than 5.2 mg/kg/day (lowest dose
tested).  The LOAEL is less than or equal to 5.2 mg/kg/day, based on
significant decreases in body weight gain, and food and water
consumption.

	4.3	Prenatal Developmental Toxicity

Adequacy of database for Prenatal Developmental Toxicity: The database
for developmental toxicity is considered complete.

870.3700a 	Developmental Toxicity – Rat

A prenatal developmental toxicity study (Bates, et al.) was designed to
evaluate postnatal growth and viability of 104 prenatally exposed mated
female CD rats.  Sodium fluoride (purity >99%) was administered ad
libitum in drinking water to groups of 26 rats/dose at dose levels of 0,
7, 18, or 27 mg NaF/kg/day (0, 50, 150, and 300 ppm, respectively) from
gestation days (GD) 6 to 15.  Females were weighed,and observed for
clinical signs of toxicity daily starting on GD 6.  Food and water
weights were recorded every other day during GD 0 to 20.  The maternal
body, liver, right kidney, and intact uterus were weighed and corpora
lutea were counted.  Each live fetus was weighed and examined for
external, visceral, and skeletal malformations.

There were no treatment-related clinical signs, increases in mortality
(100% survival), or decreases in body weight in rats dosed with sodium
fluoride.  The maternal body weight gain during the first two days of
exposure (GD 6 to 8) was significantly reduced (55%) at 300 ppm (27
mg/kg/day) relative to controls.  The mean maternal body weight gain and
water consumption during the treatment period was also significantly
reduced, possibly due to a decrease in palatability.  The Maternal
toxicity NOAEL is 18 mg/kg/day.  The Maternal toxicity LOAEL is 27
mg/kg/day, based on reduced maternal body weight gain.

There were no changes in reproductive parameters in treated animals
compared to controls.  The Reproductive toxicity NOAEL is greater than
or equal to 27 mg/kg/day (highest dose tested).  The Reproductive
toxicity LOAEL is greater than 27 mg/kg/day (not established).

There were no treatment-related effects observed in mean live fetal body
weight/litter or the number of live fetuses.  A dose-related increase in
the percent of litters with one or more externally malformed fetuses,
externally malformed fetuses/litter, and skeletally malformed
fetuses/litter occurred, however these increases were not statistically
significant.  The Developmental toxicity NOAEL is greater than or equal
to 27 mg/kg/day (highest dose tested).  The Developmental toxicity LOAEL
is greater than 27 mg/kg/day (not established).

870.3700a 	Developmental Toxicity – Rat

A prenatal developmental toxicity study (Collins, Sprando, et al.  1995)
was designed to evaluate the developmental toxicity effects of sodium
fluoride (100% purity) administered to caesarean-derived, viral
antibody-free CD-CRL:CD-BR, VAF + female rats, 33-37 animals/dose, in
drinking water at concentrations of 10, 25, 100, 175, or 250 ppm (1.4,
3.9, 15.6, 24.7, or 25.1 mg/kg/day, respectively).  Animals were also
fed a low-fluoride diet (7.95 ppm fluoride).  Prior to treatment with
sodium fluoride, 1 male for every 2 female rats were mated and once
females were pregnant treated drinking water was administered.  On
gestation day (GD) 20, a caesarean section was performed and the uterus
was opened and examined for resorption sites, implantation sites and
live or dead fetuses.

There were no incidences of maternal mortality, changes in behavior,
clinical signs, or mottled teeth in dams treated with sodium fluoride.
In the 100 ppm dose group, there was 1 female rat that exhibited
multiple, apparently random, clinical findings (exudate from the eye and
nose, and overgrown teeth) that were not associated with treatment.  The
250 ppm dose group experienced significant decreases in food and water
consumption, and body weight gain that were 7, 30, and 11% less than
controls, respectively.  A significant reduction (10.7%), from control,
in fluid consumption was observed in animals treated with 175 ppm sodium
fluoride; however, there were no other treatment-related changes found
at this dose level.  The Maternal toxicity NOAEL is 175 ppm (24.7
mg/kg/day).   The Maternal toxicity LOAEL is 250 ppm (25.1 mg/kg/day),
based on significant reductions in food and water consumption and body
weight gain. (The similarity in the mg/kg/day concentrations can be
attributed to decreases in water consumption, which was greater at 250
ppm.  At 175 ppm, there was a decrease in water consumption; however,
there were no significant changes in feed consumption and body weight
gain.)

The pregnancy rate was greater than 90% for all groups.  There was a
significant decrease in the mean number of corpora lutea/female in dams
of the 250 ppm dose group; however, because the number of corpora lutea
is determined at birth, this decrease is considered to be random.  There
were no significant changes in reproductive parameters in treated
animals when compared to controls.  The Reproductive toxicity NOAEL is
greater than or equal to 250 ppm (25.1 mg/kg/day; highest dose tested).
The Reproductive toxicity LOAEL is greater than 250 ppm (25.1 mg/kg/day;
not established).

There were no treatment-related effects on fetal body weight, litter
sizes, or viable fetuses.  Several external variations were observed in
control and treated animals; however, there were no significant
increases in the number of fetuses with at least 1, 2, or 3 variations,
or in the number of litters with fetal sternebral variations.  There was
no evidence of teratogenicity observed in the rats following
administration of phenol.  The Developmental toxicity NOAEL is greater
than or equal to 250 ppm (25.1 mg/kg/day; highest dose tested).  The
Developmental toxicity LOAEL is greater than 250 ppm (25.1 mg/kg/day;
not established).

870.3700a 	Developmental Toxicity – Rat

A prenatal developmental toxicity study (Heindel et al. 1996) was
designed to evaluate postnatal growth and viability of 104 prenatally
exposed mated female Sprague-Dawley rats.  Sodium Fluoride was
administered ad libitum in drinking water to groups of 26 rats/dose at
dose levels of 0, 6.6, 18.3, or 27.1 mg NaF/kg/day (0, 50, 150, and 300
ppm, respectively) from gestation days (GD) 6 to 15.  Females were
weighed, and food and water weights were recorded every other day during
GD 0 to 20 and observed for clinical signs of toxicity daily starting on
GD 6.  The maternal body, liver, right kidney, and intact uterus were
weighed and corpora lutea were counted.  Each live fetus was weighed and
examined for external, visceral, and skeletal malformations.

There were no treatment-related clinical signs, increases in mortality
(100% survival), or decreases in body weights in rabbits dosed with
sodium fluoride at the low- and mid-dose.  The maternal body weight gain
of the high dose group on GD 6-8 was 56% less than the control.  During
the treatment period, as a whole, there was not a significant difference
in mean body weight gain; however, a decreasing trend that approached
statistical significance was observed.  The water consumption during the
treatment period was significantly reduced at the high-dose.  The food
consumption was decreased  at the high-dose during GD 8-10, but was
normal thereafter.  The Maternal toxicity NOAEL is 18.3 mg/kg/day.  The
LOAEL is 27.1 mg/kg/day, based on reduced maternal body weight gain.

There were no treatment-related effects on mean live fetal body
weight/litter, live fetal number, and prevalence of malformations.  The
Developmental toxicity NOAEL is greater than or equal to 29 mg/kg/day
(highest dose tested).  The LOAEL is greater than 29 mg/kg/day (not
established).

870.3700b 	Developmental Toxicity – Rabbit

A prenatal developmental toxicity study (Heindel et al. 1996) was
designed to evaluate postnatal growth and viability of 104 prenatally
exposed mated female New Zealand White Rabbits.  Sodium Fluoride was
administered ad libitum in drinking water to groups of 26 rabbits/dose
at dose levels of 0, 10, 18, or 29 mg NaF/kg/day (0, 100, 200, and 400
ppm, respectively) from gestation days (GD) 6 to 19.  Females were
weighed, and food and water weights were recorded every other day during
GD 0 to 30 and observed for clinical signs of toxicity daily starting on
GD 6.  Pregnancy rates were 84, 87, 78, and 83% in the control to high
exposure groups, respectively.  The maternal body, liver, right kidney,
and intact uterus were weighed and corpora lutea were counted.  Each
live fetus was weighed and examined for external, visceral, and skeletal
malformations.

There were no treatment-related clinical signs, increases in mortality
(100% survival), or decreases in body weights in rabbits dosed with
sodium fluoride at the low- and mid-dose. During the treatment period,
as a whole, there was not a significant difference in mean body weight
gain.  The high-dose (400 ppm) group, during GD 6 to 8, experienced a
mean weight loss of 112 grams versus an mean weight gain of 14 grams for
the control.  During GD 10 to 12, the 400 ppm group recovered with a
mean weight gain of 71 grams versus 22 grams for the control.  The water
consumption during the treatment period was significantly reduced,
possibly due to a decrease in palatability.  Overal the maternal food
consumption was decreased at the high-dose with a significant decrease
from GD 6 to 8.  The Maternal toxicity NOAEL is 18 mg/kg/day. The LOAEL
is 29 mg/kg/day, based on reduced maternal body weight gain.

There were no treatment-related effects on mean live fetal body
weight/litter, live fetal number, and prevalence of malformations.  The
Developmental toxicity NOAEL is greater than or equal to 29 mg/kg/day
(highest dose tested).  The LOAEL is greater than 29 mg/kg/day (not
established).

	4.4	Reproductive Toxicity

Adequacy of database for Reproductive Toxicity: The database for
reproductive toxicity is considered complete.

870.3800	Reproduction – Rat

A reproduction and fertility effects study (Collins, Sprando, et al.
2001) was designed to determine any generational, possibly cumulative,
effects on the development of offspring when sodium fluoride (purity not
reported) was ingested continuously through multiple generations of
caesarean-derived CD CRL:CB-BR, viral antibody-free rats.  Sodium
fluoride was administered to 48 rats/sex/dose at concentrations of 25,
100, 175, or 250 ppm in drinking water for 10 weeks.  Animals were also
fed a low-fluoride (7.95 ppm) NIH-07 diet.  Following the treatment
period, female rats were mated 1:1 with males.  On gestation day (GD) 20
a caesarean section was performed on 8 mated F0 females to measure
reproductive and developmental parameters.  The uterus was opened and
examined for presence and position of resorption sites, implantation
sites, and live or dead fetuses.  The remaining F0 females were allowed
to litter and wean their pups.  On postnatal day (PND) 21, 36/sex/dose
group, F1 rats were randomly selected to provide the F2 generation.
Both fluoridated drinking water and the low-fluoride diet were provided
as before for 10 weeks, and animals were mated as previously described.
F1 dams were euthanized and caesarean sections were performed on GD 20.

There were no treatment-related effects on maternal mortality.  A
significant decrease from control in fluid consumption (30%) was
observed at the 250 ppm dose level.  There were no other changes in F0
maternal generation.  Significant decreases from control were observed
in fluid consumption, with reductions of 28 and 31% in the F1 dams at
the 175 and 250 ppm dose levels, respectively.  The decreases in fluid
consumption corresponded with decreased palatability of the solution.
Food consumption was significantly reduced (11%) in F1 dams when
compared to control in the 175 ppm dose group.  There was a 14% decrease
from control in the body weight gain of F1 females (dams) treated with
175 ppm.  These reductions at 175 ppm were considered random because of
the lack of effect in 250 ppm group.  Gravid uterine weight measurements
showed no dose-related differences.  The Maternal toxicity NOAEL is
greater than or equal to 250 ppm (highest dose tested).  The Maternal
toxicity LOAEL is greater than 250 ppm (not established).

There were no treatment-related effects in the mean number of corpora
lutea, mean number of implantation sites, implantation efficiency, mean
number of viable fetuses, or average percentage of early and late deaths
per litter of dams.  The Reproductive toxicity NOAEL is greater than or
equal to 250 ppm (highest dose tested).  The Reproductive toxicity LOAEL
is greater than 250 ppm (not established).

Fetal body weight was not affected by treatment with sodium fluoride.
There was no evidence of toxicity in fetuses or pups of the F1
generation.  Similarly, the F2 generation fetuses and pups were
unaffected by treatment with sodium fluoride with the exception of
decreased ossification of the hyoid bone in the F2 fetuses at the 175
(not significant) and 250 ppm (significant) dose groups.  The Offspring
toxicity NOAEL is 175 ppm.  The Offspring toxicity LOAEL is 250 ppm,
based on decreased ossification of the hyoid bone.

Non-guideline Reproduction – Rat

A special reproductive and fertility effects toxicity study (Aráibi, et
al. 1989) was designed to assess the effects of sodium fluoride (purity
not reported) on the reproductive performance of the adult male albino
rat.  Sodium fluoride was administered to 15 rats/dose at concentrations
of 100 or 200 ppm in the diet for 60 days.  Blood samples from cardiac
puncture were collected from 5 animals of each dose group and the
control group.  These animals were sacrificed (cervical dislocation) and
testes were removed.  The remaining animals were mated with normal
females (1 male to 2 females) to examine fertility.

Lesions on the teeth (mottling and erosion of enamel), a characteristic
commonly associated with sodium fluoride exposure, were observed in
animals at the end of the experiment.  Males treated with sodium
fluoride seemed to show less interest toward females when compared to
those animals of the control group.  The number of pregnant females were
decreased 10 and 40% from controls in groups treated with 100 and 200
ppm, respectively.  High-dose animals exhibited significant reductions
in the number of pregnant females.  The number of newborns produced by
the 100 and 200 ppm dose groups were significantly less than controls,
with decreases of 30 and 57%, respectively.  There was a decrease in
average litter size for both dose levels, although neither reduction was
significantly different from controls.

Mean tubular diameters were significantly less than controls with 3 and
7% decreases in diameter for the 100 and 200 ppm dose levels,
respectively.  There were 94 and 93% (significant) increases in
peritubular membrane thickness in the low- and high-dose groups,
respectively.  Treatment of animals with 200 ppm sodium fluoride
resulted in significant decreases from control in percentage of
seminiferous tubules containing spermatozoa.  There were decreases in
mean testosterone levels in the serum of treated animals with 29
(nonsignificant) and 71% (significant) reductions from controls observed
in the 100 and 200 ppm dose groups, respectively.

There was a decrease in reproductive performance of male rats exposed to
a high intake of sodium fluoride in spite of the absence (until the end
of the experiment) of clinical signs in the teeth that are
characteristic features of fluorosis.  The testes of 200 ppm sodium
fluoride-treated rats exhibited impairments of spermatogenesis and
steroidogenesis based on changes in mean diameter of seminiferous
tubules, the thickness of peritubular membranes, percentage of
seminiferous tubules containing spermatozoa, and serum testosterone
levels.  The researchers suggested that sodium fluoride appears to be
antispermatogenic and the decrease in testosterone may account for the
decrease of mated females in sodium fluoride-treated groups. However,
the results of this study could not be verified.

	4.5	Chronic Toxicity

Adequacy of database for Chronic Toxicity: There is only one chronic
toxicity study available for sodium fluoride. This study is considered
inadequate for assessment of chronic toxicity. However, chronic
toxicity/carcinogenicity data are available for sodium fluoride that
address the chronic toxicity of this chemical.

	4.6	Carcinogenicity

Adequacy of database for Carcinogenicity: The database for
carcinogenicity consists of three studies; one open literature study in
the rat and two National Toxicology Program studies.

870. 4200a	Oncogenicity – Rat

A 24-month carcinogenicity study (Maurer, et al. 1990) was designed to
assess the oncogenic potential of sodium fluoride (>99% purity) when
administered to groups of 70 male and 70 female Albino Sprague-Dawley
rats.  The treatment groups were fed a low-fluoride diet that was
supplemented with sodium fluoride at concentrations of 4, 10, or 25
mg/kg/day (groups 3-5, respectively) and the control groups were fed
either the low-fluoride diet (group 1) or the normal laboratory chow
(group 2) (Purina Certified Rodent Chow; fluoride content not
determined) for up to 99 weeks.  The study was designed to continue for
24 months or until survival for a single group of males or a single
group of females was 20% or less.  The survival was 20% or less at week
95 for males receiving 4 mg/kg/day and at week 99 for females in the
low-fluoride control group.  At 26 weeks, sufficient rats were killed to
retain 60 animals/sex/group. Ten rats/sex were killed after 52 weeks to
assess fluoride toxicity.

There was no evidence of treatment-related incidence of carcinogenicity
in Sprague-Dawley rats administered dietary sodium fluoride in
concentrations up to 25 mg/kg/day  for 2 years.  All bone neoplasms
observed were considered to be incidental and spontaneous and, not
related to sodium fluoride treatment because of their low incidence and
random distribution.  The incidence of preneoplastic and neoplastic
lesions at any site in rats of either sex was not altered by the
adminstration of sodium fluoride.  Sodium fluoride was not carcinogenic
to rats within the confines of this study.

There were no treatment-related increases in mortality in rats receiving
sodium fluoride in the feed for 95 or 99 weeks.  Survival rates for
groups 1-5 were approximately 35, 44, 20, 43, and 48% for males (95
weeks) and 20, 50, 35, 42, and 22% for females (99 weeks), respectively.
 There was no evidence of a significant, positive dose-related trend in
mortality for either sex; however, females in the 4 and 10 mg/kg/day
dose groups exhibited significantly greater survival rates than that of
the low-fluoride control.

At study termination, diet consumption for the 25 mg/kg/day (group 5)
was significantly reduced when compared to the control (group 1), with
decreases of approximately 20 and 18% for males and females,
respectively.  Body weight gain was significantly less than the control
for the 25 mg/kg/day dose group.  Both male and female rats administered
the high-dose of sodium fluoride experienced decreases of roughly 25% in
mean body weight gain.

Clear evidence of fluoride toxicity was seen in the teeth, bones, and
stomach, the severity of which was related to dose and duration of
treatment.  At sodium fluoride concentrations of 4 mg/kg/day or greater,
dental changes occurred including incisors malformations and fractures,
and enamel hypoplasia.  Treatment-related bone effects, mostly skull,
were observed at concentrations of 10 mg/kg/day and greater.  Affected
bones were white, thick, and found to have roughened surfaces and
subperiosteal hyperostosis.  There was a lack of bone marrow cavities in
the new bone.  There was an increase in incidence and severity of
chronic inflammation of the gastric glandular mucosa in rats treated
with sodium fluoride doses at or above 10 mg/kg/day.

At the 25 mg/kg/day dose level, animals experienced decreased plasma
glucose levels and specific gravity of urine.  Males exhibited a
decrease in plasma globulins that was probably related to the reduced
diet consumption.  The lack of survival effect indicates that these
small parameters did not significantly affect the general health of the
rats.

Levels of fluoride in the urine and bone of animals increased as the
level of sodium fluoride in the diet increased.  The fluoride
concentration of the urine was a linear function of the ingested dose,
regardless of whether the fluoride was endogenous to the diet or added
as sodium fluoride.  The low-fluoride diet yielded a proportionately
lower level of fluoride in bone than the added sodium fluoride.

The diet consumption for males in group 2 (lab chow control) was
significantly less (18%) than that of the control (group 1, low-fluoride
diet).  However, this same group (2, lab chow) exhibited a 10% decrease
in mean body weight gain compared to control.  Females of the lab chow
control group exhibited a survival rate that was overall greater than
the sodium fluoride-treated females and significantly greater than the
low-fluoride control.  The results associated with the animals
maintained on laboratory chow were equivocal and prevented comparisons
with the treated and control groups fed the low-fluoride diet.

870.4200 	Oncogenicity – Mouse

In a carcinogenicity study (NTP TR 393), male and female B6C3F1 mice on
a low fluoride diet were administered sodium fluoride (NaF; 99% a.i.,
Lot A022085) at concentrations in deionized drinking water of 0 (100
mice/sex), 25 (70 mice/sex), 100 (70 mice/sex), or 175 ppm (100
mice/sex) for 103 weeks.  The average daily doses of NaF corresponding
to these drinking water concentrations were estimated to be 2.4, 9.6, or
16.7 mg/kg in males, and 2.8, 11.3, or 18.8 mg/kg in females.  Including
the fluorine available from the low-fluoride diet, the estimated total
daily fluoride ion intakes for control, low-, mid-, and high-dose groups
were 0.6, 1.7, 4.9, and 8.2 mg/kg in males and 0.6, 1.9, 5.7, and 9.1
mg/kg in females.  Ten mice/sex/dose group were sacrificed at 24 and 66
weeks.  Fluoride concentrations in bone (humerus) were determined from
samples taken from all animals at interim sacrifice and 10 randomly
selected mice/sex at terminal sacrifice.  Complete hematology and select
clinical chemistry analyses were conducted on all interim sacrifice
animals.  Organ weights were taken only at interim sacrifice and only of
the liver, both kidneys, and the brain.

There were no compound-related effects on mortality, body weight, food
consumption, water consumption, hematology, or organ weights.
Treatment-related clinical findings included a dose-dependent increase
in white discoloration of the teeth (27%, 39%, 80%, and 100% in males
and 19%, 43%, 84%, and 100% in females, from control to high dose,
respectively) which occurred as early as Day 74 in the high-dose animals
compared to Day 508 in the control animals.  Serum alkaline phosphatase
was significantly increased in high-dose females at 24 (29%) and 66
weeks (88%) and in high dose-males at 66 weeks (11%).  Serum phosphorus
levels were significantly decreased (13%) in high-dose males at 66
weeks.  There was a significant increase in incisor dentine dysplasia in
high-dose males (78% in controls versus 91% at the high dose).  There
was an increase in the incidence of myelofibrosis (femoral, humerus,
maxilla, and thoracic) in female mice at all doses. Based on the
clinical chemistry changes in alkaline phosphatase (males and females)
and serum phosphorus (males) at 66 weeks and bone lesions (dentine
dysplasia, males; myelofibrosis, females), the LOAELs in males and
females were 16.7 and 18.8 mg/kg/day, respectively and the NOAELs in
males and females were 9.6 and 11.3 mg/kg/day, respectively.

At the doses tested, a treatment-related increase in any tumor incidence
was not noted when compared to controls.  Dosing was considered adequate
based on the overall changes noted in the high-dose animals.  However,
it should be noted that the mice may have been able to tolerate a higher
dose.

This carcinogenicity study in mice is ACCEPTABLE-GUIDELINE and satisfies
the guideline requirement for a carcinogenicity study [OPPTS 870.4200;
OECD 451] in mice.

870.4300	Combined Chronic Toxicity/ Oncogenicity – Rodent

A 24-month study (NIH Publication No. 91/2848, 1990) was

designed to examine the chronic and carcinogenic effects of sodium
fluoride (99% purity) on male and female F344/N rats fed a low fluoride
diet.  Sodium fluoride (NaF) was administered in deionized drinking
water to animals at concentrations of 0 (100 rats/sex), 25 (70
rats/sex), 100 (70 rats/sex), or 175 ppm (100 rats/sex) for 103 weeks.
These concentrations were equivalent to average daily doses of NaF from
drinking water of 0, 1.3, 5.2, or 8.6 mg/kg for male rats and 0, 1.3,
5.5, or 9.5 mg/kg for females.  Including the fluoride content in the
diet, the estimated average daily doses of fluoride ion at these
concentrations were 0.2, 0.8, 2.5, and 4.1 mg fluoride/kg for males and
0.2, 0.8, 2.7, and 4.5 mg fluoride/kg for females.  An additional 50
age-matched control rats served as concurrent controls for animals dying
prior to terminal sacrifice in the exposed groups.

Mortality, body weight, body weight gain, food consumption, water
consumption, hematology, and organ weights were not affected by exposure
to NaF.  Fluoride concentration increased with dose in blood (serum) at
Weeks 27 and 66, and bone and urine at Weeks 27, 66, and 105.  Analysis
of bone fluoride revealed an increase with dose and age.  Urinary
calcium was observed to be significantly increased in high-dose females.

Tooth discoloration (whitening and mottling) was noted in all treated
animals with attrition, deformity, and occasional malocclusions noted in
the high- and/or mid-dose males. Histopathology of the incisors noted
dentine dysplasia (all dosed animals), degeneration of the ameloblasts
(mid- and high-dose animals), and, to a lesser extent, degeneration of
the odontoblasts (principally dosed males).  These effects were
dose-dependent in males and showed similar results in females, although
without the strong dose-dependence noted in the male rats.  Increases in
the incidence and severity of osteosclerosis of the long bones were
noted in the high-dose females (7.5% control; 22% high-dose, P=0.04).

The Chronic Toxicity NOAEL is less than 1.3 mg/kg/day.  The Chronic
Toxicity LOAEL is 1.3 mg NaF/kg/day in both sexes, based on dentine
dysplasia in males and females, and ameloblast degeneration in males.

Three bone osteosarcomas were noted in high-dose males and one in a
mid-dose male, with none in controls.  A fourth osteosarcoma, not
originating in the bone, was observed in an additional high-dose male.
Dosing was considered adequate based on tooth deformities and
discoloration; dentine dysplasia and degeneration in the ameloblasts and
odontoblasts, bone osteosarcomas in males and osteosclerosis in females.
 Trend analyses revealed that, at the doses tested, there was a
significant treatment-related increase in the incidence of bone
osteosarcomas in males but the incidence was not significantly increased
in the high-dose males as compared to controls when comparisons were
made either within the animals scheduled for terminal sacrifice or all
animals (including the interim sacrifice and concurrent control
animals).  In those animals scheduled for terminal sacrifice,
statistical analysis of all organ osteosarcoma in dosed animals as
compared to controls also failed to show significance.  The study
authors failed to perform the statistical analysis all osteosarcoma
analysis among all animals. That analysis, done by the contractor, did
reveal a significant difference between the high dose and control
groups.  Due to the fact that bone osteosarcoma incidence of the
high-dose as compared to the control group was not significant, but
displayed a significant positive trend, the occurrence of these rare
tumors was considered equivocal evidence of carcinogenicity in male rats
by the study authors.  Such a conclusion was bolstered by the fact that
bone osteosarcomas were not observed in treated females or in the
parallel study in B6C3F1 mice (TR393, NIH Publication No. 91/2848).
However, with the significant difference between high dose animals and
controls in the all organ osteosarcoma incidence analysis when all
animals are considered, the reviewer believes that the occurrence of
osteosarcomas in the male rats should have been considered some
evidence, if not clear evidence, of the carcinogenic activity of sodium
fluoride.

This chronic/carcinogenicity study in the rat is ACCEPTABLE-GUIDELINE
and satisfies the guideline requirement for a chronic/carcinogenicity
study [OPPTS 870.4300; OECD 453] in rats.

	4.7	Mutagenicity

Adequacy of database for Mutagenicity Toxicity: The database for
mutagenicity is considered complete. Several open literature studies are
available as well as data from the National Toxicology Program.

The mutagenicity data for sodium fluoride is summarized below in Table
3.

Table 3. Genotoxicity Profile for Sodium Fluoride

Guideline No./Study type/Citation	Results	Positive or Negative for
Mutagenicity

Gene Mutation

870.5100

Bacterial reverse mutation test

Gocke et al. (1981). Mutagenicity of Cosmetics Ingredients Licensed by
the European Communities.

Mutation Research 90.2:91-109.

Open Literature

5 Tester strains of Salmonella typhimurium

There was no evidence of induced mutant colonies over background
following administration of sodium fluoride in the presence or absence
of metabolic activation. The numbers of his+ revertants observed with
treatment were not significantly different from control with any of the
study concentrations of sodium fluoride. Sodium fluoride was not
mutagenic to any of the 5 Salmonella typhimurium bacterial strains in
the presence or absence of metabolic activation.

NEGATIVE

870.5100

Bacterial reverse mutation test

Haworth et al. (1983). Salmonella Mutagenicity Test Results for 250
Chemicals. Env. Mutagenesis Supplement 1:3-142.

Open Literature

Incubation concentration up to 10 mg/plate

There was no evidence of induced mutant colonies over background.
Positive controls produced appropriate responses in corresponding
strains of the bacterial reverse mutagenesis test. S. typhimurium did
not show mutagenic activity in the presence or absence of metabolic
activation following administration of sodium fluoride.

NEGATIVE

870.5100

Bacterial reverse mutation test

Li, Y., Dunipace, A., Stookey, G. (1987). Absence of Mutagenic and
Antimutagenic Activities of Fluoride in Ames Salmonella

Assays. Mutation Res 190:229-236.

Open Literature

Bacterial Tester Strains TA97a, TA98, TA100, TA102, and TA1535

0.44, 4.42, 44.2, 88.4m 221.1, 442.1, 1105.3, 2210.5, or 4421.0
µg/plate NaF

Sodium fluoride was not mutagenic in the Salmonella typhimurium
bacterial strains in the presence or absence of metabolic activation.

ic effects were first observed at concentrations ≥ 1100 µg/plate in
various strains. The strains ranged from the most sensitive to least
sensitive; 97a, 102, 100, 1535 and 98. The incorporation of metabolic
activation increased the number of revertants, but did not significantly
influence the toxic effects of sodium fluoride on the bacteria. There
was no evidence of induced mutant colonies over background following
administration of sodium fluoride in the presence or absence of
metabolic activation. The numbers of his+ revertants observed with the
treatment were not significantly different from control with any of the
study concentrations of sodium fluoride.

NEGATIVE

870.5100

Bacterial reverse mutation test

Martin, G. et al. (1979). Lack of Cytogenic Effects in Mice or Mutations
in Salmonella Receiving Sodium Fluoride. Mutation Res 66:159-167.

Open Literature

Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, and TA 100

0.1-500 µg/plate NaF

There was no evidence of induce mutant colonies over background.
Positive controls produced appropriate responses in corresponding
strains of the bacterial reverse mutagenesis test. S. typhimurium did
not show mutagenic activity in the presence or absence of metabolic
activation following administration of sodium fluoride.

NEGATIVE

870.5100

Bacterial reverse mutation test

Tong et al. (1988). The Lack of Genotoxicity of Sodium Fluoride in a
Battery of Cellular Tests. Cell Biology and Toxicology 4.2:173-186.

Open Literature

Bacterial Tester Strains, Salmonella typhimurium

TA 1535, TA 1537, TA 1538, TA 98, and TA 100

10, 20, 40, 80, 160, or 320 µg/plate

Sodium fluoride was not mutagenic to any of the 5 Salmonella typhimurium
bacterial strains in the presence or absence of metabolic activation.

The higher doses (80-320 µg/plate) were slightly cytotoxic. However at
all doses there was no evidence of induced mutant colonies over
background following administration of sodium fluoride in the presence
or absence of metabolic activation. The numbers of his+ revertants
observed with treatment were not significantly different from control
with any of the study concentrations of sodium fluoride.

NEGATIVE

870.5100

Bacterial reverse mutation test

Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No.
7681-49-4) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies).
National Toxicology Program, Technical Report Series No. 393, NIH
Publication No. 91-2848, December 1990, Pgs. 1-477.

Acceptable

Guideline

Strains TA98, TA100, TA1535, and TA1537 of S. typhimurium

0, 100, 333, 1000, 3333, and 10000 µg/plate

There was no clear evidence, or a concentration related positive
response, of induced mutant colonies over background.

NEGATIVE

870.5300

In Vitro mammalian cell gene mutation test

Caspary, W. et al. (1987). Mutagenic Activity of Fluorides in Mouse
Lymphoma Cells. Mutation Res 187:165-180

Open Literature

-S9 Trial 1-200, 300, 400, 500, 600, 0r 800 µg/mL

Trial 2-50, 100, 200, 300, 400, 500, or 600 µg/mL

+S9: Trial 1-100, 200, 300, 400, 500, or 600 µg/mL

Trial 2-50, 100, 200, 300, 400, 500 or 600 400, 500, or 600 µg/mL

Sodium fluoride was mutagenic in L5179Y mouse lymphoma cells in the
presence and absence of metabolic activation.

There was evidence of general toxicity in the 300-500 µg/mL sodium
fluoride concentration range and lethality usually occurred at higher
concentrations (600-800 µg/mL). In the absence of metabolic activation,
cytotoxicity was apparent at the 800 µg/mL sodium fluoride dose level
in Trial 1. Cytotoxicity was observed in the presence of metabolic
activation at 600 µg/mL.

There were significant increases in mutation frequency following
administration of sodium fluoride. Trial 1 (-S9) experienced 1.8-, 1.6-,
1.9-, and 2.9-fold increases at sodium fluoride concentrations of 300,
400, 500 and 600 µg/mL, respectively, while Trial 2 (-S9) exhibited a
1.6-fold increase at the 500 µg/mL dose. In the presence of metabolic
activation, 1.5-, 3.1-, and 3.6- fold increases in Trial 1 and 1.6-,
1.9-, and 2.3-fold increases in Trial 2 occurred at sodium fluoride 400,
500, and 600 µg/mL and 300, 400, and 500 µg/mL, respectively.

Sodium fluoride had a significant effect in gene mutations at the TK
locus, although the addition of metabolic activation ha no apparent
effect on either the toxicity or mutagenic activities of sodium
fluoride. The measured mutant colony size was predominantly small.

POSITIVE

870.5300

In Vitro mammalian cell gene mutation test

Oberly et al. (1990). An Evaluation of the CHO/HGPRT Mutation Assay
Involving Suspension Cultures and Soft Agar Cloning: Results for 33
Chemicals. Environmental and Molecular Mutagenesis 16:260-271.

Open Literature

Chinese Hamster Ovary (CHO)/HGPRT+ cells, Strain K1-BH4

250, 500, 600, 700, or 800 µg/mL in –S9

200, 400, 450, 500, 550, 600, or 700 µg/mL in +S9

All doses greater than 450 µg/mLwith and without activation were toxic,
as is evident by the relative total growth of 38% or less. Sodium
fluoride was not mutagenic at the HGPRT locus of Chinese hamster ovary
cells.

NEGATIVE

870.5300

In Vitro mammalian cell gene mutation test

Tong et al. (1988). The Lack of Genotoxicity of Sodium Fluoride in a
Battery of Cellular Tests. Cell Biology and Toxicology 4.2:173-186.

Open Literature

ARL 1 (rat liver epithelial cell line)

2, 10, 20, 40, 80, or 160 µg/mL

The higher doses (80 and 160 µg/mL) were toxic and were not analyzed
for gene mutatins. Sodium fluoride at doses up to 40 µg/mL did not
result in any significant increase in TGR mutants above the control.
Sodium fluoride was not mutagenic at the HGPRT locus.

NEGATIVE

870.5300

In Vitro mammalian cell gene mutation test

Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No.
7681-49-4) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies).
National Toxicology Program, Technical Report Series No. 393, NIH
Publication No. 91-2848, December 1990, Pgs. 1-477.

Acceptable

Guideline

Trifluorothymidine (TFT)-resistant cells at the thymidine kinase (TK)
locus exposed to sodium fluoride in lymphoma L5178Y cells cultured in
vitro in either distilled water or culture medium

There was evidence of a concentration related positive response of
induced mutant colonies over background.

Sodium fluoride was tested up to cytotoxic concentrations (the maximum
dose tested was 1000 µg/mL in one laboratory and 800 µg/mL in
another).  Mutant frequencies increased in a dose-related manner.
Statistically significant (p<0.05) responses were observed in all
trials, ±S9, at the high doses.  Mutant fractions (vs. the solvent
control response) at the highest doses tested in each trial were
reported to be 83.0x10-6 vs. 29.5x10-6, 41.3x10-6 vs. 24.3x10-6,
134.0x10-6 vs. 58.0x10-6, and 195.5x10-6 vs. 51.0x10-6 in cultures
tested in the absence of metabolic activation, and 94.0x10-6 vs.
25.8x10-6 and 75.7x10-6 vs. 33.0x10-6 in cells tested in the presence of
metabolic activation.

POSITIVE

Chromosome Aberration

870.5375

In Vitro mammalian chromosome aberration test

Aardema MJ, et al.  (1989). Sodium Fluoride-Induced Chromosome
Aberrations in Different Stages of the Cell Cycle: A Proposed Mechanism.
 Mutation Research 223:191-203.

Open Literature

Cells exposed to 465, 650, 911, 1276, or 1786 ug/mL NaF

In separate experiments, cells exposed to 100 ug/mL of sodium fluoride
for 1 or 2 hours or concentrations of 0.1, 1.0, 10, 25, 50, 75, and 100
ug/mL for 3 hours

A high level of toxicity was observed at 1786 go/mL (high-dose) that
limited chromosome aberration analysis to the lower dose groups.  There
was an increase in average cell generation time (AGT) as the
concentration of sodium fluoride increased; indicating a
treatment-related cell-cycle delay.  At 20 hours after treatment,
greater than 50% of the cells were in their first mitosis at 911 and
1276 go/mL +/-S9 and at 465 and 650 go/mL +S9.

Overall, there was a significant increase in the percentage of aberrant
cells in sodium fluoride treated groups at the 8 and 20 hour
post-treatment harvesting of cells.  There were 4.5- and 3.5-fold
increases in the percentage of aberrant cells, at the 8 hour harvest
time, in the 465 and 911 go/mL dose group, respectively, in the absence
of metabolic activation.  In the presence of S9, the 465, 650, and 1276
go/mL dose groups exhibited 6.3-, 3.7-, and 5.0-fold increases,
respectively, in the number of aberrant cells.  In the 20 hour
harvesting assay there was only one significant increase in the number
aberrant cells; a 6.5-fold increase at the 1276 go/mL sodium fluoride in
the presence of metabolic activation.  In both harvest (8 or 20 hours)
the aberrations were almost exclusively chromatid-type deletions and
gaps.

There was evidence of endoreduplicated cells observed in the chromosome
aberration screening assay.  At the 465, 650, 911, and 1276 go/mL dose
levels, the percentages of endoreduplicated cells were 2, 3, 5, and 11%
for -S9 and 4, 12, 15, and 14% for +S9, respectively.  These cells all
had an M0/M1 staining pattern indicating they had gone through 0 or 1
round of DNA synthesis.  Endoreduplicated cells were also observed in
the CHO 8 and 20 hour harvest time assays.  At the

 8 hour timepoint in the absence of metabolic activation there was a 6%
increase in endoreduplicated cells with the 1276 go/mL dose of sodium
fluoride.  There were endoreduplicated cell increases of 6, 16, and 22%
in -S9 and 28, 26, and 34% in +S9 at the 20 hour harvest time point for
the sodium fluoride concentrations of 650, 911, and 1276  go/mL,
respectively.

In separate experiments cells were exposed to 100 go/mL of sodium
fluoride for 1 or 2 hours or concentrations of 0.1, 1.0, 10, 25, 50, 75,
and 100 go/mL for 3 hours.  There were no significant changes from
control in chromosome aberrations until the 3 hour incubation assay.
Sodium fluoride at doses greater than 10 go/mL induced increases in the
percentage of aberrant cells that were significant at concentrations
greater than or equal to 50 go/mL.  The types of aberrations were
chromatid deletions, isochromatid deletions, and a large number of gaps
but not chromatid exchanges.In this CHO assay, cell-cycle kinetic
studies indicated that aberrations were induced in cells exposed to
sodium fluoride at the 20 hour harvest time (G1/S phase) but the
increases in aberrant cells were greater at the 8 hour time point where
most of the metaphases were from cells exposed to sodium fluoride in the
G2 stage of the cell cycle.  This sensitivity of the G2 cells was
evident in the 3 hour exposure assay with increases in aberrant cells at
concentrations greaterthan 10 go/mL; concentrations that are relatively
much greater than levels present in water or dentifrices.  The
researchers suggest that the level of sodium fluoride-induced
mutagenicity is dependent on both the cell-cycle stage that cells are in
during exposure and the length of time until harvest.  Sodium fluoride
induced positive mutagenic results in CHO cells at concentrations
greater than or equal 50 go/mL when exposed for 3 hours in the presence
and absence of metabolic activation.  However, longer exposure times (8
or 20 hours) required greater concentrations of sodium fluoride (greater
than or equal to 465 go/mL) to achieve mutagenic results.

POSITIVE

870.5375

In Vitro mammalian chromosome aberration test

Albanese.  (1987). Sodium Fluoride and Chromosome Damage (In Vitro Human
Lymphocyte and In Vivo Micronucleus Assays).  Mutagenesis 2:497-499.

Open Literature

Cells exposed to 20 or 40 ug/mL NaF for 28 or 2 hours, +/-S9

Sodium fluoride was mutagenic in human peripheral blood lymphocytes in
both the presence and absence of metabolic activation.  However,
mutagenicity appeared to be dependent on exposure time and
concentration.  There were significant dose-dependent increases in
chromosome aberrations in the experiment without metabolic activation;
with 6- and 18-fold increases in total number of damaged cells at the 20
and 40 go/mL dose levels, respectively, after 28 hours of incubation.
In the presence of metabolic activation, there was a significant
2.5-fold increase in total number of damaged cells over control at the
40 go/mL dose level, after 2 hours of incubation.  The chromosome
aberrations observed following the administration of sodium fluoride
were predominantly gaps, breaks, and fragments.  No exchange-type
aberrations (the type thought to correlate better with the carcinogenic
potential of chemicals) were found at any dose level or exposure period.

POSITIVE

870.5375

In Vitro mammalian chromosome aberration test

Khalil.  (1995). Chromosome Aberrations in Cultured Rat Bone Marrow
Cells Treated with Inorganic Fluorides.  Mutation Research 343:67-74.

Open Literature

Bone marrow cells of Sprague-Dawley rats

0.1, 1.0, 10 or 100 uM

Chromosomal aberrations in bone marrow cells increased following the
administration of sodium fluoride in a dose- and time-dependent manner.
Sodium fluoride was mutagenic in Sprague-Dawley rat bone marrow cells
within the confines of this study.

Overall, there was a significant increase in the percentage of aberrant
cells in sodium fluoride treated groups for the 12-, 24-, and 36-hour
exposures.  Only the 0.1 uM treatment, 12-hour exposure cells did not
have a significant increase in breaks/cell or in the percent of aberrant
cells compared to controls.  The increased aberrations at the other
treatment levels mainly consisted of simple aberrations, such as breaks
and fragments.  Small number of complex aberrations, such as chromatid
exchanges and rings, occurred sporadically, at doses 1.0, 10.0, and
100.0 uM.

The number and percentage of aberrations increased with the increasing
concentrations of sodium fluoride and with the prolongation of
treatment.  The only significant exposure time-related effects observed
were between the 12- and 36-hour exposures at concentrations of 1.0,
10.0, and 100.0 uM.

POSITIVE

870.5375

In Vitro mammalian chromosome aberration test

Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No.
7681-49-4) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies).
National Toxicology Program, Technical Report Series No. 393, NIH
Publication No. 91-2848, December 1990, Pgs. 1-477.

Acceptable

Guideline

Chinese hamster ovary cells exposed to sodium fluoride concentrations
between 50 and 1600 µg/mL with metabolic activation or between 16 and
800 µg/mL without metabolic activation

There was evidence of a concentration related positive response of
chromosome aberrations induced over background in one laboratory testing
without metabolic activation; no other evidence of a positive response.

POSITIVE

870.5375

In Vitro mammalian chromosome aberration test

Tsutsui T, et al.  (1984). Sodium Fluoride-Induced Morphological and
Neoplastic Transformation, Chromosome Aberrations, Sister Chromatid
Exchanges, and Unscheduled DNA Synthesis in Cultured Syrian Hamster
Embryo Cells.  Cancer Research 44.3:938-941

Open Literature

Syrian hamster embryo (SHE) cells exposed to sodium fluoride at 50 or
100 ug/mL for 16 hours or  100 or 200 ug/mL for 28 hours

There was a dose- and time-dependent increase in chromosomal aberrations
following administration of sodium fluoride in Syrian hamster embryo
cells.  Sodium fluoride was mutagenic in SHE cells within the confines
of this study.

Treatment-related effects on mortality were observed in SHE cells
administered sodium fluoride.  At sodium fluoride concentrations of 75,
100, and 125 go/mL,/mL, there were decreases in cell survival that were
10, 47, and 61%, respectively, less than control.

There were significant dose-dependent increases in chromosome
aberrations in the sodium fluoride-treated SHE cells.  Sodium fluoride
incubation of 16 hours exhibited increases in aberrant metaphases that
were 9- and 24-fold greater than the control at the 50 and 100 go/mL/mL
dose levels, respectively.  After 28 hours of exposure, the 100 and 200
go/mL/mL dose levels of sodium fluoride induced aberrant metaphases that
were 19- and 29.5-fold greater than control, respectively.  The
chromosomal aberrations were predominantly gaps and some breaks.

POSITIVE

870.5375

In Vitro mammalian chromosome aberration test

Tsutsui, T., N. Suzuki, et al.  (1984). Cytotoxicity, Chromosome
Aberrations and Unscheduled DNA Synthesis in Cultured Human Diploid
Fibroblasts Induced by Sodium Fluoride.  Mutation Research 139:193-198.

Open Literature

Cytotoxicity test: sodium fluoride administered to JHU-1 cells at
concentrations of 50, 100, or 150 go/mL/mL for 1, 2, 6, 12, or 24 hours.

Mutagenicity assay: Sodium fluoride was administered to JHU-1 cells at
concentrations of 25, 50 or 75 go/mL/mL for 12 hours of exposure and 20
or 40 ug/mL for 24 hours exposure

There was a dose- and time-dependent increase in chromosomal aberrations
following administration of sodium fluoride in cultured human diploid
fibroblasts.  Sodium fluoride was mutagenic in JHU-1 cells within the
confines of this study.

Treatment-related cytotoxic effects were observed in JHU-1 cells
administered sodium fluoride.  Cell survival decreased as the sodium
fluoride concentration and duration of exposure increased.  At sodium
fluoride concentrations of 50, 100, and 150 go/mL, there were decreases
in cell survival that were 100, 98, and 90% after 1 hour; 100, 75, and
65% after 2hours; 70, 48, and 40% after 6 hours; 55, 22, and 15% after
12 hours; and 17, 7, and 1% after 24 hours of exposure, respectively.
Sodium fluoride was cytotoxic to JHU-1 cells and cell survival decreased
linearly with increasing dose or exposure time.

There were significant dose- and time-dependent increases in chromosome
aberrations in the sodium fluoride-treated JHU-1 cells.  The 12 hour
sodium fluoride incubation exhibited increases in aberrant metaphases
that were 3.5- and 22.4-fold greater than the control at the 25 and 50
go/mL dose levels, respectively.  Sodium fluoride at 75 go/mL provided
few metaphases to analyze for chromosomal aberrations.  After 24 hours
of exposure, the 20 and 40 go/mL dose levels of sodium fluoride induced
aberrant metaphases that were 7- and 47-fold greater than control,
respectively.  The chromosomal aberrations were predominantly gaps and
some breaks.

POSITIVE

870.5380

Mammalian

Spermatogonial

Chromosomal aberration test

Li, Dunipace, and Stookey.  (1987). Effect of Fluoride on the Mouse
Sperm Morphology Test.  J. Dent. Res. 66:1509-1511.

Open Literature

B6C3F1 male mice fed a low fluoride diet (<0.2 ppm) via stomach
intubation at concentrations of 0.1, 1.0, 10, 20, 35, or 70 mg/kg.
Treated daily for 5 days.

The frequency of abnormal sperm in NaF-treated groups was not
significantly different from controls.  NaF did not cause spermatogenic
damage as determined by the frequency of sperm abnormalities and weights
of testes.

There was an increase in bone fluoride content with increasing dosage;
concentrations less than or equal to 10 mg/kg exhibited significantly
lower bone fluoride content than concentrations greater than or equal to
20 mg/kg.  The increase in bone fluoride demonstrated that fluoride was
adequately absorbed following intubation, and therefore, the route of
administration of NaF used was justified.  NaF was nonspermatogenic in
male mice and supports the view point that Fl has no adverse mutagenic
effects.

NEGATIVE

870.5380

Mammalian

Spermatogonial

Chromosomal aberration test

Mohamed and Chandler.  (1982). Cytological Effects of Sodium Fluoride on
Mice.  Dept. of Biology and School of Medicine, University of Kansas
City, Missouri.  Presented at the 12th I.S.F.R. Conference.

Open Literature

Male BALB/c mice administered a low fluoride diet (0.263 ppm) for 1 week
and sodium fluoride in drinking water (1, 5, 10, 50, 100, or 200 ppm )
for 3 or 6 weeks

There were significant treatment-related increases from controls in
aberration rates among spermatocytes.  There was evidence of cytogenetic
damage found in animals administered sodium fluoride.  Sodium fluoride
within the parameters of this study was found to be mutagenic.

In the three week study, all of the treatments demonstrated a
significantly higher frequency of chromosomal aberrations compared to
controls.  A dose-related response occurred at doses 5 to 200 ppm, but
the frequency of aberrations in the low dose (1 ppm) compared to the 5
ppm group did not express the same increasing trend.

In the three week study, all of the treatments demonstrated a
significantly higher frequency of chromosomal aberrations compared to
controls.  A dose-related response occurred at doses 5 to 100 ppm, but
the frequency of aberrations in the low dose (1 ppm) compared to the 5
ppm group and the 100 ppm group compared to the 200 ppm group did not
express the same increasing trend.

POSITIVE

870.5380

Mammalian

Spermatogonial

Chromosomal aberration test

Pati and Bhunya.  (1987). Genotoxic effect of an environmental
pollutant, sodium fluoride, in mammalian in vivo test system.  Carylogia
40:79-87.

Open Literature

Male Swiss mice administered sodium fluoride as intraperitoneal
injections

10, 20, or 40 mg/kg

 Sodium fluoride was mutagenic to spermatogonial cells in Swiss mice
within the confines of this study.

There was a dose-related increased in the number of abnormal sperm that
increased with increasing dose.  The mean percentages of sperm
abnormalities were 6.4, 6.8 and 7.6% for the 10, 20, and 40 mg/kg dose
levels, respectively, of sodium fluoride.  There were significant
dose-dependent increases in the frequency of spermatogonial aberrations
that were 3.1-, 3.3-, and 3.7-fold greater than the control at the
sodium fluoride doses of 10, 20, and 40 mg/kg, respectively.

The higher incidence of sperm abnormalities induced by sodium fluoride
may be a measure of the genetic damage caused in the germline cells.
There were significant increases over control in the number of
spermatogonial aberrations in animals receiving sodium fluoride.

POSITIVE

870.5385

Mammalian bone marrow chromosomal aberration test

Martin G, et al.  (1979). Lack of Cytogenetic Effects in mice or
mutations in salmonella receiving sodium fluoride.  Mutation Res
66:159-167.

Open Literature

Male BALB/c mice administered sodium fluoride in the diet (0.5 ppm) and
in drinking water (1,5, 10, 50, or 100 ppm) for six weeks

There were no significant treatment-related differences observed in
aberration rates among bone marrow cells.  There was no evidence of
cytogenetic damage found in animals administered sodium fluoride.

NEGATIVE

870.5385

Mammalian bone marrow chromosomal aberration test

Mohamed and Chandler.  (1982). Cytological Effects of Sodium Fluoride on
Mice.  Dept. of Biology and School of Medicine, University of Kansas
City, Missouri.  Presented at the 12th I.S.F.R. Conference.

Open Literature

Male BALB/c mice administered a low fluoride diet (0.263 ppm ) for 1
week and sodium fluoride in drinking water (1, 5, 10, 50, 100, or 200
ppm ) for 3 or 6 weeks

There were significant treatment-related differences observed in
aberration rates among bone marrow cells.  There was evidence of
cytogenetic damage found in animals administered sodium fluoride.
Sodium fluoride within the parameters of this study was found to be
mutagenic.

In the three week study, all of the treatments demonstrated a
significantly higher frequency of chromosomal aberrations compared to
controls.  A dose-related response occurred at doses 1 to 50 ppm, but
the aberration frequencies in the higher doses were not significantly
different from each other.

In the six week study, all of the treatments demonstrated a
significantly higher frequency of chromosomal aberrations compared to
controls.  A dose-related response occurred at doses 1 to 10 ppm, but
the frequency of aberrations in the higher doses were not significantly
different from each other.

POSITIVE

870.5385

Mammalian bone marrow chromosomal aberration test

Pati and Bhunya.  (1987). Genotoxic Effect of an Environmental
Pollutant, Sodium Fluoride, in Mammalian In Vivo Test System.  Carylogia
40:79-87.

Open Literature

Swiss mice administered sodium fluoride orally (40 mg/kg); ip (10 or 20
mg/kg for 24 hours; 40 mg/kg  for 6, 24, or 48 hours or 8 injections of
5 mg/kg intraperitoneally for 120 hours); subcutaneously (40 mg/kg ) for
6, 24, or 48 hours

Sodium fluoride was found to be mutagenic in Swiss mice bone marrow
cells within the confines of this study.

There were significant dose-related increases in chromosomal aberrations
in the 24 hour experiment.  Intraperitoneal injections of sodium
fluoride at concentrations of 10, 20, and 40 mg/kg were 3.3-, 4.3-, and
5.2-fold greater than control, respectively.  Similar and significant
results were observed in the oral and subcutaneous experiments with 5.5-
and 5.0-fold increases, respectively, over controls at the 40 mg/kg dose
level.  There were 1.9 and 3.3-fold increases over control at the 40
mg/kg dose levels of sodium fluoride administered intraperitoneally at 6
(not significant) and 48 hours (significant), respectively.  The
multiple, 5-time dosing of 8 mg/kg resulted in a significant 2.9-fold
increase over control in mouse chromosomal aberrations.

There were treatment-related aberrations, including chromatid gaps and
breaks, isochromatid gaps, fragments and exchanges in mouse bone marrow
cells.  Gaps were observed more frequently than breaks.  Sodium fluoride
induced dose- and time-dependent increases in the number of chromosomal
aberrations, but there was no evidence of route-sensitivity.  There was
no practical difference observed at the same dose level in the 3
administration routes employed.   Additionally, the chronic, repeated
exposure of fractionated doses induced less aberrations than that of an
equivalent dose treated once.  The increases in chromosome aberrations
were significantly greater than control in all experiments with one
exception.  Intraperitoneal injection of 40 mg/kg sodium fluoride over 6
hours failed to induce a significant number of chromosomal aberrations.

POSITIVE

870.5385

Mammalian bone marrow chromosomal aberration test

Zeiger et al.  (1994). Cytogenetic Studies of Sodium Fluoride in Mice.
Mutagenesis 9:467-471.

Open Literature

Male B6C3F1 mice administered fluoride in the diet for 1 week and sodium
fluoride in drinking water (100, 200 or 400 ppm) for 7 days/week for 6
weeks

There were no significant treatment-related differences observed in
aberration rates among metaphase and anaphase bone marrow cells.  There
was no evidence of cytogenetic damage found in animals administered
sodium fluoride.

Three of sixteen mice from the 400 ppm group died during the
sixteen-week treatment period.  A decrease in body weight gain and water
consumption occurred at the 200 and 400 ppm group.  There were no other
treatment-related signs.

NEGATIVE

Other Genotoxicity

870.5395

Mammalian erythrocyte micronucleus test

Albanese.  (1987). Sodium Fluoride and Chromosome Damage (In Vitro Human
Lymphocyte and In Vivo Micronucleus Assays).  Mutagenesis 2:497-499.

Open Literature

Oral gavage of 500 or 1000 mg/kg NaF to Male Alpk:APF Sprague-Dawley
rats

There were no significant increases in the frequency of micronucleated
polychromatic erythrocytes in rat bone marrow cells at the
concentrations of sodium fluoride used in this study.  Sodium fluoride
was not mutagenic in Alpk:APFSD rat bone marrow cells.

There were no treatment-related effects on mortality in any of the
low-dose (500 mg/kg) group; however, 4 of the 5 rats in the 1000 mg/kg
group died prior to the 48 hour sampling period.  No other abnormal
signs were observed in the remaining animals in other dose/time groups.
The ratio of normochromatic erythrocytes (NCEs) to PCEs was 1:1; which
indicated that at the doses used sodium fluoride was not cytotoxic to
the bone marrow cells.

NEGATIVE

870.5395

Mammalian erythrocyte micronucleus test

Gocke et al.  (1981). Mutagenicity of Cosmetics Ingredients Licensed by
the European Communities.  Mutation Research 90.2:91-109

Open  Literature

Male and female NMRI mice and Sprague-Dawley rats

There were no a significant increases in the frequency of micronucleated
polychromatic erythrocytes in mouse bone marrow cells at the
concentrations of sodium fluoride used in this study.  Sodium fluoride
was not mutagenic in NMRI mouse bone marrow cells.

There were no treatment-related effects on mortality (100% survival) or
MNPCEs in mice receiving administrations of phenol.

NEGATIVE

870.5395

Mammalian erythrocyte micronucleus test

Pati and Bhunya.  (1987). Genotoxic effect of an environmental
pollutant, sodium fluoride, in mammalian in vivo test system.  Carylogia
40:79-87.

Open Literature

Swiss mice administered intraperitoneal NaF (10, 20, or 40 mg/kg) 2
times over 24 hours

There were treatment-related increases in MNPCEs in animals administered
sodium fluoride, with 3-, 3.5-, and 5.15-fold increases over control at
the 10, 20, and 40 mg/kg dose levels, respectively.  These increases
were significantly greater than the control with the exception of the
low-dose (10 mg/kg).  The induction of MN in bone marrow cells increased
with sodium fluoride dose.  MN frequency was at its highest in PCEs and
least in immature white cells.  There was evidence of mutagenicity in
mouse bone marrow cells administered sodium fluoride within the confines
of this study.

POSITIVE

870.5395

Mammalian erythrocyte micronucleus test

Zeiger et al.  (1994). Cytogenetic Studies of Sodium Fluoride in Mice.
Mutagenesis 9:467-471.

Open Literature

Male B6C3F1 mice administered fluoride in the diet for 1 week and sodium
fluoride in drinking water for 7 days/week for 6 weeks.

100, 200, or 400 ppm

There were no significant increases in the frequency of micronucleated
polychromatic erythrocytes and normochromatic erythrocytes in B6C3F1
mice peripheral blood cells in the parameters of this study.  Sodium
fluoride was not mutagenic in B6C3F1 mice peripheral blood cells.

Three of sixteen mice from the 400 ppm group died during the
sixteen-week treatment period.  A decrease in body weight gain and water
consumption occurred at the 200 and 400 ppm group.  There were no other
treatment-related signs.

NEGATIVE

870.5500

Bacterial DNA damage or repair tests

Tong et al.  (1988). The Lack of Genotoxicity of Sodium Fluoride in a
Battery of Cellular Tests.  Cell Biology and Toxicology 4.2:173-186.

Open Literature

Male Fischer F-344 rat hepatocyte primary cultures (HPC) administered
sodium fluoride (2, 10, 20, 40, 80, or 160 ug/mL) for 18 hours

There was no significant increase in net nuclear grain counts at sodium
fluoride concentrations up to 160 go/mL.  Sodium fluoride did not elicit
DNA repair synthesis in the rat hepatocytes.

NEGATIVE

870.5550

Unscheduled DNA synthesis in mammalian cell culture

Tsutsui T, et al.  (1984). Sodium Fluoride-Induced Morphological and
Neoplastic Transformation, Chromosome Aberrations, Sister Chromatid
Exchanges, and Unscheduled DNA Synthesis in Cultured Syrian Hamster
Embryo Cells.  Cancer Research 44.3:938-941.

Open Literature

Syrian hamster embryo (SHE) cells exposed to sodium fluoride (10, 20, or
40 ug/mL) for 4, 8, 12, 24, or 33 hours.

A dose- and time-dependent increase in unscheduled DNA synthesis was
observed following administration of sodium fluoride in Syrian hamster
embryo cells.  Sodium fluoride was mutagenic in SHE cells within the
confines of this study.

Treatment-related effects on mortality were observed in SHE cells
administered sodium fluoride.  At sodium fluoride concentrations of 75,
100, and 125 go/mL, there were decreases in cell survival that were 10,
47, and 61%, respectively, less than control.

There was no evidence of UDS at any dose of sodium fluoride in the 4 or
8 hour exposure time period.  However, significant dose- and
time-dependent increases in UDS were observed in the SHE cells treated
with all three doses of sodium fluoride for 12 hours or greater.  After
12 hours of exposure, the UDS (as measured by [3H]dThd cpm/culture well
(x 10-2)) was at a level of 0.55 and 1.30 [3H]dThd cpm/culture well (x
10-2) for the 20 and 40 go/mL, respectively, dose groups.  Sodium
fluoride induced UDS levels of 0.75, 1.45, and 2.30 after 24 hours of
exposure and 0.45, 2.25, and 5.55 after 33 hours of exposure at
concentrations of 10, 20, and 40 go/mL, respectively.

POSITIVE

870.5550

Unscheduled DNA synthesis in mammalian cell culture

Tsutsui, T., N. Suzuki, et al.  (1984). Cytotoxicity, Chromosome
Aberrations and Unscheduled DNA Synthesis in Cultured Human Diploid
Fibroblasts Induced by Sodium Fluoride.  Mutation Research 139:193-198.

Open Literature

Cytotoxicity Test: cytotoxicity test sodium fluoride administered to
JHU-1 cells at concentrations of 50, 100, or 150 go/mL for 1, 2, 6, 12,
or 24 hours.

Mutagenicity Assay: Sodium fluoride was administered to JHU-1 cells at
concentrations of 50, 70, 100, 150, 200, 300, or 400 go/mL for 4, 8, 12,
16, or 24 hours

There was a significant dose-dependent increase in unscheduled DNA
synthesis following administration of sodium fluoride in cultured human
diploid fibroblasts. Sodium fluoride was mutagenic in JHU-1 cells within
the confines of this study.

Treatment-related cytotoxic effects were observed in JHU-1 cells
administered sodium fluoride.  Cell survival decreased as the sodium
fluoride concentration and duration of exposure increased.  At sodium
fluoride concentrations of 50, 100, and 150 go/mL, there were decreases
in cell survival that were 100, 98, and 90% after 1 hour; 100, 75, and
65% after 2hours; 70, 48, and 40% after 6 hours; 55, 22, and 15% after
12 hours; and 17, 7, and 1% after 24 hours of exposure, respectively.
Sodium fluoride was cytotoxic to JHU-1 cells and cell survival decreased
linearly with increasing dose or exposure time.

UDS was not induced by sodium fluoride treatment over the dose range of
50-5000 go/mL for 1 hour.  There were increases in the level of UDS
after 4 hours of exposure; however, none exceeded 7 [3H]TdR cpm/culture
well (x 10-2) and were not significantly different from untreated cells.
 No significant UDS was detected until the cells were treated for longer
than 4 hours.  The UDS levels were 9, 22, 29, and 41 for 8 hours and 3,
22, 44, and 52 for 12 hours of exposure at the sodium fluoride
concentrations of 100, 150, 200, and 300 go/mL, respectively.  The UDS
levels increased with dose after 16 hours of exposure, with 15, 39, and
47 [3H]TdR cpm/culture well (x 10-2) at 150, 200, and 300 go/mL sodium
fluoride, respectively.  The inducibility was markedly decreased in
cells treated for 24 hours; most likely a result of cytotoxicity.

POSITIVE

870.5900

In Vitro sister chromatid exchange assay

Khalil A, Da'Dara A.  (1994). The Genotoxic and Cytotoxic Activities of
Inorganic Fluoride in Cultured Rat Bone Marrow Cells.  Arch Environ
Contam Toxicol 26:60-63.

Open Literature

Bone marrow cells of Sprague-Dawley rats from tibia and femurs

0.1, 1, 10, 100, 1,000 and 10,000 uM for 12, 24, and 36 hours

Cell survival and cell division was significantly reduced at the
high-doses (1,000 and 10,000 uM).  However, Sodium fluoride did not
induce a SCE increase in bone marrow cells; there was no evidence of
mutagenicity.

NEGATIVE

870.5900

In Vitro sister chromatid exchange assay

Li Y, et al.  (1987). Genotoxic Effects of Fluoride Evaluated by
Sister-Chromatid Exchange.  Mutation Res 192:191-201.

Open Literature

Male CHO cells

0.05, 0.5, 1.0, 2.10, 4.20, 5.30, or 6.30 mM

The 5.30 and 6.30 mM dose levels of sodium fluoride were toxic and were
not evaluated.  There were no significant increases from controls in
SCEs in CHO cells exposed to 0.05 to 4.20 mM sodium fluoride.  Sodium
fluoride did not induce a SCE increase in CHO cells; there was no
evidence of mutagenicity.

NEGATIVE

870.5900

In Vitro sister chromatid exchange assay

Tong et al.  (1988). The Lack of Genotoxicity of Sodium Fluoride in a
Battery of Cellular Tests.  Cell Biology and Toxicology 4.2:173-186.

Open Literature

Human peripheral blood lymphocytes (HPBL) exposed to sodium fluoride (2,
10, 20, 40, 80, or 160 ug/mL) for 72 hours

The 160 go/mL dose of sodium fluoride was toxic as indicated by total
lack of cell entering the mitotic cycle.  SCEs of cells exposed to
sodium fluoride concentrations of 80 go/mL or lower did not differ
significantly from the control.  Sodium fluoride did not induce an
increase in SCEs in HPBL cells; there was no evidence of mutagenicity.

NEGATIVE

870.5900

In Vitro sister chromatid exchange assay

Tong et al.  (1988). The Lack of Genotoxicity of Sodium Fluoride in a
Battery of Cellular Tests.  Cell Biology and Toxicology 4.2:173-186.

Open Literature

Chinese hamster cells (CHO) exposed to sodium fluoride (2, 10, 20, 40,
80, or 160 ug/mL ) for 24-27 hours

The 80 and 160 go/mL dose levels of sodium fluoride were toxic.  There
were no significant increases from control in SCEs in CHO cells exposed
to 2-40 go/mL sodium fluoride.  Sodium fluoride did not induce a SCE
increase in CHO cells; there was no evidence of mutagenicity.

NEGATIVE

870.5900

In Vitro sister chromatid exchange assay

Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No.
7681-49-4) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies).
National Toxicology Program, Technical Report Series No. 393, NIH
Publication No. 91-2848, December 1990, Pgs. 1-477.

Acceptable

Guideline

There was evidence of a concentration related positive response in SCEs
induced over background in one of two studies performed, while the
second did not find any evidence of a positive response.

POSITIVE

870.5900

In Vitro sister chromatid exchange assay

Tsutsui T, et al.  (1984). Sodium Fluoride-Induced Morphological and
Neoplastic Transformation, Chromosome Aberrations, Sister Chromatid
Exchanges, and Unscheduled DNA Synthesis in Cultured Syrian Hamster
Embryo Cells.  Cancer Research 44.3:938-941

Open Literature

Syrian hamster embryo (SHE) cells  exposed to sodium fluoride (20, 40,
or 80 ug/mL) for 24 hours

A dose-dependent increase in sister chromatid exchanges was observed
following administration of sodium fluoride in Syrian hamster embryo
cells.  Sodium fluoride was mutagenic in SHE cells within the confines
of this study.

Treatment-related effects on mortality were observed in SHE cells
administered sodium fluoride.  At sodium fluoride concentrations of 75,
100, and 125 go/mL, there were decreases in cell survival that were 10,
47, and 61%, respectively, less than control.

There were significant dose-dependent increases in SCE frequency in the
sodium fluoride-treated SHE cells. After 24 hours of exposure, the
frequency of SCEs increased 1.4-, 1.6-, and 2.1-fold over control at the
20, 40, and 80 go/mL dose levels of sodium fluoride, respectively.

POSITIVE

870.5915

In Vivo sister chromatid exchange assay

Li Y, et al.  (1987). Genotoxic Effects of Fluoride Evaluated by
Sister-Chromatid Exchange.  Mutation Res 192:191-201.

Open Literature

Male Chinese hamsters

0.1, 1, 10, 60 or 130 mg/kg

Sodium fluoride did not induce a SCE increase in CHBM cells; there was
no evidence of mutagenicity.   Death occurred in three out of the eight
hamsters in the 130 mg/kg/day group. Although toxic effects were seen in
the high dose group, there were no treatment-related increases in SCE.

NEGATIVE

4.8	Neurotoxicity

Adequacy of database for Neurotoxicity: The database for neurotoxicity
is considered incomplete.

Special Developmental Neurotoxicity Study

A developmental neurotoxicity study (Mullenix, et al. 1995) was designed
to determine critical periods of CNS susceptibility at various stages of
development and to evaluate the effects of sodium fluoride (purity not
reported) on the developing brain of Sprague-Dawley rats.  Seven and
nine pregnant dams were administered 0.13 mg/kg sodium fluoride via
subcutaneous injection two to three times daily on gestation days (GD)
14-18 and 17-19, respectively, (a total of nine injections per group, at
least four hours apart).  At birth, litters were culled to ten pups/dam
(5 male and 5 female, where possible).  At 21 days of age, 19-27
pups/sex/dose were administered 75, 100, 125, or 175 ppm fluoride in the
drinking water for 6 or 20 weeks.  Behavior tests were performed on the
rats at 9-, 14-, or 19-weeks of age and plasma fluoride levels were
measured thereafter.  The behavior was tested in a computer recognition
system that classified acts in a novel environment and quantified act
initiations, total times, and time structures.  In an additional, adult
exposure study, 21-24 adult (12-week old) rats/sex/dose were
administered 0 or 100 ppm sodium fluoride in deionized water for 5 to 6
weeks.  Body weights were recorded weekly for all test groups.

No maternal or offspring toxicity was indicated by reduced body weight
in dams during prenatal treatment or in their pups soon after birth.
However, prenatal exposure to sodium fluoride altered the behavioral
outcome in male offspring when exposure occurred on GD 17-19 and
consisted of time structure changes in eleven behaviors and behavioral
sequences.  The behavioral differences did not coincide with the plasma
fluoride levels.

Body-weight was significantly reduced from the control group in 3-week
old rats administered 125 ppm fluoride.  Concentrations below 125 ppm
did not affect body weight gain during 6-week exposures.  Plasma
fluoride levels were significantly increased in all test groups compared
to control groups.  The same direction of behavioral change (initiation
and total time) occurred in treated animals when compared to controls.
This change was independent and unrelated to sex of the animal, exposure
time (6 or 16 weeks), or dose level (100 or 125 ppm).  The act of
standing and the related attention cluster tended to increase in total
time, while the other acts consistently decreased in initiations and
total times.

The adult exposure to 100 ppm sodium fluoride had a significant effect
on female behavior consistent with the behavioral change in the 3-week
old rats.  Similar behavioral time structure effects occurred when adult
and weanling exposed rats approached 5 months of age.

The effect on behavior varied with the timing of exposure during CNS
development.  There were differences between behavioral changes in
weanling and adult exposure when compared to prenatal exposures.
Prenatally induced behavioral effects were unaccompanied by changes in
body weight or elevated plasma fluoride levels.  The behavioral effects
induced by weanling and adult exposures were accompanied often by weight
reduction and always by elevated plasma fluoride levels.

Rats were exposed to sodium fluoride at concentrations ranging from
75-125 ppm for 6 or 20 weeks.  Plasma fluoride levels reached
0.059-0.640 ppm and after 6 weeks of consuming 75 and 100 ppm of sodium
fluoride animals exhibited greater plasma fluoride levels than animals
treated with 125 ppm.  The researchers suggest that there was a taste
aversion that limited the water consumption at the 125 ppm level;
prolonging the period needed to attain plasma levels that were achieved
in 6 weeks by the two lower exposure levels. The levels of fluoride in
plasma, best predicted effects on behavior.

	4.9	Metabolism and Pharmacokinetics

The database for metabolism consists of one study from the open
literature. In a study by Hall et al. 1977, 6 adult male New Zealand
rabbits were administered sodium fluoride in the diet (15 ppm), water (1
ppm), and  in a single oral dose injected (0.5 mg/kg ) directly into
stomach through nasal catheter. Urine excretion following oral
administration of sodium fluoride was 5 and 13% for 60 and 600 minutes,
respectively.  Under steady state conditions approximately 15% of
fluoride ingested in food and water was absorbed by the animals. 15% was
excreted in urine and 85% of ingested fluoride was removed via fecal
excretion.

5.0	TOXICITY ENDPOINT SELECTION

5.1	See Section 7.1, Summary of Toxicological Doses and Endpoint
Selection, Table 2.

6.0	FQPA CONSIDERATIONS

	FQPA considerations are not applicable to sodium fluoride.  There are
no food use tolerances for this chemical and indirect food contact is
not expected from the current uses of this chemical.

7.0	SUMMARY OF TOXICOLOGICAL DOSES AND ENDPOINTS FOR Sodium Fluoride FOR
USE IN HUMAN RISK ASSESSMENT

7.1	Summary Table of Toxicological Dose and Endpoint Selection (Table 4)

Table 4. Sodium Fluroide for Use in Human Risk Assessment

Exposure

Scenario	Dose (mg/kg/day) used in risk assessment

UF	Special FQPA SF and Level of Concern for Risk Assessment	Study and
Toxicological Effects

Dietary Risk Assessments

Acute Dietary

(general population and females 13-49)

	No appropriate endpoints were identified that represent a single dose
effect.

Therefore, this risk assessment is not required.

Chronic Dietary

	No appropriate endpoints were identified that represent a single dose
effect.

Therefore, this risk assessment is not required.

Non-Dietary Risk Assessments

Short -Term Dermal

(1 - 30 Days)

LOAEL = 20 mg/kg/day

Target MOE=300 (10x inter-species extrapolation, 10x intra-species
variation, 3x for use of LOAEL)

Oral Subchronic Toxicity – Rat (Sodium Fluoride)

LOAEL = 20 mg/kg/day, based on significant reductions in body weight
gain and suppressed spontaneous motor activity.

Intermediate -Term Dermal

(30 Days- 6 months)

NOAEL = 1.5 mg/kg/day

Target MOE=100 (10x inter-species extrapolation, 10x intra-species
variation)

6-month NTP oral toxicity study-mouse

LOAEL = 7.5 mg/kg/day based on  histopathology observed in bone with
degeneration in tibias and femurs of animals

Long-Term Dermal (> 6 months)

LOAEL = 1.3 mg/kg/day

TARGET MOE = 100 (10x inter-species extrapolation, 10x intra-species
variation)

2-year NTP chronic toxicity/carcinogenicity study in rats

LOAEL = 1.3 mg/kg/day, based on   dentine dysplasia in males and
females, and ameloblast degeneration in males

Short-term Inhalation

(1-30 days)

LOAEL = 20 mg/kg/day

Target MOE=300 (10x inter-species extrapolation, 10x intra-species
variation, 3x for use of LOAEL)

Note:  10x route extrapolation for confirmatory inhalation study.

Oral Subchronic Toxicity – Rat (Sodium Fluoride)

LOAEL = 20 mg/kg/day, based on significant reductions in body weight
gain and suppressed spontaneous motor activity.

Intermediate-term Inhalation

NOAEL = 1.5 mg/kg/day

Target MOE=100 (10x inter-species extrapolation, 10x intra-species
variation)

Note:  10x route extrapolation for confirmatory inhalation study.

6-month NTP oral toxicity study-mouse

LOAEL = 7.5 mg/kg/day based on  histopathology observed in bone with
degeneration in tibias and femurs of animals

Long-term Inhalation

LOAEL = 1.3 mg/kg/day

TARGET MOE =300 (10x inter-species extrapolation, 10x intra-species
variation, 3x for use of LOAEL)

Note:  10x route extrapolation for confirmatory inhalation study.

2-year NTP chronic toxicity/carcinogenicity study in rats

LOAEL = 1.3 mg/kg/day, based on   dentine dysplasia in males and
females, and ameloblast degeneration in males

Cancer

Sodium fluoride has been classified as a “Group D” (not classifiable
as to  carcinogenicity).  This conclusion is consistent with the recent
report by the National Academy of Sciences which concluded that ‘the
evidence on the potential of fluoride to initiate or promote cancers,
particularly of the bone, is tentative and mixed.’

8.0	TOXICITY PROFILE TABLES

8.1	Acute Toxicity Profile Table - (See Section 4.1, Acute Toxicity,
Table 2).

8.2	Subchronic, Chronic and Other Toxicity Profiles Table (Tables 5).

Table 5.  Subchronic, Chronic, and other Toxicity Profiles for Sodium
Fluoride

Guideline Number/

Study Type/

Test Substance (% a.i.)	MRID Number (Year)/

Citation/

Classification/ Doses	Results

870.3100 (§ 82-1)

90-Day oral toxicity in rodents

Purity: 99%

Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No.
7681-49-4) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies).
National Toxicology Program, Technical Report Series No. 393, NIH
Publication No. 91-2848, December 1990, Pgs. 1-477.

Acceptable

Guideline

10 F344/N rats/sex/dose administered sodium fluoride at doses of 0, 10,
30, 100, or 300 ppm for 6 months

NOAEL = 30 ppm

LOAEL = 100 ppm, based on the presence of hyperplasia in the glandular
stomach

There were no treatment-related effects on mortality.
Treatment-related effects were noted in 300-ppm treated rats including
clinical observations of dental fluorosis (chalk white appearance of
teeth, overgrowth of upper incisors, occlusal surface of the lower
incisor worn to the gum, unusual wear pattern of incisors) and rough
hair coat, decreased food and water consumption, and

reductions in mean body weight and body weight change.

Measurement of fluoride content revealed a dose-dependent increase in
fluoride concentration in bone and urine, while elevated levels of
fluoride in plasma were only observed in 300-ppm treated rats.

Treatment-related macroscopic effects were observed in the 300-ppm
treated rats.  A majority of the high-dose males exhibited thickened
stomachs.  Focal or multifocal punctate hemorrhages, perforated ulcer of
the glandular stomach, and multiple, small, nonperforated ulcers were
also observed in several 300-ppm males and/or females.  These
macroscopic changes were supported by microscopic evidence of pathology.
 Dose-dependent increases in incidence and severity of hyperplasia and
necrosis of the glandular stomach was observed in rats treated with
≥100 ppm.  Diffuse hyperplasia of the mucosal epithelium of the
glandular stomach was noted in 5/10 males and 2/10 females treated with
100 ppm, and in 10/10 males and 9/10 females treated with 300 ppm.  This
effect was accompanied by minimal individual cell necrosis (apoptosis)
in the pyloric region in 300-ppm treated

rats, and by evidence of acute inflammation in several males at 300 ppm.
 Focal basal cell hyperplasia of the stratifiedsquamous epithelium was
located adjacent to the limiting ridge in nearly all 300-ppm treated
rats.   Microscopic evidence of the effects of the test article on the
incisors included focal or multifocal degeneration of the enamel organ
in 300-ppm males (5/10), localized in the maturation zone near the
apical end of the tooth.

870.3100 (§ 82-1)

90-Day oral toxicity in rodents

Purity: 99%

Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No.
7681-49-4) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies).
National Toxicology Program, Technical Report Series No. 393, NIH
Publication No. 91-2848, December 1990, Pgs. 1-477.

Acceptable

Guideline

8-12 B6C3F1 mice/sex/dose administered deionized water at doses of 0,
10, 50, 100, 200, 300 or 600 ppm for 6 months

NOAEL = 50 ppm in female mice, and could not be determined in males
based on the observation of increased osteoid of the tibia in 5/10 males
dosed at 50 ppm.

LOAEL = 50 ppm in male mice and 100 ppm in female mice based on
histopathology observed in bone.

Clinical signs in surviving animals included chalky white incisors
(≥100 ppm) and chipped teeth (≥300 ppm).  The effects on the
incisors correlated with microscopic findings, which included focal or
multifocal degeneration of the enamel organ. Mean body weight was
significantly decreased in 600-ppm treated males and in 200- and 300-ppm
treated females.  Mean body weight gain was significantly decreased in
≥200-ppm males and in 200- and 300-ppm females.  These parameters were
also decreased in the 600-ppm females, but did not reach statistical
significance, likely due to the reduced number of animals in this group
as a result of premature deaths.  Food consumption in 600-ppm males was
approximately 77% of controls.  Food consumption in the other treatment
groups, and water consumption in all treatment groups were within 20% of
control values.

There was a dose-dependent increase in fluoride content in bone and
urine.  Due to the pooling of plasma samples for sufficient volume for
analysis, meaningful statistical analyses in this fluid could not be
performed.  The data indicate that there was generally a dose-dependent
increase in fluoride concentration in the plasma.

t be determined if these effects were due to treatment with sodium
fluoride or secondary to the mice dying from other unrelated causes.
Bone lesions, in the form of increased osteoid, were observed in the
femurs of treated mice at ≥100 ppm, and in the tibias at 50 ppm.  This
histopathology is indicative of altered rates of bone deposition and
remodeling as a result of treatment.  Further, treatment with sodium
fluoride induced degeneration of the incisors as evidenced by
degeneration (dysplasia) in ≥300-ppm treated mice.

Non-Guideline

Oral Subchronic (Rodent)

Purity  not reported

Bohatyrewicz, A. (1999). Effects of Fluoride on Mechanical Properties of
Femoral Bone in Growing Rats. Fluoride 32:47-54.

Open Literature

10 female 6-week-old Wistar rats/group administered NaF at levels of 0,
8, 30, and 60 mg of fluoride/L in drinking water for 6 weeks.

Femoral bones from each rat were assayed for bending strength

High fluoride intake (30 and 60 mg/L) significantly decrease bone
quality of the femoral shaft and neck of young rats.

NaF administered in lower concentrations (8 mg/L) significantly
increases the strength of the femoral neck from the control.

Non-Guideline

Oral Subchronic (Rodent)

Purity  not reported

Paul, V. et al. (1998). Effects of Sodium Fluoride on Locomotor Behavior
and a Few Biochemical Parameters in rats. Environmental Toxicol and
Pharmacol 6:187-191.

Open literature

10 female Wistar rats/dose administered Sodium Fluoride via oral
intubation at dose levels of 20 or 40 mg/kg/day for 60 days.

Subchronic Toxicity:

NOAEL < 20 mg/kg/day (lowest dose tested)

LOAEL ≤ 20 mg/kg/day based on significant reductions in body weight
gain and suppressed spontaneous motor activity.

There were significant dose-dependant decreases of 17 and 30% for food
intake and 14 and 37% for body weight gain at the 20 and 40 mg/kg/day
dose levels, respectively. Total protein concentrations in serum
(low-dose, 13%; high-dose, 38%), liver (low-dose, 22%; high-dose, 42%),
and skeletal muscle (low-dose, 15%; high-dose, 31%) were also
significantly reduced in a dose-related manner in animals treated with
sodium fluoride.

Spontaneous motor activity was suppressed in a dose-dependant manner
with decreases of 15 and 29% at the 20 and 40 mg/kg/day dose levels,
respectively. However

, motor co-ordination was

not altered in treated animals. Total blood cholinesterase activity was
reduced at the low- and high-dose, although there was no evidence of
change in acetyl-cholinesterase activity of the cerebral cortex, brain
stem, or cerebellum.

Food intake reductions may account for the decrease in protein
concentration of a direct deleterious action of fluoride on protein
metabolism can also play a role in depleting protein in sensitive
tissues. Thus, a decreased food intake together with a depletion of
protein in soft tissues accounted for an inhibition of body growth in
sodium fluoride-treated animals. Sodium fluoride deprived skeletal
muscle of total protein and suppressed blood cholinesterase activity;
although, these effects are unlikely to have a deteriorating action on
neuromuscular function. However, similar sodium fluoride doses can
produce neurobehavioral deficit resulting in an inhibition of
spontaneously occurring locomotor activity.

Non-Guideline

Oral Subchronic (Rodent)

Purity  not reported

Pillai et al. (1988). Effect of Subacute Dosage of Fluoride on Male
Mice. Toxicology Letters 44:21-29.

Open Literature

5 Male Swiss albino mice administered 5.2 mg F/kg/day for 35 days.

NOAEL ≤ 5.2 mg/kg/day (lowest dose tested)

LOAEL ≤ 5.2 mg/kg/day, based on significant decreases in body weight
gain, and food and water consumption.

There were significant changes in hematological analyses with decreases
in red blood cells, lymphocytes, hemoglobin, albumin, total protein,
cholesterol, glucose, and alkaline phosphatase. Statistically
significant increases were observed in white blood cells, monocytes,
basophils, and eosinophils. Food and water consumption was significantly
decreased in treated animals compared to controls. There were
significant treatment-related decreases from controls in body weight
gain of sodium fluoride-treated mice after day 19 of the treatment
period. A significant relationship between food and water consumption
and the body weight was observed in the controls, but not in the treated
animals.

Significant increases in fluoride content were measured in the kidneys,
stomach, brain, liver, and intestines of the sodium-fluoride-treated
animals when compared to the controls. The increases were 3.5- and 1.5
fold greater than control in the kidneys and stomach, respectively,
while the brain, intestines, and liver exhibited 2-fold increases over
control. There was no evidence of sperm abnormalities following
treatment with sodium fluoride.

Non-Guideline

Oral Subchronic (Rodent)

Purity: 99%

Chinoy and Patel. (2001). Effects of Sodium Fluoride and Aluminum
Chloride on Ovary and Uterus of Mice and Their Reversal by Some
Antidotes. Fluoride 1:9-20.

Open Literature

20 Adult female albino mice administered 10 mg/kg/day NaF for 30 days.

Significant decline of ovarian protein and 3-beta- and
17-beta-hydroxysteroid dehydrogenase activities, which could be related
to increased cholesterol levels in the ovary suggesting altered
steroidogenesis.

Special Study

Subchronic (subcutaneous injection) Toxicity

Purity  not reported	Shahshi et al. (1994). Effect of Long-term