UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

                                        WASHINGTON, D.C.  20460

OFFICE OF

PREVENTION, PESTICIDES

          AND TOXIC SUBSTANCES

MEMORANDUM

DATE:		May 14, 2008

SUBJECT:	Revised   SEQ CHAPTER \h \r 1
5-Chloro-2-(2,4-dichlorophenoxy)phenol (Triclosan): Toxicology Chapter
for the Reregistration Eligibility Decision (RED) Document.  Case No
2340.

 PC Code: 054901.  DP Barcode: 373536

FROM:	Tim McMahon, Ph.D., Senior Toxicologist

		Antimicrobials Division (7510P)

TO:		Diane Isbell, Team Leader

		Heather Garvie, Chemical Review Manager

		Regulatory Management Branch II

		Antimicrobials Division (7510P)

Attached is the Toxicology Disciplinary chapter for Triclosan for the
purpose of issuing a Reregistration Eligibility (RED) Decision.    SEQ
CHAPTER \h \r 1 TABLE OF CONTENTS

 TOC \f

1.0	HAZARD CHARACTERIZATION	3

2.0	TOXICOLOGY DATA	8

3.0	DATA GAP(S)	9

4.0	HAZARD ASSESSMENT	9

4.1	Acute Toxicity	9

4.2	Subchronic
Toxicity…………………..………………………………….1
0

4.3	Developmental
Toxicity…………………………………………………16

4.4	Reproductive Toxicity	18

4.5	Chronic Toxicity	19

4.6	Carcinogenicity	20

4.7	Mutagenicity	24

4.8	Neurotoxicity	28

4.9	Metabolism	28

4.10	Special Studies (optional section)	36

5.0	TOXICOLOGY ENDPOINT SELECTION	43

5.1	Summary of Endpoint Selection 	43

5.2	Dermal Absorption	44

6.0	FQPA CONSIDERATIONS	45

6.1	Reproductive Toxicity Conclusions	45

6.2	Pre- and Post-natal Toxicity	46

6.3	Recommendation for Developmental Neurotoxicity	47

7.0	Toxicology Endpoint Selection Table	47

8.0	Toxicity Profile Tables	49

9.0	References	56

                                           3 1.0  HAZARD
CHARACTERIZATION

Triclosan (2,4,4’ –trichloro-2’-hydroxydiphenyl ether) is a
chlorinated aromatic compound that has functional groups representative
of both phenols and ethers.  It is used as a synthetic broad-spectrum
antimicrobial agent in the form of a white to off-white powder.  It is
practically insoluble in water but is soluble in most organic solvents.
Uses of triclosan include common everyday products, such as soaps,
deodorants, toothpastes, laundry detergents, fabric softeners, facial
tissues, and adult diapers.  Triclosan is also impregnated in products
such as kitchen utensils, toys, bedding, socks, and trash bags.

Acute toxicity studies in experimental animals with technical grade
triclosan show that by the oral and dermal routes triclosan is of low
acute toxicity (Toxicity Category IV;   MRIDs 43206501 and 100178).  By
the inhalation route of exposure triclosan was assigned Toxicity
Category II for acute exposures and is thus of higher acute toxicity by
inhalation exposure than by oral or dermal exposures (MRID 42306902 and
43310501).  Triclosan produces moderate irritation to the eyes (MRID
43429) and skin (MRID 42306903) with a Toxicity Category III assigned
for these acute exposures.  Triclosan was not a dermal sensitizer in
guinea pigs using the Buehler method (MRID 43206502).

Liver toxicity in mice and rats was noted after 90 day repeated oral
exposure to triclosan, characterized by fatty metamorphosis and
cytomegaly, hypertrophic hepatocytes, vacuolization, inflammation, and
pigmentation of Kupffer cells  (MRID 43022605, 99.7% a.i.; MRID 133545,
% a.i. not stated).  A LOAEL of 25 mg/kg/day was obtained for the mouse
study, and a NOAEL of 1000 ppm was obtained for the 90-day rat study.
Similar effects on the liver including liver cell necrosis and an
increase in the liver-body weight ratio were observed in a 28-day oral
toxicity study in mice (MRID 44389707, > 99% a.i.).  Hematological
effects including significant decreases in hemoglobin and increases in
thrombocytes, clinical chemistry alterations, and elevated serum enzyme
activities were also evident in this study.  The 28-day oral mouse
systemic NOAEL was considered to be 6.48 mg/kg/day in males, and 8.25
mg/kg/day in females.  In a 90-day oral toxicity study in dogs (MRID
96102, % a.i. not stated), histopathologic examination of tissues from
dogs that were sacrificed or died showed evidence of hepatotoxicity
resulting in obstructive jaundice with the NOAEL determined to be 12.5
mg/kg/day.

In a 90-day dermal toxicity study in rats (MRID 43328001, 99.7% a.i.),
dose-related dermal irritation was present at all dose levels (10, 40,
and 80 mg/kg/day) but was also reversible after a recovery period of 20
days.  In addition, an increase in the incidence of occult blood in the
urine was found in males and females at the 80 mg/kg/day dose level.
Under the conditions of this study, the systemic LOAEL was 80 mg/kg/day;
the systemic NOAEL was 40 mg/kg/day.

In two 14-day repeated dose dermal toxicity range-finding studies in
mice and rats (MRID 44389708, 99.3% a.i.; MRID 44389710, 99.3% a.i.),
signs of dermal toxicity were observed at application sites.  A LOAEL of
6.0 mg/animal/day was obtained in the rat study.  The NOAEL was 3.0
mg/animal/day.  In the mouse study, systemic responses were noted
including a dose-dependent increase in plasma levels of the test
substance and treatment-related increases in absolute and relative liver
to body and brain weights, correlated with centrilobular hepatocellular
hypertrophy.  Body weight gain and food consumption were affected as
well.  The LOAEL for this study was 1.5 mg/animal/day, based on
treatment-related dermal irritation and on increased liver weights in
this treatment group.  The NOAEL was 0.6 mg/animal/day.

In a 21-day inhalation toxicity study (MRID 0087996), 9 rats per dose
were exposed to concentrations of 0, 50, 227/115, or 1300/301 mg/m3  for
2 hours per day, 5 days per week. The 227 and 1300 mg/ m3 doses were
reduced to 115 and 301 after the first day, due to severe clinical
signs.  Twelve high-dose animals (5 males and 7 females) died during the
course of the study.  Toxicity was observed at all dose levels and
included  dyspnea, nasal discharge, muscle spasms, pallor, and diarrhea,
decreased body weight, decreased body weight gain, decreased food
consumption, statistically-significant increased total leukocyte count,
statistically-significant increased percentage of neutrophils and
decreased lymphocytes, statistically-significant increased serum
glutamic-pyruvic transaminase (GPT) activity, statistically-significant
increased alkaline phosphatase (AP), statistically-significant decreased
serum proteins (males), and increased incidence of respiratory
inflammation.  Additional statistical analyses also showed a
statistically-significant decrease in thrombocytes. Acute purulent
inflammation with focal ulceration of the mucous membrane in the nasal
cavity and in the trachea were also observed at the high concentration.
The LOAEL is 50 mg/m3 (3.21 mg/kg/day) for males based on changes in
thrombocytes, total blood proteins, and alkaline phosphatase; the LOAEL
for females is 115 mg/m3 ( 9.91 mg/kg/day).  A NOAEL could not be
established for males; the NOAEL for females is 50 mg/m3 (4.51
mg/kg/day).

 In developmental toxicity studies in rats and rabbits (MRID
43817502/43817503, 99.8% a.i., MRID 43820401/43022607, 99.8% a.i.),
maternal toxicity consisted of transient diarrhea, retarded body weight
gain during the period of treatment, reduced food consumption, and
increased water consumption.  The maternal LOAEL was 300 mg/kg/day in
the rat study; maternal NOAEL was 100 mg/kg/day.  In the rabbit study,
the maternal toxicity LOAEL was 150 mg/kg/day, and the maternal NOAEL
was 50 mg/kg/day.  No evidence of pre- or postnatal developmental
toxicity was identified at any dose level in either study.
Developmental LOAELs were therefore not identified.  The developmental
NOAEL in the rat study was ≥ 300 mg/kg/day, and the developmental
toxicity NOAEL in the rabbit study was ≥150 mg/kg/day.

In a two-generation reproduction study in rats (MRID 40623701, ≥ 99%
a.i.), reproductive and systemic effects were noted at the high dose
only (150 mg/kg/day).  Body weights were significantly lower in the
high-dose F1 pups on Days 14 through 21 of lactation and throughout the
growth phase.  The viability index was decreased in the high-dose group
in both generations, and the weaning index was slightly lower in the
high-dose group compared to control in the F2 generation.  The NOAEL for
both reproductive and systemic effects was 1000 ppm (50 mg/kg); the
LOAEL was 3000 ppm (150 mg/kg).

In a chronic toxicity study in baboons (MRID 251773, % a.i. not stated),
vomiting, failure to eat, and diarrhea were noted in test animals orally
administered triclosan, which occurred 4-6 hours after dosing or during
the night.  At necropsy, an effect on the lining of the stomach was
observed at the high dose. The systemic NOAEL was determined to be 30
mg/kg/day, and the systemic LOAEL was 100 mg/kg/day, based on clinical
signs of toxicity.

In a chronic toxicity/oncogenicity feeding study in rats (MRID 42027906,
99% a.i.), rats administered triclosan in the diet had decreases in
erythrocyte count, hemoglobin concentration, and hematocrit.  Serum
alanine and aspartate aminotransferase activities were increased in
males at 168.0 mg/kg/day, and blood urea nitrogen was increased in
females at 217.4 mg/kg/day. Hepatocellular hypertrophy was observed in
males at all dose levels. The predominant residue of triclosan observed
in blood and kidney was the sulfate conjugate of triclosan, while
unconjugated triclosan was predominant in the liver.  No carcinogenic
potential was demonstrated for triclosan in this study. The systemic
NOAEL was determined to be 52.4 mg/kg/day, based on the increase in
non-neoplastic liver pathology observed in male rats at the 168.0
mg/kg/day dose.

No carcinogenic potential was demonstrated for triclosan in a chronic
toxicity/carcinogenicity study in hamsters (MRID 44874001/44751101,
99.5% a.i.) as well.  Beginning at 80 weeks into the study, high-dose
males had an increase in mortality which correlated with deterioration
in their clinical condition.  Plasma urea nitrogen and urine volume were
significantly increased with corresponding decreases in specific gravity
and protein concentration.  Microscopically, a significantly increased
incidence of nephropathy was observed and was considered the main factor
contributing to death in animals that died before study termination.  In
males tested at the high dose, a significantly increased incidence of
absent spermatozoa and abnormal spermatogenic cells was observed.
Increased incidence of partial depletion of one or more generations of
germ cells within the testis was also observed.  Also noted were lesions
in the stomach, focal atypical hyperplasia of the fundic region, and
distended gastric glands with or without debris.  The LOAEL was 250
mg/kg/day for male and female hamsters based on decreased body weight
gains, increased mortality (males), nephropathy, and histopathologic
findings in the stomach and testes.  The corresponding NOAEL was 75
mg/kg/day.

Triclosan was positive for carcinogenicity in the liver of mice in an
oral carcinogenicity bioassay [See,  1996].  A dose-related increase in
the activity of alanine aminotransferse and alkaline phosphatase, and
significant decreases in albumin, total protein, and serum cholesterol
suggest that triclosan can interfere with liver function.
Treatment-related hematological effects included increased reticulocyte
count and platelet count.  Increases in mean liver weight, in the
severity of hepatocellular hypertrophy, and in the incidence of nodules,
discoloration, hepatocellular vacuolation/vesiculation, hepatic
inflammation, necrosis, and microgranulomas was observed.  After 18
months of exposure, there was a statistically significant increase in
the incidence of hepatocellular adenoma and carcinoma.  A systemic NOAEL
of 10 mg/kg/day was established from the data in this study, based on
increased incidence of liver neoplasms in male and female mice at 30
mg/kg/day.

In two independently performed microbial preincubation assays (MRID
43533301, ≥99% a.i.) and a microbial mutagenicity assay (MRID
44389705, 100.5% a.i.), there was no indication of a mutagenic response
in any strain at any dose compared to the vehicle controls.  Likewise,
in a mammalian cell gene mutation assay at the thymidine kinase locus
(MRID 44389704, > 99% a.i.), triclosan was negative for inducing forward
mutations both with and without metabolic activation.  Negative results
for mutagenicity were also obtained in a chromosome aberration assay
[MRID 47276601, >99% a.i.], an in vivo bone marrow cytogenetic assay
(MRID 43740802, 99%-100%), and an in vitro DNA synthesis assay [MRID
47276602, 100.5% a.i.)].

However, in an in vitro cytogenetic assay (MRID 43740801, 99-100% a.i.),
there was a dose-related increase in the yield of cells with abnormal
chromosome morphology.  In the presence of S9 activation, nonsignificant
but concentration dependent increases in cells bearing exchange figures
were also seen.

In a metabolism study in hamsters (MRID 45307501/45307502, 99% a.i.),
urine was the major route of elimination for triclosan radioactivity.
Peak plasma and blood concentrations of triclosan-derived radioactivity
occurred at one hour post-dose.  Area Under the Curve (AUC) measurements
indicated that saturation may have been achieved at the high dose, as
AUC was not proportional to dose.  The major urinary metabolite detected
after oral administration was the glucuronide conjugate of triclosan
(U7).  The major fecal metabolite was parent triclosan.  The plasma,
kidney, and liver eliminated triclosan equivalent rapidly.  Tissue
metabolite analysis showed that the glucuronide and sulfate conjugates
of triclosan were the major metabolites detected.  Four non-parent
conjugates (M5, M6, M8, and M9) were also identified in tissues.  All
conjugates were acid labile and resulted in the parent compound, M2, or
M3.  After 1-2 hours (Cmax), males dosed singly or repeatedly, parent
glucuronide and sulfate were found in the plasma without detection of
the free parent compound.  The kidneys at Cmax had parent glucuronide
and free parent with little sulfate conjugate; the liver at Cmax had
free parent and sulfate conjugate with little glucuronide levels.

In an absorption, distribution, metabolism, and elimination study in
mice (MRID 45307503, ~99% a.i.), triclosan was eliminated primarily
through the feces, via biliary excretion.  Bioretention studies indicate
that values from Cmax to 1/8Cmax in the liver were higher than those in
plasma following repeated administration at both dose levels, indicating
that the liver is the target organ.  Primary excreted compounds in the
urine following single oral exposures included the unmetabolized parent
compound and two parent conjugates; fecal excretion was primarily that
of the free parent compound.  Four conjugated metabolites (M5, M6, M8,
and M9) were detected in kidney, plasma, and liver extracts in minor
amounts (< 5.3%) as well.  Parent and parent conjugates were rapidly
eliminated and/or metabolized with half-lives ranging from 1-13 hours in
the plasma, liver, and kidney.  Cmax values occurred at 4-11 hours.
Mice with enlarged livers exhibited parent and parent sulfate half-lives
of 13 and 14 hours, respectively.  Non-parent conjugates M5, M6, M8, and
M9 showed similar half-lives as parent and parent compound (<4-13
hours); however, Cmax values were greater, occurring at 12-32 hours.

 A value of 50% dermal absorption for triclosan was selected on the
basis of a dermal absorption study conducted in rabbits (MRID 34335).
Since that time, additional dermal absorption data on triclosan  have
been submitted and reviewed.  In vitro dermal absorption studies using
human skin preparations and various formulations containing triclosan
(MRIDs 47261408 through 47261411) showed dermal absorption values for
triclosan ranging from 11-20% in these formulations.  A paper published
in 2000 by Moss et al.  (Food and Chemical Toxicology, Volume 38, pages
361-370) examined dermal absorption of triclosan both in vivo and in
vitro using rats as well as an in vitro human skin study. These data
supported the conclusion of dermal absorption of 21-23% in the rat
studies, and showed in vitro dermal absorption through human skin in
vitro of 6.3%. Taken together, the available data  on dermal absorption
suggest a lower value, around 20% for rat skin and possibly lower for
human skin.  Additional verification is needed.

In a liver biochemical induction study in mice (MRID 44389702, % a.i.
not stated), administration of triclosan to the mouse resulted in
significant hepatic effects. The biochemical alterations observed appear
to support the conclusion of a barbituate-type induction with peroxisome
proliferation effects. Induction of certain liver enzyme activities as
measured in this study appear to occur at the lowest dose tested in male
mice, including significant increases in microsomal protein, lauric acid
hydroxylation, and EROD and PROD activities.  Increases in lauric acid
hydroxylation, and an increase in EROD and PROD activities were also
noted in a non-guideline feeding study in rats (MRID 44389703, % a.i.
not stated).  However, animals allowed to recover for 28 days following
the 14-day administration of test chemical in this study showed no
significant induction or inhibition of enzyme activities.  Absolute and
relative liver weights were increased at the end of the study.
Cytochrome P-450 content was approximately doubled in the high dose
group, while activity of glutathione-S-transferase was increased by 65%.
 In a third liver biochemical induction study (MRID 44389706, 99.5%
a.i.), a systemic NOAEL of 700 ppm was established in hamsters, with a
systemic LOAEL of 5000 ppm, based on induction of total cytochrome
P-450, EROD, and PROD, and induction of Mab clo4 immunoreactive protein
(CYP4A peroxisome proliferator inducible P-450) in males.  The data
suggest that triclosan acts as a peroxisome proliferator.

Two cell proliferation studies were conducted in mice (MRID 44389701, %
a.i. not stated; Eldridge, 1995, % a.i. not stated).  In the first
study, hepatocellular hypertrophy was the most consistent and prominent
observation.  At the higher dose levels, necrosis of hepatocytes was
observed.  Hepatocytes were also swollen and bile stasis was evident.
According to the report, cell proliferation was significantly increased
over control in male mouse liver at 200 mg/kg/day and higher, and the
increase was sustained from 45 to 90 days.  A reviewer of this study
agreed with the sustained increase, but noted that it appears that cell
proliferation (as judged by labeling index and fold increase over
control) is also increased significantly at the 75 mg/kg/day dose level
for male mice. This result is consistent with the apparent differences
in sensitivity to the hepatic effects of triclosan between male and
female mice.  According to the report, the distribution of
hepatocellular labeling was panlobular in both sexes.  The 25 mg/kg/day
dose level was identified as the NOAEL for male mice, while the 75
mg/kg/day dose level was considered the NOAEL for female mice by the
authors.  The results of this study support a mode of action consistent
with cellular regeneration as a result of hepatocellular cytotoxicity.
This conclusion was also reached in the second cell proliferation study
based on an increase in the labeling index at 200 mg/kg/day, in
conjunction with other data which show toxicity to the liver of rats and
mice.  In the report, it was noted that the mode by which a chemical
induces cell proliferation is an important consideration.  In the case
of triclosan, the evidence suggests a hepatotoxic effect followed by
regenerative cell turnover, in contrast to agents which act as direct
mitogens.  For chemicals producing increased cell turnover through
cytolethality, a threshold can be inferred below which these effects
would not occur.

  TOXICOLOGY DATA

The Toxicology data available for triclosan are shown in Table 1.

Test

Technical

Required

Satisfied

870.1100	Acute Oral Toxicity MRID 43206901

870.1200	Acute Dermal Toxicity MRID 100178

870.1300	Acute Inhalation Toxicity MRID 42306902/43310501

870.2400	Primary Eye Irritation MRID 94045

870.2500	Primary Dermal Irritation MRID 42306903

870.2600	Dermal Sensitization MRID 43206502

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

870.3100	Oral Subchronic (Rodent) MRID  133545, 43022605, 44389707

870.3150	Oral Subchronic (Non-Rodent) MRID 96102

 870.3250	90-Day Dermal MRID 43328001

870.3465	21-Day Inhalation MRID 0087996

Y

Y

 Y

Y

Y

Y

Y

N

870.3700	Developmental Toxicity (rodent) MRID 43817502 and  43817503

870.3700	Developmental Toxicity( non-rodent) MRID 43022607, 43820401,
43787101

870.3700    Developmental Toxicity (mouse) MRID 43787102 and
43817501……………………………………….

870.3800	2-Gen. Reproduction  MRID 40623701

Y

Y

Y

Y

Y

Y

Y

N

870.4100a	Chronic Toxicity (Hamster) MRID  44874001; 44751101

870.4100b	Chronic Toxicity (baboon) MRID 133230

870.4300	Chronic/Oncogenicity (rat) MRID 42027906;161332

870.4200    Carcinogenicity (mouse-FDA review)……........

Y

Y

Y

Y

Y

Y

Y

Y

870.5100	Mutagenicity—Gene Mutation - MRID 43533301, 44389705

870.5300	Mutagenicity—Gene Mutation - MRID 44389704

870.5375	Mutagenicity—Structural Chromosomal Aberrations in vitro MRID
43740801, 47276601

870.5385    Murtagenicity- Structural Chromosome Aberrations in vivo
MRID 43740802

870.5500	Mutagenicity—UDS in vitro – MRID 47276602

Y

Y

Y

Y

Y

Y

Y

Y

870.7485	General Metabolism MRID 45307501/45307502, 45307503

870.7600	Dermal Penetration (MRID 34335)

Y

Y

Y

Y

Y - Yes;   N - no

3.0 	DATA GAPS

Although the 21-day inhalation toxicity study for triclosan has been
used for regulatory purposes, a new 21-day inhalation toxicity study
toxicity is being requested in order to properly assess inhalation risk.
 There was not enough data in the current study to convert to a Human
Equivalent Concentration in accordance with the Agency’s current
method to assess inhalation risk.

4.0	HAZARD ASSESSMENT

Acute Toxicity

Adequacy of database for Acute Toxicity:   The acute toxicity database
for triclosan is considered adequate, although percent active ingredient
information for the primary dermal irritation study should be supplied.

Acute toxicity studies in experimental animals with technical grade
triclosan show that by the oral and dermal routes, triclosan is of low
acute toxicity (Toxicity Category IV;   MRIDs 43206501 and 100178).  By
the inhalation route of exposure, triclosan was assigned Toxicity
Category II for acute exposures and is thus of higher acute toxicity by
inhalation exposure than by oral or dermal exposures (MRID 42306902 and
43310501).  Triclosan produces moderate  irritation to the eyes (MRID
43429) and skin (MRID 42306903) with a Toxicity Category III assigned
for both.  Triclosan was not a dermal sensitizer in guinea pigs using
the Buehler method (MRID 43206502).

The acute toxicity data for triclosan is summarized in Table 1.

Table 1.  Acute Toxicity Profile for Triclosan

Guideline Number	Study Type/

Test substance (% a.i.)	MRID Number/

Citation	Results	Toxicity Category

870.1100

(§81-1)	Acute Oral- Rat triclosan (99.7% a.i.)	43206501	LD50: >5000
mg/kg	IV

870.1200

(§81-2)	Acute Dermal- Rabbit

triclosan (97% a.i.)	94044, 92084037	LD50: >9300 mg/kg	IV

870.1300

(§81-3)	Acute Inhalation- Rat

triclosan (100.5% a.i.)	42306902, 43310501	LC50: >0.15 mg/L	II

870.2400

(§81-4)	Primary Eye Irritation- Rabbit

Triclosan (97% a.i.)	94045	moderately irritating	III

870.2500

(§81-5)	Primary Dermal Irritation- Rabbit

triclosan (% a.i.not provided)	42306903	PII: 3.5 at 72 hours 	III

870.2600

(§81-6)	Dermal Sensitization- Guinea Pig          triclosan (99.7%
a.i.)	43206502	Not a Sensitizer	NA

4.2	Subchronic Toxicity

Adequacy of database for Subchronic Toxicity:  Acceptable subchronic
toxicity data for triclosan include two 90-day oral toxicity studies in
rodents (MRID 133545 and MRID 43022605), a 28-day oral toxicity study in
the mouse (MRID 44389707), a 90-day oral toxicity study in dogs (MRID
96102),  a 90-day dermal toxicity study in rats (MRID 43328001), in
addition to non-guideline 14-day repeat dose studies in mice (MRID
44389708) and rats (MRID 44389710).  An acceptable  non-guideline 21-day
inhalation toxicity study (MRID 0087996) is also available for
triclosan.

90-day Oral Toxicity Study (Rat)

In a 90-day feeding study in rats (MRID 133545), groups of
Sprague-Dawley rats (25/sex/dose) received Irgasan (triclosan) at
dietary concentrations 0, 1000, 3000, and 6000 ppm.  The 6000 ppm
animals showed signs of liver damage as characterized by “fatty
metamorphosis and cytomegaly.”  Similar to a lesser degree of liver
effects were also seen in the 3000 ppm groups.  The low dose, 50
mg/kg/day, was a NOAEL.

This study is classified as Acceptable/Guideline.   It satisfies the
minimum guideline requirements for a subchronic feeding study in rats.

	90-day Oral Toxicity Study (Mouse)

In a subchronic feeding study (MRID 43022605), CD-1 mice were fed
triclosan (99.7% a.i.) daily at dietary levels of 0, 25, 75, 200, 350,
750, or  900 mg/kg/day for 13 weeks (main groups, 15 mice per group) or
0, 25, 350, or  900 mg/kg/day for 7 weeks (satellite groups, 20 mice in
the control group and 10 mice per treatment group). Satellite groups
were run concurrently with the  main groups and were mainly used to
provide clinical pathology data. Animals from the satellite groups were
sacrificed after- 7 weeks of exposure.

Systemic toxicity was observed at all dose levels in a dose-related
manner as evidenced by clinical pathology, organ weight changes, and
increased incidence or severity of histopathological lesions (especially
of the liver). Clinical pathology included significantly decreased
erythrocytes, hemoglobin, and hematocrit at > 25 mg/kg/day in males
(68%—92% of controls) and at > 75 mg/kg/day in females (73%-91%).
Enzyme changes, indicative of liver injury, included increased alkaline
phosphatase (at > 25 mg/kg/day;  1.5-4.4 fold increases in both sexes),
alanine aminotransferase (at > 200 mg/kg/day; 1.3-6.2 fold increases in
both sexes), and aspartate aminotransferase (at > 200 mg/kg/day; 1.5-2.4
fold increase in males). Absolute and relative liver/gallbladder weights
increased 1.3-3.0 fold at  > 75 mg/kg/day in both sexes. Increased
incidence or severity of histopathological lesions in the liver included
hypertrophic hepatocytes, vacuolization, inflammation, necrosis,
pigmented Kupffer cells and/or macrophages, mineralizition, and chronic
bile duct inflammation. These lesions were evident in males at > 25
mg/kg/day and in females at > 200 mg/kg/day. The severity of
extramedullary hematopoiesis in the spleen increased in males ( > 200
mg/kg/day) and in females (> 750 mg/kg/day).

Additional findings at higher dose levels included organ weight changes
(kidney, adrenal gland, Uterus, ovary, and salivary gland); clinical
signs (hunched posture, thin appearance, and hypoactivity, pale
appearance, and cold  to touch); changes in body weight gain (a decrease
to 60% and 83% in males and females, respectively, for weeks 1—6 in
the satellite groups and to 83% and 67% in males and females,
respectively, for weeks 1-13 in the main groups); and increased
incidence or severity of cystic stomach hyperplasia, subacute kidney
inflammation, uterine hypoplasia, hypertrophic adrenal cortex (males);
uterine hypoplasia; chronic inflammation of the kidney (females); tubule
regeneration of the kidney, mammary gland dilatation and epithelial
hypoplasia (females), chronic heart inflammation (females); pigmented
macrophages in the mandibular lymph node (males); hypercellularity of
the marrow of the femur (males); and lymphoid hyperplasia in the cecum
(females).

Based on changes in clinical chemistry and hematology parameters as well
as lesions in the liver at the lowest dose level, the systemic toxicity
LOAEL was 25 mg/kg/day; a NOAEL could not be determined.

This study is classified as acceptable/guideline and satisfies the
minimum guideline requirements for a subchronic feeding study (OPPTS
870.3100) in mice.

870.3100	28-day Oral Toxicity Study (Mouse)

In a 28-day oral toxicity study (MRID 44389707), a total of 40 mice
(MAGf [SPF], 5/sex/dose) received technical triclosan admixed into
pelleted feed at dose levels of 0, 50, and 1000 ppm (6.48 and 135.59
mg/kg/day in males, 8.25 and 168.78 mg/kg/day in females) for 4 weeks.
Five males and 5 females were given the high dose and allowed to
“recover” (feeding of non-treated feed) for 2 weeks.  There were no
reported effects on mortality, body weight, or food consumption.
Hematological effects were observed in the high dose (135.59 mg/kg/day
in males; 168.78 mg/kg/day in females), and included significant
decreases in erythrocytes, hemoglobin, and hematocrit in males, and a
significant decrease in hemoglobin in females.  Both sexes showed
significant increases in thrombocytes at this dose, the effects of which
were not fully reversible after two weeks recovery.  Clinical chemistry
alterations (significant increases in alkaline phosphatase, alanine
aminotransferase, aspartate aminotransferase; significant decrease in
globulin fraction) were observed at the high dose in male and female
mice.  Elevated serum enzyme activities were evident after the two week
recovery period.  Absolute weight of the liver and liver/body weight
ratio were significantly increased at the high dose in male and female
mice.  Histopathological examination of the liver showed an increased
incidence of liver cell necrosis (as single cells or small cell groups),
hemosiderosis of Kupffer cells in the vicinity, cytoplasmic vacuoles in
hepatocytes, and liver cell hypertrophy.  The presence of necrosis was
still evident (2/5 males and 3/5 females) after the recovery period.

Based on the biochemical and morphological effects of triclosan
treatment on the liver of male and female mice, a systemic LOAEL of
135.59 mg/kg/day for males and 168.78 mg/kg/day for females is assigned.
 The systemic NOAEL is considered to be 6.48 mg/kg/day in males, and
8.25 mg/kg/day in females.

This study is classified as Acceptable/Guideline and provides relevant
toxicologic data on the effects of triclosan treatment to the liver of
male and female mice.  This study was not conducted to fulfill a
specific guideline requirement, but provides useful data for the risk
assessment of triclosan.

870.3150	Oral Subchronic (Non-rodent)

In a subchronic oral toxicity study (MRID 96102) conducted in male and
female Beagle dogs, triclosan was administered orally via a gelatin
capsule at doses of  0, 12.5, 25, 50, or 100 mg/kg/day seven days per
week for 13 weeks. An additional test group of 2 male and 2 female dogs
were given the test material at 50 mg/kg/day for 13 weeks and allowed a
four week recovery period prior to termination. Body weight gain in
females at 12.5 mg/kg/day was significantly lower in relation to
untreated controls but body weight decrements were not observed at
higher doses in either sex. One male in the 100 mg/kg/day dose group
died after day 23 of dosing, and one male in the 100 mg/kg group and one
female in the 50 mg/kg group were sacrificed in extremis on day 26 and
day 57, respectively. Each of above three animals displayed weight loss,
anorexia, lethargy, and symptoms of jaundice three to five days prior to
death. Upon autopsy of the animals that died or were sacrificed during
the study, histopathological examination of tissues revealed that the
jaundice was a result of hepatotoxicity. Upon examination of all the
study animals at necropsy, it was noted that there were
treatment-related morphological changes in the livers of most animals at
the 25, 50, and 100 mg/kg/day dose groups. The changes included focal
acidophilic to granular degeneration of the cytoplasm of hepatocytes.
The mean overall body weight for females receiving 12.5 mg/kg/day was
significantly lower than the weights for the control group while weights
for all other treatment groups showed no significant deviation from the
control group. Serum alkaline phosphatase activity was elevated in dogs
receiving 50 and 100 mg/kg/day. One group of animals receiving 100
mg/kg/day and then allowed a 28-day recovery period showed serum
alkaline phosphatase that returned to normal after the recovery period.
No other blood chemistry studies revealed any other abnormalities. On
the basis of this study the NOAEL was determined to be 12.5 mg/kg/day in
Beagle dogs.

This study is classified as Acceptable/Guideline.

870. 3250	90-day Dermal Toxicity Study (Rat)

In a 90-day dermal toxicity study (MRID 43328001), groups of rats
(10/sex/group) received triclosan in propylene glycol by dermal
application at dose levels of 10, 40, and 80 mg/kg for 6 hrs/day for 90
days, followed by a 28 day recovery period.  Dermal irritation at the
application site was found in all dose groups.  At the 10 mg/kg/day
dose, animals were observed with erythema and edema beginning on day 21
of the study.  At the 40 and 80 mg/kg/day dose levels, animals were
observed with dermal reactions beginning on day 4 of the study and a
greater number of animals were observed with  dermal scores of +3 and +4
for erythema and edema.   In the satellite group given test material at
80 mg/kg/day and allowed a 28 day recovery period beyond the 90-day
dosing period, dermal irritation scores had subsided by the end of the
28-day recovery period.   Systemically, an increase in the incidence of
occult blood in the urine of 80 mg/kg males and females was found.  No
other systemic toxicity was observed from the data in this study.  Under
the conditions of this study, the LOAEL for systemic toxicity was 80
mg/kg; the NOAEL was 40 mg/kg.

 This study is classified as acceptable/guideline and satisfies the
870.3250 guideline for a 90-day dermal toxicity study in rats.

Non-Guideline 14-Day Repeated Dose Dermal Toxicity (Mouse)

In a repeated dose dermal toxicity study (MRID 44389708), triclosan
(99.3% a.i.) was applied daily in acetone to the clipped skin of ten
CD-1 mice/sex/dose at dose levels of 0, 0.3, 0.6, 1.5, 3.0, or 6.0
mg/animal/day for 14 days.

(p≤0.05) at 6.0 mg/animal for females (↓32%) and significantly
increased for males at 3.0 mg/animal/day (↑64%).  Food consumption was
significantly increased (p≤0.05) for the 3.0 and 6.0 mg/animal/day
groups during Week 1 (females only), Week 2 (both sexes), and overall
for females only.  The LOAEL for this study is 1.5 mg/animal/day, based
on treatment-related dermal irritation at the treatment site and on
increased liver weights in this treatment group.  The NOAEL is 0.6
mg/animal/day.  Based on the results of this study, the highest
recommended level for a 90-day dermal study was judged to be 1.2
mg/animal/day with inclusion of at least one level below 0.3
mg/animal/day.

This dermal toxicity study is classified as Acceptable/Non-Guideline and
was intended only as a range-finding study for a 90-day dermal study.

Non-Guideline 14-Day Repeated Dose Dermal Toxicity (Rat)

In a repeated dose dermal toxicity study (MRID 44389710), triclosan
(99.3% a.i.) was applied daily in acetone to the clipped skin of ten
Crl:CD®BR rats/sex/dose at dose levels of 0, 0.3, 0.6, 1.5, 3.0, or 6.0
mg/animal/day for 14 days.

Treatment-related dermal irritation was observed in both sexes of the
6.0 mg/animal/day group consisting of erythema, scaling, and eschar.
Dose-related, non-neoplastic histopathological changes to skin were
observed at application sites in the 6.0 mg/animal/day treatment group
which consisted of treatment-related acanthosis of eschar in 4/10 males
and 3/10 females and hyperkeratosis in 10/10 females.  No unscheduled
deaths occurred and no clinical signs of toxicity were observed in any
treatment group.  There were no treatment-related changes in organ
weights.  There were no significant differences between the terminal
body weights or in food consumption in the treated and control groups
nor were there any overall differences in body weight changes in treated
males and females.  The LOAEL for this study is 6.0 mg/animal/day, based
on treatment-related dermal irritation at the treatment site.  The NOAEL
is 3.0 mg/animal/day.

This dermal toxicity study is classified as Acceptable/Non-Guideline and
was intended only as a range-finding study for a 90-day dermal study.

21-Day Inhalation Toxicity Study in Rats

In a subchronic inhalation toxicity study (MRID 0087996), triclosan
(purity not reported) was administered to 9 rats/dose/sex at dose levels
as described in the table below:

Group	No. of Rats	Mean Concentration (mg/m3) Air

	Males	Females	1st Day	2nd-15th Day

1	9*	9*	0	0

2	9	9	50	50

3	9*	9*	227	115

4	9	9	1300	301

		*8 animals (2 males/2 females) from each group were kept for a 17-day
recovery

                          period, following the 21-day exposure.

Group

Mean bodyweight (kg)	Equivalent Dose (mg/kg/day)#

	Males	Females	Males 	Females

50 mg/m3 (4.22 ppm)	.271	.193	3.21	4.51

115 mg/m3

(9.71 ppm)	.251	.202	7.97	9.91

301 mg/m3

(25.4 ppm)	.217	.170	24.14	30.81

* = (mg/m3 x 24.45)/mw = ppm

# = ((0.0087 m3/hr *mg/m3*hr/day)/bw), where 0.0087 m3/hr is a default
inhalation rate for young rats.

A 10% ethanol suspension of triclosan was administered “nose only”
as an aerosol (5 days per week , 2 hrs per day) for 21 days.  Dose
levels of 0, 50, 115, or 301 mg/m3 are equivalent to 0, 0.36, 0.77, 1.77
mg/kg/day for males, respectively, and 0, 0.36, 0.64 and 1.08 mg/kg/day
for females, respectively).  Treatment groups 3 and 4 initially received
concentrations of 227 and 1300 mg/m3, respectively.  These
concentrations were reduced after the first day of treatment because
they were not tolerated well by the animals.

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Data\\EPA SMART Meeting\\Joint Comments\\21-day Inhalation Particle Size
Distribution.xls" "" \a \f 0 \p

Twelve high-dose animals (5 males and 7 females) died during the course
of the study.  Toxicity was observed at all dose levels.
Treatment-related effects at 1300/301 mg/m3 included clinical signs of
toxicity (dyspnea, nasal discharge, muscle spasms, pallor, and
diarrhea), decreased body weight, decreased body weight gain, decreased
food consumption, statistically-significant increased total leukocyte
count, statistically-significant increased percentage of neutrophils and
decreased lymphocytes, statistically-significant increased serum
glutamic-pyruvic transaminase (GPT) activity, statistically-significant
increased alkaline phosphatase (AP), statistically-significant decreased
serum proteins (males), and increased incidence of respiratory
inflammation.  Additional statistical analyses also showed a
statistically-significant decrease in thrombocytes.  Macroscopic
findings for the high-dose animals that died prior to scheduled
sacrifice included severe acute congestion and numerous hemorrhages in
all organs.  Acute purulent inflammation with focal ulceration of the
mucous membrane in the nasal cavity and in the trachea were also
observed.  Treatment-related effects at 227/115 mg/m3 included slightly
decreased body weight and body weight gain, slightly decreased food
consumption, increased leukocytes, statistically-significant decreased
thrombocytes, statistically-significant increased alkaline phosphatase,
statistically-significant decreased serum proteins (males), and slight
incidences of respiratory irritation in one male and two females.  At
the low dose, a statistically-significant decrease in thrombocytes and
total serum proteins and a statistically-significant increase in
alkaline phosphatase were observed in the males.  Consequently, the
LOAEL is 3.21 mg/kg/day for males based on changes in thrombocytes,
total blood proteins, and alkaline phosphatase; the LOAEL for females is
9.91 mg/kg/day.  A NOAEL could not be established for males; the NOAEL
for females is 4.51 mg/kg/day.

This 21-day rat inhalation study is classified acceptable-non-guideline.
 The LOAEL value established in male rats in this study may be used as a
tentative LOAEL for regulatory purposes. However, there were several
deficiencies in this study as noted in the full review.

4.3	Prenatal Developmental Toxicity

Adequacy of database for Prenatal Developmental Toxicity:  Acceptable
developmental toxicity studies (MRID 43817502/43817503 and
43820401/43022607; 43787101) in rats and rabbits are available for
triclosan.

870.3700	Prenatal Developmental Toxicity (Rat)

In an oral prenatal developmental toxicity study in rats (MRID 43817502
and 43817503), triclosan (99.8%) was administered by gavage to pregnant
female Colworth Wistar rats (30 rats/treated group and 60 rats in the
control group) on days 6-15 of gestation at dose levels of 30, 100, or
300 mg/kg/day, with the day of mating designated as gestation day 0.
The rats were observed for signs of toxicity; body weight and food
consumption values were recorded.  On day 21 of gestation, 25 rats per
treated group and 50 control rats were sacrificed and necropsied;
uterine weights were recorded.  The uteri were examined, implantation
sites were counted, and the numbers of corpora lutea were determined.
The fetuses were removed, weighed, sexed, and examined for external
anomalies.  They were then processed for visceral and skeletal
evaluation.  Five rats per treated group and ten control rats were
allowed to deliver their litters.  Litter weight, pup mortality, and
developmental milestones (presence of vibrissae, pinna unfolding,
incisor eruption, eyelid opening, and completion of fur growth) were
recorded.  The pups were killed and necropsied on lactation day 21, and
all pups were processed for skeletal examination.

At 300 mg/kg/day, maternal toxicity consisted of transient diarrhea,
retarded body weight gain during the period of treatment, and reduced
food consumption and increased water consumption from the onset of
treatment, throughout the gestation period.  Based on these findings:
Maternal LOAEL = 300 mg/kg/day; Maternal NOAEL = 100 mg/kg/day.

No evidence of pre- or postnatal developmental toxicity was identified
at any dose level under the conditions of this study.  Developmental
NOAEL > 300 mg/kg/day); Developmental LOAEL > 300 mg/kg/day.

This study is classified as Acceptable/Guideline and satisfies the
870.3700 guideline for a developmental toxicity study in rats.

870.3700	Prenatal Developmental Toxicity (Rabbit)

In a developmental toxicity study in rabbits (MRIDs 43820401, 43022607,
and 43787101), triclosan (Lot No. 19851206, purity 100%) was
administered by gavage to groups of 18 pregnant New Zealand white
rabbits at doses of 0, 15, 50, or 150 mg/kg/day on gestation days (GD)
6-18, inclusive.  The dosing solutions were administered as suspensions
in 1% carboxymethylcellulose in an aqueous glycerin solution.  All
surviving does were sacrificed on GD 30 and subjected to gross necropsy.
 All fetuses were examined for external and visceral
malformations/variations and processed for skeletal examination.

ased body weight gain for the overall treatment period.  Mean body
weights for the 150 mg/kg/day does were reduced 3.3% (GD 6) to 7.9% (GD
16) compared to controls, and significantly (p ≤ 0.05) reduced at GD
14 and 16.  For GD 6-8 and 12-14. the high-dose animals showed a
significant (p≤ 0.01) mean body weight loss.  Over the entire GD 6-19
interval, the mean weight gain for the high dose group was significantly
lower (p ≤ 0.01, -65.8%) than controls.  Mean food consumption
(reported as g/kg day) during GD 6-19 in the high-dose group decreased
compared to controls (-7% at GD 11 to -41.1% at GD 14), with significant
differences (p ≤ 0.01) on GD 6-8 and GD 12-15.

The maternal toxicity LOAEL is 150 mg/kg/day based on significant mean
body weight loss and decreased body weight gain, and the maternal
toxicity NOAEL is 50 mg/kg/day.

No does aborted during the study.  There were no statistically
significant differences in the mean number of resorptions/doe or the
resorption/implant ratio between the control and treated groups.  Fetal
body weights of both sexes were comparable between the control and
treatment groups.

The total number of fetuses/litters examined in the 0, 15, 50, and 150
mg/kg/day groups were 143/16, 126/15, 129/15, and 124/16, respectively.
No treatment-related external, visceral, or skeletal
malformations/variations were observed in fetuses from any treated
group.

Therefore, the developmental toxicity LOAEL was not identified, and the
developmental toxicity NOAEL is > 150 mg/kg/day.

This study is classified acceptable/ guideline and satisfies the
870.3700 guideline for a developmental toxicity study in rabbits.

870.3700      Developmental toxicity in mice

In a developmental toxicity study in mice (MRID 43817501), triclosan
(99.0% a.i.) was administered to 25 Charles River CD-1 (ICR)BR female
mice/dose via the diet at target dose levels of 0, 10, 25, 75, or 350
mg/kg/day (calculated achieved doses of 0, 11.2, 26.1, 81.8, and 372.1
mg/kg/day) from days 6-15 of gestation. Maternal toxicity was observed
at 75 mg/kg/day by increases in absolute and relative liver weight and
tan areas in the liver of one dam. At 350 mg/kg/day, maternal toxicity
was evident as increases in absolute and relative liver weight and
observations of tan areas of the liver in 14/22 dams. The maternal LOAEL
is 75 mg/kg/day (81.6 mg/kg/day) based on increased liver weights. The
Maternal NOAEL is 25 mg/kg/day (11.2 mg/kg/day).

Developmental effects were noted at the 75 and 350 mg/kg/day target dose
levels as increased incidence of variations (characterized as reversible
irregular ossification of the skull at 75 and 350 mg/kg/day, and
phalanges at 350 mg/kg/day). Decreased fetal weight was also observed
*14 and 18% decrease at 75 and 350 mg/kg/day target dose levels,
respectively). The developmental LOAEL is 75 mg/kg/day (81.6 mg/kg/day)
based on irregular ossification of the skull. The developmental NOAEL is
25 mg/kg/day (11.2 mg/kg/day).

This study is classified  acceptable/guideline and satisfies the
requirement for a developmental toxicity study in mice.  .

4.4	Reproductive Toxicity

Adequacy of database for Reproductive Toxicity:  One study is available
(MRID 40623701) on the reproductive toxicity of triclosan.

870.3800	Reproduction

In a 2-generation reproduction study (MRID 40623701) triclosan was
administered in the diet to groups of either 25 or 30 (Fo and Fl,
respectively) male and female Crl:CD(BR) rats at dose levels of 0, 300,
1000, and 3000 ppm (0, 15, 50, and 150 mg/kg/day) for 10 weeks prior to
mating and through post-natal day 21 for both generations. In the Fo
generation, there were no significant decreases in parental body weight
during pre-mating, but a significant increase in mean body weight was
observed in 50 mg/kg/day males. Body weight in Fo females during
lactation was significantly decreased on post-natal day 7, with a
significant negative trend in mean body weight gain for the high dose
group of for days 0-7. Increased incidence of liver discoloration in 50
and 150 mg/kg/day parental Fo males was observed. No effects on
reproductive performance were observed in the Fo generation. Pups of the
Fo generation (Fl pups) showed decreased mean body weight on post-natal
days 14 and 21 at the 150 mg/kg/day dose. Increased pup mortality was
observed on postnatal days 0-3 in high dose pups. Decreased viability
index was also observed at the 150 mg/kg/day dose in Fl pups, as was an
increased incidence of dilated renal pelvis. In Fl parental animals,
significantly lower group mean body weights were observed during
pre-mating at the 150 mg/kg/day dose. Gestational group mean body weight
in Fl females was significantly decreased by 12% during the period of
gestation, with a significant negative trend for gestational days 1, 7,
14, and 20. There were no differences in number of pregnant animals,
mean gestation duration and mean precoital interval in Fl females. In
pups of the Fl parental generation (F2 pups), an increase in number of
pups found dead or missing was increased at the 150 mg/kg/day dose.
Weaning index was decreased at the high dose in F2 pups, and increased
total liter deaths was increased. The Parental Systemic NOAEL is l000
ppm (50 mg/kg/day), and the Parental Systemic LOAEL is 3000 ppm (150
mg/kg/day), based on reduced mean body weights. The
Reproductive/Developmental NOAEL is l000 ppm (50 mg/kg/day); the
Reproductive/ Developmental LOAEL is 3000 ppm (150 mg/kg/day), based on
reduced viability of pups and reduced body weights.

This reproductive study in the rat is classified
unacceptable/upgradable.  Information must be provided by the registrant
on the meaning of unadjusted and adjusted pup body weights and a
clarification of the random card draw procedure used to select parents
of the F2 generation.

4.5	Chronic Toxicity

Adequacy of database for Chronic Toxicity:    Chronic toxicity data for
triclosan were submitted and consist of a one-year oral toxicity study
in baboons (MRID 133230), a chronic toxicity/carcinogenicity study in
hamsters (MRID 44874001; 44751101), and a chronic
toxicity/carcinogenicity feeding study in rats (MRID 42027906).   The
baboon and hamster study were considered acceptable.

870.4100b	Chronic Toxicity (Baboon)

In a chronic toxicity study (MRID 133230), groups of 7 baboons/sex/dose
received triclosan orally at doses of 30, 100, and 300 mg/kg/day by
capsule for 52 weeks. Two males and 2 females from each dose group were
sacrificed at six months, 3 males and 3 females from each dose group at
52 weeks, and the remainder of the animals after a six week recovery
period following cessation of treatment. At the 100 and 300 mg/kg/day
dose levels, test animals were observed with signs of vomiting, failure
to eat, and diarrhea, which occurred 4-6 hours after dosing or during
the night. At necropsy, an effect on the lining of the stomach was
observed at the high dose. The systemic NOAEL was determined to be 30
mg/kg/day, and the systemic LOAEL was determined to be 100 mg/kg/day,
based on clinical signs of toxicity.

This study is classified as Acceptable/Guideline.  It satisfies the
minimum guideline requirements for a non-rodent chronic toxicity study.

870.4300	Chronic Toxicity/Carcinogenicity (Rat)

In a chronic toxicity/oncogenicity feeding study (MRID 42027906)
conducted in male and female Sprague-Dawley rats, [FAT 80'023
(triclosan, 99.0 % a.i.)] was administered in the diet at doses of 0,
300, 1000, or 3000 ppm (0, 15.3, 52.4, and 168.0 mg/kg/day in males ; 0,
20.0, 66.9, and 217.4 mg/kg/day in females) for 104 weeks.  An
additional group of 20 male and 20 female rats received triclosan in the
diet at 6000 ppm (415.0 mg/kg/day [males] and 519.3 mg/kg/day [females])
for 52 weeks.  No treatment related effects on mortality, clinical
toxicity, opthamalogy, urinalysis, gross pathology, or neoplastic
pathology were observed at any dose level tested. Erythrocyte count,
hemoglobin concentration, and hematocrit were decreased in males at the
15.3, 52.4, and 168.0 mg/kg/day dose levels, and erythrocyte count was
decreased in females at 66.9 and 217.4 mg/kg/day. Serum alanine and
aspartate aminotrasferase activities were increases in males at 168.0
mg/kg/day, and blood urea nitrogen was increased in females at 217.4
mg/kg/day. Hepatocellular hypertrophy was observed in males at all dose
levels. Increased incidence of liver necrosis was observed in males at
the 300, 1000, and 3000 ppm dose levels (5/85, 4/85, and 4/85
respectively compared to 1/95 in concurrent controls). The predominant
residue of triclosan observed in blood and kidney was the sulfate
conjugate of triclosan, while unconjugated triclosan was predominant in
the liver. Residual levels of triclosan were proportional to the dose
administered. No carcinogenic potential was demonstrated for triclosan
in this study. The systemic LOAEL was determined to be 3000 ppm (168.0
mg/kg/day) based on significant decreases in body weight in male and
female rats and non-neoplastic changes of the liver (cytoplasmic
inclusions and hepatocellular hypertrophy) in males at 3000 ppm (168.0
mg/kg/day).  The systemic NOAEL was determined to be 1000 ppm (52.4
mg/kg/day).

This study is classified as Core minimum.

4.6	Carcinogenicity

Adequacy of database for Carcinogenicity:  Carcinogenicity data for
triclosan include a chronic/carcinogenicity study in hamsters (MRID
44874001, 44751101),   a chronic toxicity/carcinogenicity feeding study
in rats (MRID 42027906), and  an oral carcinogenicity study in mice, a
summary of which was obtained from the Food and Drug Administration’s
review.  This FDA review was obtained and verified to be publicly
citable.

870.4100a	Chronic Toxicity (Rat)

 In a chronic toxicity/oncogenicity feeding study (MRID 42027906)
conducted in male and female Sprague-Dawley rats, [FAT 80'023
(triclosan, 99.0 % a.i.)] was administered in the diet at doses of 0,
300, 1000, or 3000 ppm (0, 15.3, 52.4, and 168.0 mg/kg/day in males ; 0,
20.0, 66.9, and 217.4 mg/kg/day in females) for 104 weeks.  An
additional group of 20 male and 20 female rats received triclosan in the
diet at 6000 ppm (415.0 mg/kg/day [males] and 519.3 mg/kg/day [females])
for 52 weeks.  No treatment related effects on mortality, clinical
toxicity, opthamalogy, urinalysis, gross pathology, or neoplastic
pathology were observed at any dose level tested. Erythrocyte count,
hemoglobin concentration, and hematocrit were decreased in males at the
15.3, 52.4, and 168.0 mg/kg/day dose levels, and erythrocyte count was
decreased in females at 66.9 and 217.4 mg/kg/day. Serum alanine and
aspartate aminotrasferase activities were increases in males at 168.0
mg/kg/day, and blood urea nitrogen was increased in females at 217.4
mg/kg/day. Hepatocellular hypertrophy was observed in males at all dose
levels. Increased incidence of liver necrosis was observed in males at
the 300, 1000, and 3000 ppm dose levels (5/85, 4/85, and 4/85
respectively compared to 1/95 in concurrent controls). The predominant
residue of triclosan observed in blood and kidney was the sulfate
conjugate of triclosan, while unconjugated triclosan was predominant in
the liver. Residual levels of triclosan were proportional to the dose
administered. No carcinogenic potential was demonstrated for triclosan
in this study.  The systemic LOAEL was determined to be 3000 ppm (168.0
mg/kg/day) based on significant decreases in body weight in male and
female rats and non-neoplastic changes of the liver (cytoplasmic
inclusions and hepatocellular hypertrophy) in males at 3000 ppm (168.0
mg/kg/day).  The systemic NOAEL was determined to be 1000 ppm (52.4
mg/kg/day).

This study is classified as core minimum.

870.4200	Carcinogenicity (Mouse)

In an 18-month carcinogenicity bioassay in mice, 5 groups of male and
female CD-1 mice (70 mice/sex/dose) received triclosan in the diet at
dose levels of 0, 10, 30, 100, or 200 mg/kg/day.  Fifty mice/sex/dose
received dietary triclosan for 18 months, while the remaining 20
mice/sex/dose received dietary triclosan for only 6 months, after which
time these mice were sacrificed.  Blood samples were obtained from 10
mice/sex/dose from both the 6 month and 18 month dose groups at
sacrifice, for determination of triclosan plasma levels.  Time of blood
sampling relative to the last dose of triclosan was not stated.
Parameters monitored during this study included mortality, clinical
observations, body weight, food consumption, ophthalmology, clinical
chemistry, urinalysis, hematology, gross and microscopic pathology, and
organ weights.  Reduced survival was observed in female mice receiving
200 mg/kg/day for 18 months (34/50 vs. 45/50 in control). There were no
significant signs of clinical toxicity at any dose level, and no
significant effects of treatment on group mean body weight, food
consumption, ophthalmology, or urinalysis. A dose-related increase in
activity of alanine aminotransferse and alkaline phosphatase was
observed in male and female mice at 100 mg/kg/day triclosan and above in
both the 6 month and 18 month dose groups. Significant decreases in both
albumin and total protein were observed in males at 6 months and in
females at 18 months at doses of 100 mg/kg/day and above. Serum
cholesterol was markedly reduced at all dose levels including the 10
mg/kg/day dose. Treatment-related hematological effects included
increased reticulocyte count and platelet count in males and females at
the 200 mg/kg/day dose.  Mean liver weight (absolute and relative) was
increased in both male and female mice at 30 mg/kg/day and above at both
6 and 18 months.  An increased incidence of nodules and discoloration of
the liver was observed in both male and female mice at 100 mg/kg/day and
above.  A dose-related increase in severity of hepatocellular
hypertrophy was observed in both male and female mice at 30 mg/kg/day
and above. Dose-related increases in incidence or severity of
hepatocellular vacuolation/vesiculation and hepatic inflammation,
necrosis, and microgranulomas was also observed.

After 18 months of exposure, a statistically significant increase in
the incidence of hepatocellular adenoma and/or carcinoma was observed in
male and female mice at 100 mg/kg/day triclosan and above. The incidence
was dose-related in both sexes.  Combined incidence of adenoma and
carcinoma was 12%, 20%, 34%, 64%, and 84% for males, and 0%, 2%, 6%,
12%, and 40% for females at the 0, 10, 30, 100, and 200 mg/kg/day dose
levels, respectively. The incidence of adenoma / carcinoma combined
exceeded historical control incidence at the 10 mg/kg/day dose level
(17% for males, 1% for females), but became statistically significant at
the 30 mg/kg/day dose level.  Therefore, a systemic NOAEL of 10
mg/kg/day can be established from the data in this study, based on
increased incidence of liver neoplasms in male and female mice at 30
mg/kg/day.

870.4300	Chronic Toxicity/Carcinogenicity (Hamster)

In a chronic toxicity/oncogenicity study (MRID 44874001), FAT 80'023/S
(triclosan: 99.5% a.i.; Batch # 505017) was administered in the diet to
groups of 70 male and 70 female Bio F1D Alexander Syrian hamsters at
concentrations delivering doses of 0 (control 1), 0 (control 2), 12.5,
75, or 250 mg/kg/day. Actual achieved doses were: 0, 0, 12.6, 75.4 [75.5
F], and 251 mg/kg/day for males and females. Groups of 10 hamsters per
sex per dose were killed after 52 weeks for interim evaluations; the
remaining 60 hamsters per sex per dose were maintained on treated or
control diets for up 90 weeks for females and 95 weeks for males.

No treatment-related clinical signs of toxicity were observed during the
first 80 weeks of the study.  After this time, high-dose males showed
deterioration in their general clinical condition with signs such as
lethargy, hunched posture, pallor, thin appearance, and unsteady gait.
At termination of the females (week 91) the percent survival in the
control 1, control 2, low-, mid-, and high-dose groups was 40%, 38%,
47%, 58%, and 48%, respectively.  In contrast, high-dose males had an
increase in mortality after week 80 which correlated with their
deteriorating clinical condition.  At termination of the males (week 96)
the percent survival in the control 1, control 2, low-, mid-, and
high-dose groups was 65%, 72%, 75%, 80%, and 35%, respectively.

 females were significantly (p≤0.05 or 0.01) less than one or both
control groups throughout the study.  Overall body weight gains by the
high-dose animals through week 90 were 46-53% of the control levels.
Final absolute body weights of the high-dose males and females were
84-85% and 89-90%, respectively, of the control groups.  Body weights
and body weight gains by the mid- and low-dose animals were unaffected
by treatment.  High-dose males and females had significantly (p≤0.01)
reduced food consumption during weeks 1-3 as compared with both control
groups.  Food conversion ratios during the first 16 weeks of the study
for animals in the control 1, control 2, low-, mid-, and high-dose
groups were 30.5, 29.0, 29.2, 30.1, and 38.5 mg/kg/day, respectively,
for males and 33.2, 32.2, 36.6, 36.5, and 50.1 mg/kg/day, respectively,
for females.  Water consumption was highly variable between individuals
and between groups.  However, for the high-dose groups, water
consumption tended to be slightly increased throughout the study.

Plasma urea nitrogen was significantly (p≤0.05 or 0.01) increased to
119-156% of the control levels in high-dose males and females as
compared to one or both control groups at interim sacrifice and at
termination.  Statistically significant changes were observed for other
clinical chemistry parameters and for hematologic parameters and organ
weights, but none were considered treatment related.

≤0.05 or 0.01) was attained for these parameters at almost every time
point when compared to one or both controls.

At interim sacrifice, irregular cortical scarring of the kidney was
observed at gross necropsy in 4/10 high-dose males and 9/10 high-dose
females compared with none in the control male groups and 3/19 in the
control female groups combined.  This corresponded to microscopic
findings in the kidneys of the high-dose groups of both sexes consisting
of distended medullary tubules and radial areas of dilated basophilic
tubules with or without eosinophilic colloid/fibrosis.

0.01) increased incidence of nephropathy was observed in high-dose
males and females (decedents and survivors combined) as compared to both
control groups and was considered the main factor contributing to death
in animals that died before study termination.  The severity of
nephropathy, as calculated by the reviewer, in high-dose males and
females was 3.2 and 2.8, respectively, compared with control values of
2.5-2.7 and 2.1-2.3, respectively.  The incidence of nephropathy in the
control 1, control 2, low-, mid-, and high-dose groups was 41/60, 38/60,
35/60, 36/60, and 56/60, respectively, for males and 19/60, 21/60,
26/60, 19/60, and 50/60, respectively, for females.

In males tested at the high dose of triclosan, a significantly increased
incidence of absent spermatozoa, abnormal spermatogenic cells, and
reduced numbers of spermatozoa was observed in males that died and those
that were sacrificed at the end of the study.  Increased incidence of
partial depletion of one or more generations of germ cells within the
testis was also observed in high dose male hamsters that died during the
study or were sacrificed at study termination.

≤0.01) increased in high-dose males and females at termination; focal
atypical hyperplasia of the fundic region was observed in 11/60 males
and distended gastric glands with or without debris were observed in
17/60 females.  These lesions were observed in none of the control males
and only one of the control females, respectively.  In addition,
high-dose males killed at termination and dying during the study had
significantly (p0.01) increased incidences of abnormal spermatogenic
cells and reduced numbers of spermatozoa in the epididymides and partial
depletion of germ cells in the testes.

The LOAEL is 250 mg/kg/day for male and female hamsters based on
decreased body weight gains, increased mortality (males), nephropathy,
and histopathologic findings in the stomach and testes.  The
corresponding NOAEL is 75 mg/kg/day.

No evidence of potential carcinogenicity of the test material was
observed at the doses given in this study.  Neoplastic lesions did not
occur in treated groups at incidences significantly higher than the
incidences in control animals.  The doses administered were adequate for
testing carcinogenicity as evidenced by the systemic toxicity described
above.

This chronic toxicity/carcinogenicity study in the hamster is
Acceptable/Guideline and it satisfies the guideline requirement for a
chronic toxicity/carcinogenicity oral study [OPPTS 870.4300 (§83-5)] in
hamsters.

Mutagenicity

Triclosan has been tested for mutagenic activity in several assays,
including bacterial reverse mutation tests (MRID 43533301 and MRID
44389705), an in vitro mammalian cell gene mutation test (MRID
44389704), two in vitro mammalian chromosome aberration tests
         ( MRIDs 43740801 and 47276601), a mammalian bone marrow
chromosomal aberration test (MRID 43740802), and an unscheduled DNA
synthesis assay in mammalian cells in culture (MRID 47276602).

Bacterial Reverse Mutation Test

In two independently performed microbial preincubation assays (MRID
43533301), Salmonella typhimurium strains TA1535, TA1537, TA98, or TA100
were exposed to triclosan doses of 0.015, 0.050, 0.15, 0.5, or 1.5
µg/plate either in the absence or the presence of 3, 10, or 30% S9
derived from Aroclor 1254-induced rat livers.  The test material was
delivered to the test system in dimethyl sulfoxide.

μg /plate -S9 and at doses ≥0.5 μg /plate with S9.  There was,
however, no indication of a mutagenic response in any strain at any dose
either without or with increasing concentrations of S9.  All strains
responded in the expected manner to the nonactivated and S9-activated
positive controls.

The study is classified as Acceptable/Guideline.  It satisfies the
guideline requirement for a gene mutation assay (§84-2).

	Bacterial Reverse Mutation Test

0.5% a.i.) in dimethylsulfoxide (DMSO) at concentrations of 0.005-5,000
μg/plate without mammalian metabolic activation (-S9) and 0.005-50
μg/plate with mammalian metabolic activation (±S9).  Strains TA98,
TA100, TA1535, TA1537, and TA2538 were evaluated for mutagenicity at
0.05-5.0 μg/plate (+S9) and all except TA100 at 0.00167-0.167 μg/plate
(-S9).  Without S9, TAl00 was evaluated for mutagenicity at
0.00167-0.167 μg/plate.  The standard plate incorporation test was
performed.  S9 homogenates for metabolic activation were made from
Aroclor induced rat livers.

Triclosan was tested to cytotoxic concentrations.  The test article
precipitated from solution at 5,000 μg/plate (-S9).  In pre-screen
cytotoxicity tests triclosan was not toxic to strain TA1538 at doses of
0.005 to 1.67 μg/plate with S9 activation and 0.005 μg/plate without
S9 activation and was not toxic to strain TA100 at doses of 0.005 to
0.50 μg/plate +S9 and at 0.005 and 0.0167 μg/plate -S9.  There were no
reproducible, dose-related differences in the number of revertant
colonies in any tester strain at any dose level/condition compared to
the vehicle controls.  The positive control substances induced marked
increases in revertant colonies in their respective strains.

This study is classified as Acceptable/Guideline (§84-2) and satisfies
the requirement for FIFRA Test Guideline for in vitro mutagenicity
(bacterial reverse gene mutation) data.

In Vitro Mammalian Cell Gene Mutation Test

In a mammalian cell gene mutation assay at the thymidine kinase locus
(MRID 44389704), L5178Y TK +/- mouse lymphoma cells cultured in vitro
were exposed to triclosan (>99% a.i.) in dimethylsulfoxide (DMSO) at
concentrations ranging from 1 to 25 μg/mL without metabolic activation
(-S9) and from 1 to 20 μg/mL with mammalian metabolic activation (+S9).
 Treatment levels were selected based on a preliminary cytotoxicity test
conducted at 1 to 250 μg/mL with and without activation.

Triclosan was tested up to toxic concentrations.  Mutation frequencies
were determined for concentrations selected on the basis of relative
growth.  The first mutation assay was initiated at concentrations
ranging from 1 to 25 and 1 to 20 μg/mL without S9 activation and in a
second mutation assay at 1 to 20 and 0.5 to 15 μg/mL with metabolic
activation.  Redundant or highly cytotoxic concentrations were
eliminated during the assays.  Only dose levels that resulted in ≥10%
survival were used to assess mutagenicity.  For the final concentrations
tested, relative growth ranged from 8 to 100% without activation and
from 7 to 88% with activation.

In order for the test material to be considered a mutagen, it had to
produce both a mutant frequency at one or more dose levels that was at
least twice that of the vehicle control, as well as a dose or toxicity
relationship; in addition, the effects had to be reproducible.  By these
criteria triclosan was negative for inducing forward mutations at the TK
locus in mouse L5178Y cells both with and without metabolic activation.
In both the nonactivated and activated conditions, the positive controls
induced the appropriate responses.

This study is classified as Acceptable/Guideline (§84-2), and satisfies
the requirements for FIFRA Test Guideline for in vitro mammalian forward
gene mutation data.

870.5375	In Vitro Mammalian Chromosome Aberration Test

In a mammalian cell cytogenetics chromosome aberration assay (MRID
47276601), Chinese hamster ovary cells (CHO strain K1-BH4) were exposed
to triclosan (>99% pure; Unilever sample number S15155 T01) and
dissolved with DMSO.  Concentrations of 0.1, 0.3, 0.5, and 1.0 µg/mL
and 4.8, 9.5, 19.0, 30.0, and 38.0 µg/mL were tested for the cultures
without and with metabolic activation from Aroclor 1254-induced rat
livers for 24 and 6 hours, respectively.  Cells were harvested 24 hours
after treatment and analyzed for chromosomal aberrations.

Triclosan was tested up to the toxicity limit of 1.0 and 38.0 µg/mL,
-S9 and +S9, respectively, based on a preliminary toxicity test using
CHO cells that were treated at dose concentrations of 6.3, 12.5, 25.0,
50.0, 100.0, 200.0, and 400.0 µg/mL.  No live cells were observed at
≥50 and ≥100 µg/mL in -S9 and +S9 cultures, respectively.  There
were no aberrant cells at 12.5 and 25.0 µg/mL, -S9, but the mitotic
index was declined to ~29% at 6.3 µg/mL compared to the solvent
control.  For the cultures with +S9, the mitotic index was reduced by 27
and 77% for 50 and 25 µg/mL, respectively, but was comparable to the
solvent control at 6.3 and 12.5 µg/mL.  The EC50 value for cultures
with +S9 and –S9 were estimated to be 38 and 1 µg/mL.  Hence,
concentrations of 1 and 38 µg/mL were used as the highest dose for the
cultures without and with S9, respectively, for the cytogenetic assay.

In the cytogenetic assay, toxicity was noted at 38 µg/mL, +S9, and was
not analyzed for chromosomal aberrations.  Precipitation, if observed,
was not reported for any dose level.  Cultures treated with 0.1, 0.3,
0.5, and 1 µg/mL (-S9) and 4.8, 9.5, 19, and 30 µg/mL (+S9) were
evaluated for chromosomal aberrations.  No statistically-significant
increases in the number of aberrant cells or chromosomal aberrations
were reported at any dose level compared to the concurrent
solvent/negative control.  The percentage mean number of aberrant cells
with gaps (excluding and including type) was P>0.05 comparable to the
solvent and untreated controls for all dose levels and conditions.  The
positive controls of mitomycin-C and cyclophosphamide displayed
significant increases in the percentage of aberrations, hence eliciting
a clear positive response.  There was no evidence of chromosome
aberration induced over the background.

This study is classified as Acceptable/Guideline because it satisfies
the guideline requirement (OPPTS 870.5375; OECD 473) for in vitro
cytogenetic mutagenicity data.

	In Vitro Mammalian Chromosome Aberration Test

In an in vitro cytogenetic assay (MRID 43740801), Chinese hamster lung
fibroblasts were exposed to triclosan (99-100%) nonactivated doses of 1
µg/ml (7-hour cell harvest), 0.1-3 µg/ml (18-hour harvest), or 3
µg/ml (28-hour harvest) and S9-activated concentrations of 3 µg/ml (7-
and 28-hour cell harvests) or 0.1-3 µg/ml (18-hour harvest).  The S9
fraction was derived from Aroclor 1254 induced Wistar male rat livers
and triclosan was delivered to the test system in ethanol.

No mitotic cells were recovered at any harvest time from cultures
treated with ≥6 µg/ml -S9 or ≥ 10 µg/ml +S9.  Findings with the
positive controls confirmed the sensitivity of test system to detect
clastogenesis.  However, nonactivated triclosan at 1 and 3 µg/ml
(18-hour harvest) induced a dose-related increase in the yield of cells
with abnormal chromosome morphology.  The response was significant
(p≤0.001) at the higher concentration.  A significant increase (p≤
0.001) was also seen at 3 µg/ml (28-hour harvest).  The most frequently
observed type of chromosome damage was exchange figures.  In the
presence of S9 activation, nonsignificant but concentration dependent
increases in cells bearing exchange figures were also seen at 1 and 3
µg/ml (18-hour harvest).  The data are, therefore, sufficient to
conclude that triclosan is active in this test system.

This study is classified as Acceptable/Guideline and satisfies the
guideline requirements for an in vitro mammalian cell cytogenetic assay
(§84-2).

870.5385	Mammalian Bone Marrow Chromosomal Aberration Test

In an in vivo bone marrow cytogenetic assay (MRID 43740802), groups of
six male and six female Wistar rats received a single oral gavage
administration of 4000 mg/kg triclosan (99-100%).  The test material was
delivered to the animals as suspensions prepared in 1%
carboxymethyl-cellulose.  Animals were sacrificed 6, 24, and 48 hours
following compound administration and bone marrow cells from ten animals
per group (5 males and 5 females) were harvested and examined for the
incidence of structural chromosome aberrations.

No signs of overt toxicity or cytotoxic effects on the target organ were
seen in any treatment groups.  The positive control induced the expected
high yield of cells with structural chromosome aberrations.  There was
also no indication of a clastogenic effect at any sacrifice time.

The study is classified as Acceptable/Guideline and satisfies the
requirements for FIFRA Test Guideline §84-2 for in vivo cytogenetic
mutagenicity data.

Unscheduled DNA Synthesis in Mammalian Cells in Culture

In an in vitro DNA synthesis assay (MRID 47276602), rat hepatocytes were
exposed to triclosan (batch/lot#: CC # 14663-09) dissolved in DMSO.
Hepatocytes were isolated from the liver of two male Fischer 344 rats by
the two-step in-situ perfusion.  Concentrations of 0, 0.05, 0.1, 0.25,
1.0, 2.5, 5.0, 10, 25.0, 50.0, 100.0, or 250 µg/mL were tested for
18-20 hours.  Cells were autoradiographed, and unscheduled DNA synthesis
was evidenced by a net increase in black silver grain counts using an
Artek 880 automated colony counter with microscope and connected to an
Apple II computer for data analysis.  The difference between the
cytoplasmic grain count and the corrected grain count was calculated and
the net nuclear grains (NNG) and the percentage of hepatocytes in repair
were calculated.

Triclosan was tested up to the toxicity limit of 2.5 µg/mL based on the
preliminary toxicity test using rat hepatocytes that were treated at
dose concentrations of 10.0, 25.0, 50.0, 100.0, 250.0, and 500.0 mg/mL.
Precipitation was observed ≥50 mg/mL, and turbidity was noted at 25
mg/mL.  Hence 25 mg/mL was selected as the highest dose concentration
for the UDS assay.

In the UDS assay, triplicate cultures were exposed to the test article,
untreated control, solvent control or a positive control (2AAF).
Toxicity was observed at ≥5 ug/mL in the form of low grain count.
Hence dose concentrations of 0.25, 0.5, 1.0, and 2.5 µg/mL were
evaluated for UDS assay.  No significant increases in mean NNG counts
were reported at any dose levels and the percent of cells in repair
ranged from 0-6%, comparable to the solvent and untreated controls.  The
positive control yielded 88.7% of cell in repair and a mean NNG count of
21.2, hence eliciting a clear positive response.  There was no evidence
of induction of unscheduled DNA synthesis in rat primary hepatocytes
over the background.

This study is classified as Acceptable-Guideline and satisfies the
guideline requirement (OPPTS 870.5550) for an in vitro UDS assay.

4.8	Neurotoxicity

There is one older 14-day neurotoxicity study for triclosan (HED
document # 001968).

A 14-day neurotoxicity study was conducted in rats with triclosan at
doses of 0, 100, 300, 1000, and 2000 mg/kg/day. Slight inhibition of
movement, decreased muscular tone, polydypsia and polyuria were observed
at 300 mg/kg/day, with more pronounced signs at 1000 mg/kg/day. No brain
weight changes or histopathology were observed at any dose level tested.
 No peripheral nerve changes were observed at any dose level tested.

4.9	Metabolism and Pharmacokinetics

Adequacy of database for Metabolism and Pharmacokinetics:  Data are
available on metabolism and pharmacokinetic for triclosan that include
absorption, distribution, metabolism, and elimination studies in
hamsters (MRID 45307501, 45307502), mice (MRID 45307503), mouse, rat,
rabbit, and dog (  SEQ CHAPTER \h \r 1 MRID 149464),  mouse, rat, dog,
and baboon (  SEQ CHAPTER \h \r 1 MRID 68161), rat (MRID 47261405), and
dogs and baboons (MRID 79590).   A dermal absorption study in rabbits
(MRID 34335) is also available.

General Metabolism (Hamster)

In a metabolism study (MRID 45307501 and MRID 45307502) triclosan [99%
a.i., EN275927.26 (FAT 80’023/R)], and 14C-triclosan
[5-chloro-2-[2,4-dichlor-[14C(U)]-phenoxy]-phenol; 98% a.i., Batch 0542]
was administered to hamsters following the exposure scheme below.  Four
absorption and distribution studies (i.e., balance studies) were
performed, each using 5 hamsters/sex/dose at target dose levels of 2.0
or 200 mg/kg.  The first study administered a single oral dose via
gavage at calculated doses of 2.0 mg/kg in both males (group 1) and
females (group 3) or 213 and 203 mg/kg in males (group 2) and females
(group 4), respectively.  In the second study, animals were administered
2 or 200 mg/kg unlabeled triclosan via the diet for 13 days, then dosed
with a comparable single oral dose of labeled triclosan via gavage (2.0
mg/kg in both males [group 5] and females [group 7], respectively, or
200 and 203 mg/kg in males [group 6] and females [group 8],
respectively).  A single intravenous dose was utilized with both males
(group 9) and females (group 10) receiving 2.0 mg/kg 14C-triclosan.  A
13-day feeding regime of unlabeled triclosan was followed by a single
intravenous dose of 14C-triclosan at a level of 2.0 and 2.1 mg/kg in
males (group 11) and females (group 12), respectively.  Pharmacokinetic
studies were performed on 12 hamsters/sex/dose at dose levels of 2.0
mg/kg in both males (group 13) and females (group 15) or 199 and 201
mg/kg in males (group 14) and females (group 16), respectively.
Bioretention and metabolite distribution studies were performed on 16
hamster males/dose with doses of 2.0 or 201 mg/kg of 14C-triclosan via
gavage (groups 17 and 18, respectively) and on 16 hamster males/dose fed
unlabeled triclosan for 13 days before receiving gavage doses of 2.0 or
202 mg/kg of 14C-triclosan (groups 19 and 20, respectively).

After single or repeated oral doses of 2.0 or 200 mg/kg 14C-triclosan,
urine was the major route of elimination for triclosan radioactivity
(60-80% of the administered radioactivity).  Fecal elimination
represented from 12-35% of administered radioactivity across the oral
dose groups.  Compared to the low dose, administration of a single high
or repeated dose resulted in a shift toward urinary elimination and a
decrease in fecal elimination.

Intravenous administration at the low dose resulted in a similar pattern
of elimination in male and female hamsters as those receiving the low
oral dose.  Administration intravenously after feeding of unlabelled
triclosan for 13 days also did not appear to have any significant effect
on the general pattern of elimination.  Greater than 90% of the
administered radioactivity was excreted in 7 days post-dose after oral
or intravenous administration.  Organ/tissue radioactivity levels were
<1% of the dose received after 7 days, which also included the plasma.

After oral administration  of a single low or high dose (2.0 or 200
mg/kg), peak plasma and blood concentrations of triclosan-derived
radioactivity occurred at one hour post-dose for both the low and high
oral doses.  Concentration in plasma appeared higher than in whole blood
at each sampling time.  There did not appear to be any significant sex
differences in blood or plasma kinetics at the low or high dose.
However, Area Under the Curve (AUC) measurements indicated that
saturation may have been achieved at the high dose, as AUC was not
proportional to dose (in males, AUC increased 34x from the low to high
dose; in females, AUC increased 54 times from the low to high dose).

The major urinary metabolite detected after oral administration was the
glucuronide conjugate of triclosan (U7) at 32-37% of the administered
radioactivity at the low single and low repeated dose level in males;
55-57% of the administered RA in high single and high repeated dose
males; 9.5-43% of the administered RA in low single and low repeated
dose females; and 60% in high single and high repeated dose females.
The major fecal metabolite was parent triclosan in all oral dose groups
(1-8).  After intravenous administration, triclosan glucuronide was also
detected as the major urinary metabolite.  Notably, in female hamsters,
intravenous administration of triclosan resulted in a significant
increase in the percentage of the glucuronide metabolite found in urine
compared to oral dosing (41.4% vs 9.5%).  This is similar to what
occurred after the single oral dose was increased from 2.0 to 200 mg/kg
in female hamsters (60.2 vs. 9.5%).

Distribution patterns in the orally and intravenously dosed animals were
similar between the single- and repeat-dosed groups with the highest
residual activity found in the kidney, liver, lung, and plasma; however,
the levels were low.  No organ demonstrated accumulation of triclosan
with highest levels of triclosan equivalent in the plasma 7 days after
dosing.  The plasma, kidney, and liver eliminates triclosan equivalent
rapidly with 1/8 of the concentration eliminated by hours 48-56.

Tissue metabolite analysis showed that the glucuronide and sulfate
conjugates of triclosan were the major metabolites detected at the low
and high oral single doses.  The percentage of the sulfate conjugate
increased significantly in the plasma and liver from the low to high
dose while the percentage of the glucuronide conjugate decreased only in
liver.  This pattern was also observed in the single orally dosed female
hamsters.  Four non-parent conjugates (M5, M6, M8, and M9) were also
identified in tissues.  All conjugates were acid labile and resulted in
the parent compound, M2, or M3.  The two dose levels suggest reduced
phase 1 metabolism of triclosan in hamsters at the higher dose level of
200 mg/kg as compared to the low dose of 2.0 mg/kg 14C-triclosan.

After 1-2 hours (Cmax), males dosed single or repeatedly parent
glucuronide and sulfate were found in the plasma without detection of
the free parent compound.  The kidneys at Cmax had parent glucuronide
and free parent with little sulfate conjugate; the liver at Cmax had
free parent and sulfate conjugate with little glucuronide levels.  The
major urinary metabolite was the parent glucuronide conjugate with
little free parent compound detected; the major feces metabolite was the
free parent compound with a little parent glucuronide conjugate; neither
urine or feces had detectable levels of the parent sulfate conjugate.
Comparing the low- to the high-dose groups, the liver and kidney levels
of free parent compound were similar between the dose groups.  The
percentage of parent conjugates increased in the plasma, liver, and
kidneys after 200 mg/kg/day compared to the 2 mg/kg/day.  The parent
sulfate was the most pronounced in the plasma with levels of 7-9% in the
low-dose group and 28-59% in the high-dose group and liver with levels
of 8-16% in the low-dose group and 36-38% in the high-dose group.
Parent glucuronide increased after feeding for 13 days in the plasma
from 24 to 56% and kidneys from 48 to 60% in the low- to high-dose
groups, indicating the hamsters have an increased conjugating capacity
with the higher-dose group (200 mg/kg).  Triclosan sulfate conjugate
concentrations in the liver were enhanced by 250-600 times when
comparing the high- to the low-dose groups.

The metabolite data suggest that the sulfate conjugate produced in the
liver is transported to the kidney where it undergoes hydrolysis and
re-conjugation with glucuronic acid and then is eliminated in urine.
However, sulfation is usually predominant at lower doses while
glucuronidation normally increases with increasing dose.

This metabolism study in the hamster is classified Acceptable/Guideline
and satisfies the guideline requirement for a metabolism study [OPPTS
870.7485, OECD 417] in hamsters.

870.7485	General Metabolism (Mouse)

In an absorption, distribution, metabolism, and elimination study (MRID
45307503), 14C-triclosan (98% pure) was administered to mice in various
separate experiments.  These separate experiments were as follows:

Balance and distribution studies, in which male and female mice were
administered either (1) a single oral low (2 mg/kg) or high (200 mg/kg)
dose (groups 1-4) of 14C-triclosan or (2) a repeated (13X) food supply
of non-labeled test article followed by a single oral administration of
14C-triclosan at the low or high dose (groups 5-8).

Balance and distribution studies, in which male and female mice were
intravenously administered a low dose (2 mg/kg) of 14C-triclosan with
and without repeated (13X) food supply of non-labeled test article
(groups 9-12).

Blood/plasma level studies, in which male and female mice were
administered a single oral low (2 mg/kg) or high (200 mg/kg) dose of
14C-triclosan (groups 13-16).

Bioretention studies from Cmax to 1/8Cmax, in which male mice received a
single oral administration of 14C-triclosan at either the low (2 mg/kg)
or high (200 mg/kg) dose with or without repeated (13X) food supply of
non-labeled test article (groups 17-20).

Metabolite characterization was determined by using mice excreta from
balance study groups (1-12).  Plasma from the bioretention study groups
(17-20) and liver and kidney pools from the bioretention study groups
(17-20) were analyzed for metabolite characterization.

The test material was rapidly absorbed following oral administration,
and was eliminated primarily through the feces, via biliary excretion.
Urinary excretion was secondary to that in the gastrointestinal tract.
This excretory pattern was consistent following I.V. administration as
well.  Most organs/tissues showed comparable levels of radioactivity
after oral and intravenous administration.  Only organs with the highest
blood supply (lung, liver, and spleen) showed higher levels of
radioactivity (~3-15 times) following I.V. administration.  Bioretention
studies indicate that values from Cmax to 1/8Cmax in the liver were
higher than those in plasma following repeated administration at both
dose levels, indicating that the liver is the target organ.  Primary
excreted compounds in the urine following single oral exposures included
the unmetabolized parent compound and two parent conjugates (parent
sulfate and parent glucuronide); fecal excretion was primarily that of
the free parent compound, as small amounts of glucuronide were detected,
and no sulfate was detected.  It was speculated that intestinal
microflora were responsible for deconjugating the parent glucuronide
prior to elimination in the feces. Interestingly, parent glucuronide was
detected in the fecal matter of females, but not males, following I.V.
dosing.

Because parent sulfate was detected in plasma, liver, and kidney, and
the parent glucuronide was absent in the liver and kidney, but present
in the plasma and urine, it is assumed that the glucuronide, once
formed, is rapidly eliminated from both the liver and kidney. It was
also suggested that the kidney would reconjugate any sulfate conjugate
that was transported there from the liver, to the glucuronide, prior to
excretion in the urine.  This seems to happen predominantly in females
following a low single oral dose, in females at low and high single oral
doses, and in both sexes following repeated high oral doses.

Additionally, 4 conjugated metabolites (M5, M6, M8, and M9) were
detected in kidney, plasma, and liver extracts in minor amounts (<5.3%).
The compounds were generally present in plasma only after a single, low
oral dose, increasing slightly in kidney (and potentially liver—the
values were illegible in the study report) at the high single oral dose.
M6 and M9 were not present in these tissues after repeated dosing at
either dose level.  In general, as the dose increased, the urinary
excretion shifted to free parent and parent glucuronide.

Parent and parent conjugates were rapidly eliminated and/or metabolized
with half-lives ranging from 1-13 hours in the plasma, liver, and
kidney.  Cmax values occurred at 4-11 hours.  Mice with enlarged livers
(i.e., group 20) exhibited parent and parent sulfate half-lives of 13
and 14 hours, respectively.  Non-parent conjugates M5, M6, M8, and M9
showed similar half-lives as parent and parent compound (<4-13 hours);
however, Cmax values were greater, occurring at 12-32 hours.

This absorption, distribution, metabolism and elimination study is
Acceptable/Guideline and satisfies the guideline requirement for a
metabolism study OPPTS 870.7485.

  SEQ CHAPTER \h \r 1 Metabolism and Pharmacokinetics - Multiple Species

CITATION:		Stierlin, H.  (1972).  GP 41 353: Study of pharmacokinetics
and metabolism in mouse, rat, rabbit, and dog.  Pharma Research,
Pharmacological Chemistry. CIBA-GEIGY, Ltd.  Basle. Project No. GP 41
353. Report No. 33/1972.   MRID 149464. December 1, 1972. Unpublished.

EXECUTIVE SUMMARY: In a metabolism study (MRID 149464), rats, mice,
rabbits, and dogs were administered [14C]-GP 41 353 or [3H]-GP 41 353
(radiochemical purity 99%; chemical purity 99.5%; batch/lot nos. not
provided) intravenously, intraduodenally, or orally (gavage) at doses of
10 mg/kg (mice), 0.4 mg/kg (rats), 5 mg/kg (rats, rabbits, dogs), or 50
mg/kg (rabbits).  Radioactivity levels in the blood, tissues, and
excreta were measured for time intervals up to 168 hours.  Additionally,
biliary elimination was also analyzed in rats given a single
intraduodenal dose.

There was no indication of any toxic effects in the test animals.
Overall recovery of administered radioactivity in rats ranged from
99.67% to 104.53% at 2 to 8 hours after dosing.  Data were unavailable
in the study report to accurately determine radioactivity inventory for
the other species tested.

Absorption of the test material was reported by the study author as
70-80% as determined by comparisons of areas under-the-blood
concentration curve for oral and intravenous administrations.
Absorption in rats could also be estimated based upon biliary and
urinary elimination data where, over a 7.5 to 10-hour period, biliary
elimination accounted for 62.5% of a 5 mg/kg gavage dose and 67% of a 5
mg/kg intraduodenal dose and urinary excretion at 6 hours accounted for
76.60% of a 5 mg/kg gavage dose.  These data affirm that absorption
exceeded 70%.  Absence of biliary secretion data for the other test
species precludes assessment of absorption.  Time-course concentration
data revealed that peak blood levels occurred within 30 minutes in rats
following a single oral or intravenous dose of 5 mg/kg and at 2-4 hours
for dogs.

Tissue distribution patterns were similar among mice and rats, and
exhibited only slight quantitative variability for intravenous versus
oral dosing.  Following a single oral dose in rats, radioactivity in
tissues was low (generally <1 µg/g tissue) with the exception of blood
and the organs associated with excretory function (e.g., liver, gall
bladder, kidneys).  Following an intravenous dose to rats and mice,
tissue levels were also greatest in highly perfused organs or those
associated with excretory function.  Based upon data from rats, tissue
burdens declined appreciably over 24 hours with no indication of
accumulation/sequestration.

Excretion of GP 41 353 was examined in two strains of rats, rabbits, and
dogs.  Biliary elimination was also assessed in rats.  Urinary excretion
appeared to be a minor route of elimination in rats and dogs, accounting
for 3-17% of the administered oral dose in rats over a 168-hour period,
and 8.3-8.8% in dogs over a 120-hour period.  Urinary elimination in
dogs was somewhat greater following intravenous administration;
12.9-17.7% over 120 hours.  For rabbits, urinary excretion was a
significant route of elimination and accounted for 74.1% of a single 50
mg/kg oral dose and 60.4% of a single 5 mg/kg oral dose over a 72-hour
period.  The biliary secretion data in rats showed that most of the
fecal radioactivity could be attributed to biliary elimination products
rather than unabsorbed test material.  Biliary elimination was also
affirmed by data from the mouse showing very high concentrations of
radioactivity in the gall bladder at 5 minutes to two hours following a
10 mg/kg, i.v. dose.

Analysis of bile samples from the rats indicated that the test material
underwent Phase II biotransformation.  Treatment of samples with
β-glucuronic acid revealed that most of the biliary product was
glucuronide conjugates while some was unchanged parent compound.

The results of this multi-species study indicated that at least 70% of
an oral dose of GP 41 353 is absorbed from the gastrointestinal tract
and that biliary secretion and subsequent fecal elimination is a major
excretory route in the rat and dog.  Urinary excretion appeared to be a
major route of elimination in the rabbit.  Tissue accumulation was
minimal and primarily associated with highly perfused tissues and organs
with excretory function.  Metabolite data in rats revealed glucuronide
conjugates and unchanged parent compound as biliary metabolites.

This metabolism study in rats is Acceptable/Guideline and satisfies the
requirements for a Metabolism and Pharmacokinetics study [OPPTS 870.7485
(85-1)].  Although information regarding dose confirmation, homogeneity,
and stability were lacking, the consistency of results across several
species by several dose routes would seem to preclude potential problems
pertaining to these parameters.  The data are also consistent with
findings from other reports in humans (MRID 68163, 68162), and other
laboratory species (MRID 68161, 79590).

  SEQ CHAPTER \h \r 1 Metabolism and Pharmacokinetics - Rats, Dogs, and
Baboons

CITATION:		Stierlin, H., K. Schmid, A. Sutter  (1977).  GP 41 353:
Comparison of pharmacokinetic and metabolic parameters of triclosan and
HCP in the mouse, rat, beagle, dog and baboon. Part A.  Survey of
findings. Part B. Detailed account of the study.  Pharma Research,
Pharmacological Chemistry. CIBA-GEIGY, Ltd.  Basle. Report No. B 1/1977.
 MRID 68161. January 27, 1977. Unpublished

EXECUTIVE SUMMARY:  In a metabolism study (MRID 68161), 14C-triclosan
(>98% radiochemical purity, lot/batch no. not specified) was
administered to male rats by gavage at doses of 5 or 30 mg/kg (single or
14-day repeated).  In addition, two male beagle dogs and two male
baboons received single 5 mg/kg doses in gelatine capsules and 10 male
mice were given a single 10 mg/kg intravenous dose.  Blood levels were
monitored in mice up to 2 hours, in rats at 24 hours postdose, in the
dogs up to 72 hours and in monkeys up to 168 hours postdose.  Urinary
and fecal excretion was monitored in monkeys and dogs for 6-10 days.
Tissue distribution was assessed in mice and rats.

There were no test article-related toxic effects reported.  Recovery of
administered radioactivity was marginal; 86.9% and 74.1% for each of two
dogs, and 83.1% and 80.5% for each of two monkeys. Administered
radioactivity was widely distributed among tissues/organs in mice after
an intravenous injection (10 mg/kg) and in rats following a single oral
administration or the last dose of a 14-day repeated oral administration
(5 or 30 mg/kg).  Based on the radioactivity distribution data, there
was no evidence indicating sequestration of the test material or its
metabolites in either mice or rats.

Time-course analysis of blood/plasma radioactivity in rats revealed that
peak concentrations were attained within three hours after a single oral
dose of 5 mg/kg and that the concentrations declined approximately
five-fold within 24 hours.  For dogs, somewhat greater blood tmax values
were observed but were variable (2- 8 hours) for the two dogs tested.
Generally, the test article did not exhibit especially rapid
partitioning into or clearance from the blood for either species.
Time-course analysis of tissues from mice given a single intravenous
dose (10 mg/kg) of 14C- triclosan showed that the highest concentrations
of radioactivity were initially associated with highly perfused
organs/tissues.  These levels significantly declined within 30 minutes
but the decline was somewhat less rapid for organs /tissues associated
with metabolism and elimination and notably increased for the gall
bladder.

Both urinary and fecal elimination were identified as major routes of
excretion.  The relative contribution of each to overall elimination of
administered radioactivity exhibited species variability.  For monkeys,
urinary excretion accounted for 56.69% of the administered dose and
fecal elimination accounted for 25.15%.  For dogs, urinary and fecal
elimination accounted for 12.16% and 68.30%, respectively, of the
administered dose.  In both species, most of the urinary and fecal
excretion occurred within 48 hours.

Definitive characterization of metabolites was not performed.
Preliminary investigations using acid and enzyme hydrolysis, indicated
that very little (<1%) of the blood/plasma radioactivity was associated
with unchanged triclosan.   In the brains of rats, however, 35-50% of
the radioactivity was attributed to parent compound.

Overall, this study demonstrated that 14C-triclosan is readily absorbed
from the gastrointestinal tract, has a potential wide volume of
distribution, and can cross the blood-brain barrier. Blood absorption
and clearance is not especially rapid, but the compound does not appear
to undergo sequestration in the species tested.  Most of the circulating
radioactivity was attributed to metabolism products (probably
conjugation products based upon preliminary experiments using acid and
enzymatic hydrolysis).  Both the urine and the feces are significant
routes of excretion with the relative importance appearing to be species
dependent.

This metabolism study in multiple species, which predates GLP
guidelines, is Unacceptable/Nonguideline and does not satisfy the
requirements for a Metabolism and Pharmacokinetics study [OPPTS 870.7485
(85-1)].  The study was properly conducted and provided data regarding
excretion and plasma/blood kinetics of triclosan in monkeys, dogs, and
rats, and tissue distribution data in rats and mice. Dose confirmation,
homogeneity, and stability were not provided, and overall recoveries of
administered radioactivity were marginal. Additionally, a quality
assurance statement was not provided. The results of experiments
assessing excretion and blood time-course, and tissue distribution,
however, do affirm findings of companion studies (MRID 149464, 79590) in
animal species and studies (MRID 68162, 68163) in human volunteers.

Metabolism and Pharmacokinetics-Rat

CITATION:	van Dijk, A. (1996) Absorption, distribution and execution
(ADE) after single oral and repeated oral administration. RCC
Umwelchemie AG (Itingen, Switzerland). RCC Project 341998, July 19,
1996. MRID 47261405. Unpublished.

EXECUTIVE SUMMARY: In a metabolism study (MRID 47261405), 14C-labeled
triclosan (99% a.i., Batch # B-71.32A) was administered to two groups of
10 Wistar rats/dose and four groups of 36 Wistar rats/dose. The levels
of radioactivity appearing in the feces, urine, plasma, liver, and
kidney were studied. Groups 1 and 2 were administered a single dose of
2.3 mg/kg or 211 mg/kg of radiolabeled triclosan, respectively, by
gavage and sacrificed at 96 hours. Groups 3 and 4 were administered a
single dose of 2.2 mg/kg or 199 mg/kg of radiolabeled triclosan,
respectively, by gavage and groups of 4 rats were sacrificed at 0.5, 1,
2, 4, 8, 24, 48, 72, or 96 hours. Groups 5 and 6 were administered
thirteen doses of 2.0 mg/kg or 201 mg/kg of non-radiolabeled triclosan,
respectively, through food followed by a single gavage dose of
radiolabeled triclosan.

The radioactive dose was rapidly excreted and elimination was nearly
complete by 72 h. Fecal excretion predominated at both dose levels and
dosing regimes. At 96 hours after administration of the low dose (Group
1), 11.2% of the administered radioactivity was found in the urine,
81.2% in the feces, 2.4% in the cage wash, 0.1% in the intestinal tract,
<0.05% in the organs/tissues, 0.3% in the carcass, and 0.2% in the
plasma. At 96 hours after administration of the high dose (Group 2),
12.2% of the administered radioactivity was found in the urine, 82.2% in
the feces, 3.5% in the cage wash, 0.1% in the intestinal tract, <0.05%
in the organs/tissues, 0.2% in the carcass, and <0.05% in the plasma.
Further single oral dose studies (Groups 3 and 4) showed that at 48
hours after administration most of triclosan or its equivalent were
excreted with, 10.7% (low dose) and 11.8% (high dose) of the
administered dose excreted in the urine and 79.5% (low dose) and 80.2%
(high dose) excreted in the feces.

At both dose levels in single and repeated oral administration, the
maximum level of radioactivity in the plasma was reached 1 to 4 hours
post administration. Half lives for triclosan and its equivalents in the
plasma ranged from 10.0-12.6 hours, single (12.6 or 10 hours, low- or
high-dose, respectively) and repeated (11.7 or 10.7 hours, low- or
high-dose, respectively) dose groups were comparable. At both dose
levels in single and repeated oral administration, the maximum level of
radioactivity in the liver was always reached at 1 to 4 hours post
administration. Half lives for triclosan and its equivalents in the
liver for all groups ranged from 9.4-11.0 hours (single dose: 11.0 or
9.8 hours, low- or high-dose, respectively; repeated dose: 9.4 or 10.0
hours, low- or high-dose, respectively). At both dose levels in single
and repeated oral administration, the maximum level of radioactivity in
the kidneys was always reached at 1 to 4 hours post administration. Half
lives for reactivity metabolism in the kidney for all groups ranged from
10.8-14.1 hours (single dose: 13.0 or 12.0 hours, low- or high-dose,
respectively; repeated dose: 14.1 or 10.8 hours, low- or high-dose,
respectively).

The majority of the metabolites found in the urine and liver were in the
form of conjugated and/or non-parent compound. Analysis of the
metabolites found in the urine revealed that 3.7% (single low dose) and
1.7% (single high dose) of the radioactivity administered was in the
form of free parent compound while 9.3% (single low dose) and 10.3%
(single high dose) was in the form of conjugated parent and/or
non-parent compound. Analysis of the metabolites found in the liver
revealed that 91.1% (single low dose) and 75.6% (single high dose) of
the metabolites found in the livers were in the form of free parent
compound while 5.2% (single low dose) and 21.7% (single high dose) were
in the form of conjugated parent or non/parent compound. Repeated dose
groups showed similar breakdowns.

Repeated doses of triclosan did not cause significant changes to the
pharmacokinetics of triclosan and/or its metabolites in the rat, as
compared to single dose administration. In addition, the results
indicate that triclosan exhibits saturation of absorption and a higher
conjugating/metabolizing capacity at approximately 200 mg/kg, as
compared to approximately 2 mg/kg.

This Tier I metabolism study in rats is classified ACCEPTABLE, GUIDELINE
and satisfies the guideline requirement for a metabolism study [OPPTS
870.7485, OECD 417] in rats. The study does not satisfy the requirements
for a Tier II study; an intravenous dose was not administered, nor was a
biliary excretion study conducted.

  SEQ CHAPTER \h \r 1 Metabolism and Pharmacokinetics - Dogs and Baboons

CITATION:		Stierlin, H.  (1976).  GP 41 353: Isolation and
identification of the main metabolites in the blood of the beagle and
baboon and in the urine of the latter following oral administration of
14C-labelled triclosan.  Pharma Research, Pharmacological Chemistry.
CIBA-GEIGY, Ltd.  Basle. Report No. B 14/1976.   MRID 79590. March 16,
1976. Unpublished.

EXECUTIVE SUMMARY: In a metabolism study (MRID 79590), two baboons and
two dogs were administered a single oral dose (5 mg/kg) of [14C]-GP 41
353 (radiochemical purity 99%; chemical purity 99.5%; batch/lot nos. not
provided).  Blood samples were taken at 3 hours postdosing from one dog
and at 8 and 12 hours postdosing from one baboon.  Another dog and
baboon were killed at 6 and 7 hours postdosing to obtain sufficient
blood samples for metabolite characterization.  Urine samples were
collected from the dog and baboon that were not sacrificed for blood
sample acquisition.

There were no adverse effects associated with the test article.  At 3
hours postdosing, total radioactivity was 6.08 µg eq./mL blood in the
dog.  For the baboon total radioactivity was 1.24 and 1.03  µg eq./mL
blood, respectively at 8 and 12 hours postdosing.  For the dog and
baboon terminated for sample acquisition, total blood radioactivity was
4.86 µg eq./mL blood for the dog at 6 hours and 2.29 µg eq./mL blood
for the baboon at ~7 hours postdosing. Urinary elimination accounted for
32% of the administered dose to the surviving baboon by 72 hours.
Urinary excretion by the dog was minimal and accounted
f牯錠湯祬愠映睥瀠牥散瑮漠桴摡業楮瑳牥摥搠獯
鑥മ

 to endogenous urinary β-glucuronidase.  No analyses were conducted for
the dog due to the minimal urinary elimination of radioactivity.

In summary the results of this study showed that the major blood
metabolites in the baboon and beagle dog were sulfate and glucuronide
conjugation products.  The major urinary metabolite in the baboon was a
glucuronide conjugate but analysis of urinary metabolites in the dog was
precluded by negligible urinary products as determined by radioactivity.

This metabolism study in rats is Acceptable/Nonguideline and does not
satisfy the requirements for a Metabolism and Pharmacokinetics study
[OPPTS 870.7485 (85-1)].  Although not designed as an 85-1 Guideline
study (no tissue distribution data), it was properly conducted and
provided data regarding the characterization of blood and urinary
metabolites in the baboon and blood metabolites in the dog following a
single oral dose.  These data complement the findings from other studies
in humans (MRIDs 68162, 68163) and laboratory species (MRID 149464,
68161).

870.7600	Dermal Absorption

An older in vivo rabbit dermal absorption study is available (MRID
34335). In this study, up to 48% of an applied dermal dose of 0.89 mg
triclosan (3% a.i.) was absorbed. The in vivo rabbit dermal absorption
data are in agreement with the estimate of dermal absorption of 50%
derived from comparison of the LOAEL’s from a rat 90-day dermal
toxicity study (MRID 43328001) and a rat 2-generation reproduction study
(MRID 401623701). This estimate was based on a reduced mean body weight
observed in the reproduction study at 150 mg/kg/day, and occult blood in
urine observed at 80 mg/kg/day in the dermal study.

 Additional dermal absorption data on triclosan  have been submitted and
reviewed.  In vitro dermal absorption studies using human skin
preparations and various formulations containing triclosan (MRIDs
47261408 through 47261411) showed dermal absorption values for triclosan
ranging from 11-20% in these formulations.  A paper published in 2000 by
Moss et al.  (Food and Chemical Toxicology, Volume 38, pages 361-370)
examined dermal absorption of triclosan both in vivo and in vitro using
rats as well as an in vitro human skin study. These data supported the
conclusion of dermal absorption of 21-23% in the rat studies, and showed
in vitro dermal absorption through human skin in vitro of 6.3%.  Several
human in vivo dermal absorption studies were cited in the 2007 CANTOX
report on the Toxicological Evaluation of Triclosan that also suggest a
lower value for dermal absorption than 50%, but none of these studies
have been reviewed by the Agency’s Human Studies Review Board for
scientific and ethical conduct and are thus not cited in this risk
assessment.    Taken together, the available data  on dermal absorption
suggest a lower value, around 20% for rat skin and possibly lower for
human skin.  Additional verification is needed.

	4.10	Special Studies

Three liver biochemical induction studies (MRID 44389702, 44389703, and
44389706) were performed with triclosan, in addition to two liver cell
proliferation studies (MRID 44389701, Eldridge, 1995).  Although these
studies do not fulfill a guideline requirement, they provide additional
data that may be used to characterize the toxicity of triclosan,
specifically the potential mode(s) of action in regard to the formation
of the liver tumors observed in the oral carcinogenicity study in mice.

Liver Biochemical Induction (Mouse)

In this study (MRID 44389702), triclosan technical was administered to
CD-1 mice in a pelleted rodent diet for a 14-day period at doses of 0,
25, 75, 350, and 900 mg/kg/day and 0, 25, 350, or 900 mg/kg/day for
males and females, respectively. An additional group of male mice
received either 0 or 900 mg/kg/day for 14 days followed by a 4-week
recovery period. Decreased absolute body weight and body weight gain was
observed in male mice receiving 900 mg/kg/day or greater following 14
days of test article administration and weight gain was decreased by 75%
vs. control and absolute group mean body weight was decreased by 16% vs.
control. No effect on body weight or body weight gain was observed in
female mice.

Liver weight in male mice receiving 75 mg/kg/day and above and in female
mice at 350 and 900 mg/kg/day was significantly increased in comparison
to the respective controls. Liver weight effects were reversible in
recovery groups.

Significant increases in the activity of all biochemical parameters were
observed at the top dose in male mice and significant increases were
observed for microsomal protein (25% increase), lauric acid
hydroxylation, and EROD (82% increase) and PROD (431% increase)
activities at the lowest dose tested in males. These effects were
reversible in the recovery group.

In female mice, there were no significant increases in liver biochemical
parameters at the low dose with the exception of PROD activity (268%
over control). Increase in liver biochemical parameters were observed
for female mice at the mid dose and above.

endant. Formation of the 2β-, 6β-, 15β-, and 16β- metabolites were
increased 11.6 fold, 10.9 fold, 5.3 fold, and 7.6 fold, respectively, at
the high dose level. Hydroxylation of testosterone at the 7 position is
associated with CYP2A1 and isoenzymes of the peroxisome proliferator
inducible P-450 family CYP4A in the rat.

In male mice, electron microscopy revealed marginal to moderate
proliferation of endoplasmic reticulum. Rough endoplasmic reticulum
membranes were distinctly reduced and disorganized at 54.7 mg/kg/day and
above, leading to a mixture of rough and smooth ER membranes.
Peroxisomes showed a moderate (at 54.7 mg/kg/day) to striking (1346.8
mg/kg/day) proliferation and were increased in size. At 54.7 mg/kg/day
and above, lipid vacuoles were encountered in hepatocyte nuclei and, at
the top dose, nearly all nuclei contained numerous lipid vacuoles of
various sizes.

In female mice, the same morphological alterations were observed at the
top dose (1105.6 mg/kg/day) as were observed in male mice.

From these data it is apparent that administration of triclosan to the
mouse results in      significant hepatic effects. The biochemical
alterations observed appear to support the conclusion of a
barbituate-type induction with peroxisome proliferation effects.
Induction of certain liver enzyme activities as measured in this study
appear to occur at the lowest dose tested in male mice, including
significant increases in microsomal protein, lauric acid hydroxylation,
and EROD and PROD activities.

Liver Biochemical Induction (Rat)

In a non-guideline feeding study (MRID 44389703), the effect of
triclosan on selected biochemical liver parameters was examined at
concentrations of 0, 300, 1500, and 6000 ppm. Reversibility of effects
was assessed in a single group of five animals, who received 6000 ppm in
the diet for 14 days followed by a 28 day recovery period.

There were no clinical signs of toxicity reported. Food consumption was
reduced in all test groups with the exception of the 6000 ppm dose group
which had a reduced food intake on day 1 and then an almost 3-fold
increase over the control group. Group mean body weight was not
significantly changed except in the 6000 ppm group which was slightly
decreased (4-8%) over the first 8 days of the study. At the end of the
study, rats receiving 6000 ppm showed a significant increased absolute
and relative liver weight.

Significant effects were observed for several biochemical parameters in
the liver at the 6000 ppm dose level. Cytochrome P-450 content was
approximately doubled in the high dose group, while activity of
glutathione-S-transferase was increased by 65%. Other enzymes affected
at the 6000 ppm dose level included an increase in lauric acid
hydroxylation and an increase in PROD and EROD activity. In general,
those animals allowed to recover for 28 days following the 14-day
administration of test chemical showed no significant induction or
inhibition of enzyme activities.

Liver Biochemical Induction (Hamster)

In a liver biochemical induction study (MRID 44389706), triclosan
(purity 99.5%) was administered to 4 groups of young adult male and
female Syrian Hamsters (five/sex/group) in a pelleted standard hamster
diet (Nafag 924) at concentrations of 0, 700, 5000, and 15000ppm
[approximately 0, 49.9, 309.8, 799.0 mg/kg/day (males) and 46, 314.3,
958.8 mg/kg/day (females)] for 14 days. Separate recovery groups of five
males and five females received either 0 or 15000 ppm triclosan in the
diet for 14 days followed by a 28 day recovery period.

Significant treatment-related effects were observed in male and female
hamsters at the 5000 (309.8 mg/kg/day in males, 314.3 mg/kg/day in
females) and 15000 ppm (799 mg/kg/day in males, 958.8 mg/kg/day in
females) treatment levels.  At 5000 ppm triclosan, significant induction
of total hepatic microsomal cytochrome P-450 and activities of
ethoxyresorufin-o-deethylase (EROD) and pentoxyresorufin-o-depentylase
(PROD) was observed, as was an increase in Mab clo4 immunoreactive
protein in male hamster liver.  At 15000 ppm, the above effects were
also observed, and in addition, abnormal histopathology of the kidneys
in females (randomly distributed spots or white patches of white
pigmentation on the surface of the kidney) was observed after 14 days of
treatment.  Total activity towards testosterone was not affected by
triclosan feeding in the diet, but specific hydroxylation reactions were
affected.  Of note in males, formation of androstenedione was increased
in a dose-related manner, as was the formation of the 16-β metabolite.
A noticeable dose-response was observed only for androstenedione
formation, however.

In female hamsters, a dose-related increase in formation of both the
7-∝ and 15-∝ hydroxyl metabolites was  noted as a result of
triclosan treatment (formation of the 7-∝ metabolite: activities of
22.16, 35.74, 39.33, and 46.66 nmol/min/g at the 0,700, 5000, and 15000
ppm dose levels, respectively; formation of the 15-∝ metabolite: 8.61,
12.78, 18.29, and 29.87 nmol/min/g at 0, 700, 5000, and 15000 ppm
triclosan, respectively).  Androstenedione formation was also slightly
increased with dose of triclosan, with a doubling of activity at the
high dose (42.72 nmol/min/g) in relation to control activity (21.14
nmol/min/g).

∝-carbonitrile, the data suggest that triclosan acts as a peroxisome
proliferator, as observed in other work with rats and mice.  Hamsters,
however, appear less sensitive to triclosan treatment relative to rats
and mice.

Based on the results of this study, a Systemic NOAEL of 700 ppm can be
established, with a Systemic LOAEL of 5000 ppm, based on induction of
total cytochrome P-450, EROD, and PROD in male and female hamsters, and
induction of Mab clo4 immunoreactive protein (CYP4A peroxisome
proliferator inducible P-450) in male hamsters).

This study is classified as Acceptable/Non-guideline.  The study
provides important information on the mechanistic basis of triclosan
induced liver toxicity, and also provides information on the relative
sensitivity of the hamster to the hepatic effects of triclosan.

Liver Cell Proliferation

A cell proliferation study (MRID 44389701) was conducted as supplemental
to a previously reviewed subchronic feeding study in mice (MRID
43022605) to determine whether cell proliferation was induced in the
liver of male and female mice after 45 and 90 days’ dietary
administration of triclosan (% a.i. not stated). Liver tissue from mice
receiving 0, 25, 75, 200, 350, or 900 mg/kg/day was obtained--as
formalin-fixed wet tissue. Slides prepared from paraffin embedded tissue
were stained with hematoxylin and eosin for histopathology evaluation,
or stained for proliferating cell nuclear antigen (PCNA) using
immunohistochemical methods.

Positive staining for PCNA was identified by uniform dark red nuclear
staining of hepatocytes in the S-phase of the cell cycle. Homogeneity of
a cell proliferative response was evaluated by perusing liver sections
from each individual animal, and found to be similar among the lobes
examined from each animal. At least 1000 hepatocellular nuclei were
counted in six fields using a 20X objective and an eyepiece containing a
10 x 10 mm grid. Labeling index was calculated by dividing the number of
labeled hepatocyte nuclei by the total number of nuclei counted, and
expressing the result as a percentage.

Histopathological evaluations are presented below. Severity of lesions
was graded from 0 to 4 with 4 being most severe.

GROUP 	Lipofuscin 	Hepatocyte Hypertrophy 	Bile Retention	Necrosis	Lipid
Vacuolization 	Biliary Hyperplasia

01 MM 	0	0	0	0	0	0

02 MM	0	0	0	0	0	0

03 MM 	0.2	1	0.2	0.2	0.2	0.2

04 MM 	1	1	0.8	0.6	0.4	0.8

05 MM 	1	1	2	1.2	0.6	0.4

07 MM 	1.4	2	1.2	1.8	0.8	1

08 MM 	0	0	0	0	0	0

09 MM 	0	1.6	0	0	0	0

10 MM 	1.2	3.6	0.4	1.2	0.4	0.8

11 MM 	2.2	3.4	1.2	3	0.8	2

01 FM 	0.2	0	0	0	0	0

02 FM 	0	0	0	0	0	0

03 FM 	0	0.4	0	0	0	0

04 FM 	0.2	0.8	1	1.2	0.4	0.6

O5 FM 	1.4	1	1.4	0.6	0.4	1

07 FM 	1	1.4	1	1.6	0.8	1.6

08 FM	0	0	0	0	0	0

09 FM 	0	1	0	0	0	0

1O FM 	1	3	0.4	1	0	0.8

11 FM 	1.8	3.4	1	2.4	1.2	2.2

MM = male mice; FM = female mice. Dose groups: (male and female

90 day dose groups): 01, 0 mg/kg/day; 02; 25 mg/kg/day; 03, 75

mg/kg/day; 04, 200 mg/kg/day; 05, 350 mg/kg/day; 07, 900

mg/kg/day. Dose groups (male and female 45 day dose groups): 08,

0 mg/kg/day; 09, 25 mg/kg/day; 10, 350 mg/kg/day; 11, 900

mg/kg/day.

                        At the 45 day time point (dose groups 08 through
11), hepatocellular hypertrophy was the most consistent and prominent
observation. This lesion could be observed in male and female mice at 25
mg/kg/day (dose group 09), and the severity generally increased with
increasing dose. At the higher dose levels of 350 and 900 mg/kg/day,
necrosis of hepatocytes was observed, usually in large areas surrounded
by proliferating bile duct epithelial cells, fibroblasts, and/or Kupffer
cells. Along with the “fibrosis” were macrophages with yellow-brown
pigment compatible with lipofuscin or ceroid, breakdown products of
cellular organelles. Bile stasis between hepatocytes, in the canaliculi
of the liver lobules, was, according to the report, not a prominent
feature at 45 days, but was more prominent at 90 days. As noted above,
male mice were scored higher for severity of this lesion than female
mice at the 350 and 900 mg/kg/day dose levels at 90 days.

        At the 90 day time point, similar liver lesions were observed.
Hepatocellular hypertrophy was observed at 75 mg/kg/day and above in
male and female mice. With increasing dose, severity of this lesion also
increased. In addition to hypertrophy, necrosis of hepatocytes was
observed at 75 mg/kg/day and above in male mice and at 200 mg/kg/day and
above in female mice, again with a dose- related increase in severity.
The report stated that with increasing dose, necrosis became more
severe, involving groups of cells, and in the most severe cases,
involved all cells of individual lobules (panlobular).  Hepatocytes were
also swollen and many had cytoplasmic yellow-brown granules at the
periphery, near bile canaliculi. This lesions was classified as bile
stasis.

For many of the pathological changes in the liver, male and female mice
at the top dose showed higher severity scores at 45 days than 90 days,
with the possible exception of bile retention, which appeared to
increase with time.

Results of cell proliferation experiments are shown below:

GROUP	Mean Labeling Index	SEM	Fold Increase Over Control	GROUP	Mean
Labeling Index	SEM	Fold Increase Over Control

01 MM	0.035	0.016

01 FM	0.042	0.036

	02 MM	0.008	0.008	0.0	02 FM	0.046	0.015	1.1

03 MM	0.167	0.082	4.8	03 FM	0.065	0.009	1.5

04 MM	0.124	0.031	3.5	04 FM	0.140	0.050	3.3

05 MM	0.398	0.063	11.0	05 FM	0.256	0.218	6.1

07 MM	0.536	0.195	15.0	07 FM	0.300	0.060	7.1

08 MM	0.090	0.018

08 FM	0.058	0.029

	09 MM 	0.112	0.089	1.2	09 FM	0.064	0.021	1.1

10 MM	0.292	0.096	3.2	10 FM	0.242	0.072	4.2

11 MM	0.726	0.218	8.0	11 FM	0.380	0.080	6.6

MM = male mice; FM = female mice. Dose groups (male and female 90

day dose groups: 01, 0 mg/kg/day; 02, 25 mg/kg/day; 03, 75

mg/kg/day; 04, 200 mg/kg/day; 05, 350 mg/kg/day; 07, 900

mg/kg/day. Dose groups (male and female 45 day dose groups: 08, 0

mg/kg/day; 09, 25 mg/kg/day; 10, 350 mg/kg/day; 11, 900 mg/kg/day.

According to the report, cell proliferation was significantly increased
over control in male mouse liver at 200 mg/kg/day and higher, and the
increase was sustained from 45 to 90 days. The reviewer would agree with
the sustained increase, but it appears that cell proliferation (as
judged by labeling index and fold increase over control) is also
increased significantly at the 75 mg/kg/day dose level for male mice.
This result is consistent with the apparent differences in sensitivity
to the hepatic effects of triclosan between male and female mice, as
cell proliferation in female mice was not affected at 75 mg/kg/day, but
was increased at 200 mg/kg/day, consistent with the observed difference
in liver histopathology.

According to the report, the distribution of hepatocellular labeling was
panlobular in both sexes.

The results of this study support a mode of action consistent with
cellular regeneration as a result of hepatocellular cytotoxicity. The 25
mg/kg/day dose level was identified as the NOAEL for male mice in this
study, while the 75 mg/kg/day dose level was considered the NOAEL for
female mice by the authors. The reviewer agrees with this
interpretation, as liver responses in female mice, while evident at 75
mg/kg/day, were not significant enough to support a true effect level.
Males, by contrast, did show hepatic responses at the 75 mg/kg/day dose
which would support an effect level.

This study is classified as Acceptable/Non-guideline.

Liver Cell Proliferation

A cell proliferation study (Eldridge, 1995; MRID 44389701) was conducted
to examine whether cell proliferation was induced in male and female
mice which had been the subject of an earlier subchronic toxicity study
in mice exposed to dietary triclosan, % a.i. not stated at dose levels
of 0, 25, 75, 200, 350, 750, or 900 mg/kg/day for 7 or 13 weeks (MRID #
430026-05).