   

DATE:	August 29, 2008

MEMORANDUM

SUBJECT:	TRICLOSAN: - Revised Report of the Hazard Identification
Assessment Review Committee and Antimicrobial Division Toxicity Endpoint
Committee. 

FROM:	 Timothy F. McMahon, Ph.D., Senior Scientist

Antimicrobials Division (7510P)

 

 

PC Code: 054901

On March 10, 1998, the Health Effects Division's Hazard Identification
Assessment Review committee evaluated the toxicology data base of
triclosan and selected doses and endpoints for acute dietary as well as
occupational and residential exposure risk assessments, re-assessed the 
Reference Dose (RfD) established for chronic dietary risk assessment,
and addressed the sensitivity of infants and children as required by the
Food Quality Protection Act (FQPA) of 1996.  In 2006, the Antimicrobials
Division Toxicity Endpoint Committee updated the endpoint document with
respect to dermal endpoints based on the dermal irritation policy
developed within the Antimicrobials Division.  In 2008, new data on the
effect of triclosan on rat thyroid homeostasis was considered. 



I.  INTRODUCTION

On March 10,  1998, the Health Effects Division's Hazard Identification
Assessment Review Committee (HIARC) evaluated the toxicology data base
of Triclosan and selected doses and endpoints for acute dietary as well
as occupational and residential exposure risk assessments, re-assessed
the Reference Dose (RfD) established for chronic dietary risk
assessment, and addressed the sensitivity of infants and children from
exposure to Triclosan  as required by the Food Quality Protection Act
(FQPA) of 1996.  The application of the 10x factor for protection of
infants and children from exposure to Triclosan, as required by FQPA,
will be determined by the FQPA Safety Factor Committee (FQPA SFC). It is
noted that a previous FQPA assessment was performed for Triclosan in
1997, with a recommendation for no additional safety factor.   The
HIARC’s conclusions are presented below.

II.  HAZARD IDENTIFICATION

A. Acute Reference Dose (Acute RfD) 				

Study Selected:         Chronic Toxicity - Baboon  

MRID. No.		257773

Executive Summaries: In a chronic toxicity study, groups of 7
baboons/sex/dose received Irgasan DP300 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 remaining 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. 

Dose and Endpoint for Risk Assessment:   NOAEL = 30 mg/kg/day, based on
diarrhea observed  4-6 hours after dosing at the LOAEL of 100 mg/kg/day.


Comments about Study and Endpoint: none

           This risk assessment is  required.B. Chronic Dietary
[Reference Dose (RfD)] 

The RfD established in 1993 was re-assessed by this Committee and is
discussed below:

Study Selected:  	Chronic Toxicity - Baboon 	§83-1

MRID No.  		257773

Executive Summary: In a chronic toxicity study, groups of 7
baboons/sex/dose received Irgasan DP300 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 remaining 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. 

Dose/Endpoint for establishing the RfD:  NOAEL= 30 mg/kg/day based on
diarrhea 	observed at 100 mg/kg/day, and hematologic alterations at 300
mg/kg/day. 

Comments about Study and Endpoint: The HIARC concurred with the RfD
established.  The committee also noted supporting evidence for selection
of the RfD from the two-year rat chronic toxicity / carcinogenicity
study (MRID # 42027906) in which hepatocellular hypertrophy was observed
at a dose of 52 mg/kg/day, consistent with several other studies on
triclosan showing the liver to be a target organ of toxicity.  

Uncertainty Factor (UF):  100 (10x for inter-species extrapolation and
10x for intra-species variation) .

RfD =	 30 mg/kg/day (NOAEL) 	=	0.30 mg/kg/day 

100 (UF)

This risk assessment is required.	

C. Occupational/Residential Exposure

1. Dermal Absorption

An older rabbit dermal absorption study was available from the one-liner
database (HED document # 001958).  In this study, up to 48% of an
applied dermal dose of 0.89 mg triclosan was absorbed. In addition,
literature data available on dermal absorption in the mouse show dermal
absorption up to 70%.  These 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 # 40623701 ).  This estimate was
based on 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%.  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 to revise the currently selected dermal
absorption value. 

	Selected Dermal Absorption Factor:    50%

	2. Incidental  Oral (short-term [1-7 days]; intermediate-term [30 days-
6months]) 

	Study selected:	Chronic Toxicity- Baboon

	MRID: 		257773

	Executive Summary: see summary under chronic dietary assessment

Dose and Endpoint for Risk Assessment:   NOAEL = 30 mg/kg/day, based on
diarrhea observed  4-6 hours after dosing at the LOAEL of 100 mg/kg/day.
 

Comments about dose and endpoint:  the selection of the NOAEL value of
30 mg/kg/day for the short-term incidental oral is appropriate for the
time frame of this risk assessment based on the response observed in the
baboon study.  For the intermediate-term risk assessment, the NOAEL
value is consistent with NOAEL values from several oral
subchronictoxicity studies that show NOAEL values of 25 and 50
mg/kg/day.  A recently completed study (US EPA, 2008) showed that
triclosan decreased circulating T4 levels in rats administered triclosan
orally for 30 days at doses of 30 mg/kg and above. Benchmark dosing
analysis derived a BMD and BMDL for the 20% response level of 14.51
mg/kg and 7.23 respectively. Although more work needs to be done in this
area, the sensitivity of the rat to thyroid hormone disruption vs.
humans, and the similarity in pharmacokinetics to humans would result in
a decrease in the total uncertainty factor from 100 to 30, based on a
reduction in the interspecies factor from 10x to 3x. Thus, an equivalent
oral endpoint of 0.24 mg/kg would be derived, supporting the selection
of the current endpoint as health protective. 

3.  Short-Term Dermal - (1-7 days)

Study Selected:  	14-Day Dermal Toxicity study -mouse

MRID No.  		44389708

Executive Summary:      SEQ CHAPTER \h \r 1 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.

Signs of dermal toxicity in both sexes at 3.0 and 6.0 mg/animal/day
included erythema, edema, alopecia, fissuring, eschar, thickening and
discoloration.  At 1.5 mg/animal/day erythema, fissuring, eschar,
thickening, and discoloration were observed in males and erythema and
fissuring in females.   Dermal irritation observed in mice in the 0.3,
and 0.6 mg/animal/day treatment groups was comparable to that observed
in the controls.  Non-neoplastic skin lesions were observed at
application sites and included superficial ulceration and suppurative
inflammation, slight or minimal acanthosis and/or hyperkeratosis, and
mild, diffuse, generally subacute/chronic inflammation of the dermis. 
The lesions were dose-related and occurred primarily in the 1.5, 3.0,
and 6.0 mg/animal/day treatment groups.  Systemic responses were
observed as dose-dependent increases in plasma levels of the test
substance.  There were treatment-related increases in absolute and liver
to body and brain weights at 1.5, 3.0, and 6.0 mg/animal/day which
correlated with centrilobular hepatocellular hypertrophy at 3.0 and 6.0
mg/animal/day.  There were no significant differences between the
terminal body weights in the treated and control groups. For males, the
overall body weight gain was significantly decreased (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 LOEL 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 NOEL 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.

 

Dose and Endpoint for Risk Assessment:    NOAEL = 0.6 mg/animal based on
treatment-related dermal irritation at the treatment site and on
increased liver weights at 1.5 mg/animal.  

Comments about Study and Endpoint:    The 14-day study duration is
appropriate for selection of a short-term dermal endpoint (i.e., 1-7
days).  The NOAEL of 0.6 mg/animal was converted to a concentration of
100 µg/cm2 by using the surface area of the applied gauze (2 x 3 cm or
6 cm2) and converted according to the formula:

	0.6mg/animal  x 1000 µg/mg /  6 cm2   =  100 µg/cm2      

This risk assessment is required.

 

Intermediate-Term Dermal (30 days-6 months) and Long-term Dermal (> 6
months)

	Study Selected: 90 Day Dermal Toxicity Study in Rats 

Executive Summary: 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.     

Comments about Study/Endpoint:   The endpoint is appropriate for the
intermediate- and long-term assessment of systemic toxicity from dermal
exposure to triclosan.  A standard Margin of Exposure (100) is applied
to the intermediate-term dermal endpoint, and a Margin of Exposure of
300 is applied to any long-term dermal risk assessments.  The extra 3x
for the long-term assessment is applied to account for extrapolation
from a 90-day time point to a chronic time point. 

 

	5. Inhalation Exposure (Short-, intermediate-, long-term)

MRID No. 0087996

Executive Summary:    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, 3.21, 7.97, and
24.14 mg/kg/day for males, respectively, and 0, 4.51, 9.91, and 30.81 
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. 

  LINK Excel.Sheet.8 "D:\\H&PC Products\\TRICLOSAN\\Triclosan Safety
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.

.

Dose and Endpoint for Risk Assessment:  LOAEL=3.21 mg/L (males) based on
increased total leucocyte count and increased serum alkaline phosphatase
at 3.21 mg/L. 

Comments about Study and Endpoint: Since this is the only study
available, the LOAEL should be used for the Short, Intermediate and
Long-Term risk assessments.

This risk assessment is required.

D.  Margin of Exposure for Occupational/Residential Exposures:

A MOE of 10 is applied to the short-term dermal risk assessment (3x
interspecies extrapolation, 3x intraspecies variation).  For
intermediate- and long-term dermal risk assessments, an MOEs of 100 is
applied.     

An MOE of 1000 was applied to the inhalation endpoint. The inhalation
toxicity study lacked sufficient data with which to convert the animal
doses to human equivalent concentrations (HECs) in accordance with
Agency policy and a LOAEL value was selected as the endpoint for
inhalation risk assessment; therefore, the use of the LOAEL value from
the animal study and uncertainty in determination of what the HEC would
be warrants the MOE of 1000. 

E.  Recommendation for Aggregate Exposure Risk Assessments

 	Dietary and incidental oral exposures can be combined based on the use
of the same study and endpoint for these risk assessments.  Dermal and
inhalation exposures are considered separately based on the use of
different studies with different endpoints for these risk assessments. 

 

III.  CLASSIFICATION OF CARCINOGENIC POTENTIAL

1.  Combined Chronic Toxicity/Carcinogenicity Study- Rats			

MRID No: 42027906

Executive Summary:  In a chronic toxicity/oncogenicity feeding study
conducted in male and female Sprague-Dawley rats, FAT 80'023 (triclosan)
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).    No treatment-related effects on mortality,
clinical toxicity, ophthalmology, 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 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 168.0 mg/kg/day, and the incidence of hepatic necrosis was
increased 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 liver. 
Residual levels of triclosan were proportional to the dose administered.
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.

Discussion of Tumor Data:  There was no evidence of carcinogenicity.  

Adequacy of the Dose Levels Tested:  The dose levels were adequately
tested.   

2.  Carcinogenicity Study- Mice							

A second carcinogenicity study for Triclosan was conducted by the oral
route and was reviewed by the Food and Drug Administration . 

In a carcinogenicity bioassay in mice conducted by Colgate-Palmolive and
submitted to the Food and Drug Administration, 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. These data suggest that Triclosan can interfere with
liver function.  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 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.  

 This study was not reviewed by the Office of Pesticide Programs but is
acceptable for purposes of carcinogenicity assessment.   		

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

 

3.  Classification of Carcinogenic Potential:    

 On March 10, 1998, the Health Effects Division’s HIARC committee
examined the available carcinogenicity data for triclosan and was unable
to assign a classification to triclosan at that time since data for only
one species (rat) were submitted for evaluation of carcinogenicity. 
Since this determination, a chronic toxicity/carcinogenicity study in
the hamster (MRID 44874001) and a carcinogenicity study in the mouse
reviewed by the Food and Drug Administration were submitted and/or
obtained by the Agency.  The Agency was not able to obtain the
individual animal data records for the mouse carcinogenicity study but
was able to obtain the FDA’s review and Expert Panel reports on the
significance of the mouse study results.  On July 25, 2007, the Health
Effects Division’s Carcinogenicity Assessment Review Committee met to
discuss the additional data submitted as well as the biochemical studies
conducted with triclosan in support of a mode of action involving
peroxisome proliferation as a causative factor in the positive
tumorigenic results observed in the mouse carcinogenicity study. 

ά activation and toxicokinetic differences between the mouse and human.

 tc "V. 	SUMMARY" 

ά activation and toxicokinetics.  The quantification of risk is not
required.  

.  

IV.  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           
         (Broker, P.C., Gray, V.M., and Howell, A., 1988 and MRID
43740801), a mammalian bone marrow chromosomal aberration test (MRID
43740802), and an unscheduled DNA synthesis assay in mammalian cells in
culture (SanSebastian, J. et al., 1993).  

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.

In Vitro Mammalian Chromosome Aberration Test

In a mammalian cell cytogenetics, chromosome aberration assay (MRID not
assigned), 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 not assigned), 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.

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V.  Susceptibility CONSIDERATIONS

There are no existing tolerances or tolerance exemptions for Triclosan
under 40 CFR 180, and there are no food additive clearances from the
Food and Drug Administration.  However, as there are expected exposures
of infants and children to this chemical as well as potential exposures
from indirect food uses, the reproductive and developmental toxicity
database is discussed. 

1. Reproductive Toxicity Study Conclusions

In a 2-generation reproduction study (MRID # 40623701), triclosan was
administered to 25 rats/sex/dose at dietary levels of 300, 1000, and
3000 ppm (nominal doses of 15, 50, and 150 mg/kg/day). Significant body
weight reduction was observed in adult rats at the high dose during
weeks 0-12, gestation, and lactation. The Systemic NOAEL = 1000 ppm, and
the Systemic LOAEL = 3000 ppm, based on reduced mean body weight. Body
weights in high dose F1 pups were significantly lover on days 14 and 21
of lactation. F2 pups displayed significantly lower body weights at
birth which did not persist at day 4 of lactation or greater. Viability
index was decreased at the high dose in both generations of pups and the
weaning index was slightly lower in high dose F2 pups vs control. The
Reproductive NOAEL = 1000 ppm, and the Reproductive LOAEL = 3000 ppm,
based on reduced pup weights and equivocal reduced pup viability in both
generations.  

2. Pre-and/or Postnatal Toxicity

In a developmental toxicity study in rabbits, triclosan (100% a.i.) was
administered by gavage to pregnant female New Zealand White rabbits
(18/group) on gestation days 6-18 at dose levels of 15, 50, or 150
mg/kg/day. Rabbits were observed for signs of toxicity; body weight and
food consumption values were recorded. On day 30 of gestation, rabbits
were sacrificed and necropsied; gravid 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, visceral and skeletal
anomalies. They were then examined by the Staple's dissection procedure.
 Evidence of treatment-related toxicity at the high dose (150 mg/kg/day)
consisted of reduced body weight gain and food consumption over the
period of treatment.  The Maternal NOAEL = 150 mg/kg/day, based on
decreased body weight gain and food consumption during treatment.  The
Maternal NOAEL = 50 mg/kg/day.   No developmental toxicity was observed
under the conditions of this study.  The Developmental LOAEL = not
determined; the developmental NOAEL = 150 mg/kg/day. 

 

Triclosan was administered by gavage to pregnant female Wistar rats (30
rats/group, 60/group in control) on days 6-154 gestation at dose levels
of 30, 100, or 300 mg/kg/day.  At 300 mg/kg/day, maternal toxicity was
evident and consisted of transient diarrhea, decreased body weight gain
during treatment, and reduced food consumption and increased water
consumption from onset of treatment through gestation. Based on these
findings, the Maternal NOAEL = 100 mg/kg/day, and the Maternal LOAEL =
300 mg/kg/day. There was no evidence of pre- or post-natal developmental
toxicity at any dose level in this study.  The Developmental LOAEL = not
determined (> 300 mg/kg/day); the Developmental NOAEL > 300 mg/kg/day. 

Determination of Susceptibility

The data base is complete and there are no data gaps pertaining to
developmental or reproductive toxicity.  The data provided no indication
of increased sensitivity of rats or rabbits to in utero and post-natal
exposure to triclosan.  Two prenatal developmental toxicity studies, one
in rats and one in rabbits, failed to show evidence of developmental
toxicity in the absence of maternal toxicity.  In the two-generation
reproduction study in rats, effects in the offspring were observed only
at or above treatment levels which resulted in evidence of parental
toxicity. 

Additional Safety Factor(s): 

 A safety factor to account for susceptibility is not needed: 

(I)	The data provided no indication of increased susceptibility of rats
or rabbits to in utero and/or postnatal exposure to triclosan. 

(ii)	No evidence of developmental anomalies, including abnormalities in
the  development of the fetal nervous system, were observed in the pre-
and/or  post-natal studies. 

(iii)	There are no data gaps for evaluation of increased susceptibility
to infants and children. 

	3. Recommendation for a Developmental Neurotoxicity Study

The committee considered the available data on triclosan for evaluation
of neurotoxicity, including the 14-day neurotoxicity study in rats,
developmental and reproductive toxicity studies in rats and rabbits, and
subchronic and chronic data in rats and mice.  There was no evidence of
a neurotoxic effect of triclosan in any of these studies.  Thus, the
committee did not recommend a developmental neurotoxicity study for
triclosan. 

VI.  DATA GAPS

There are no current data gaps for hazard evaluation of triclosan. 

VII.   SUMMARY OF TOXICOLOGY ENDPOINT SELECTION

The doses and toxicological endpoints selected and Margins of Exposures
for various exposure scenarios are summarized below. 

Exposure

Scenario	Dose Used in Risk Assessment	Uncertainty factors for Risk
Assessment	Study and Toxicological Effects

Acute Dietary

(gen. pop.)	NOAEL = 30 mg/kg/day

aRfD = 0.03 mg/kg/day	 Interspecies = 10x

Intraspecies = 10x

DBSS* = 1x

 

UF = 100	Chronic Toxicity study in Baboons

MRID 257773

Acute Dietary

(females 13+)	Endpoint not identified in the database

Chronic Dietary

(all populations)	NOAEL = 30 mg/kg/day

 	 Interspecies = 10x

Intraspecies = 10x

DBSS* = 1x

 

UF = 100	Chronic Toxicity study in Baboons

MRID 257773

Short-Term/ Intermediate-Term Incidental Oral (1-30 days; 30 days- 6
months)	NOAEL = 30 mg/kg/day

 	Interspecies = 10x

Intraspecies = 10x

DBSS* = 1x

 

UF = 100	Chronic Toxicity study in Baboons

MRID 257773

Dermal (short-term)	NOAEL (dermal) = 0.6 mg/animal (100 µg/cm2)

 	Interspecies = 3x

Intraspecies = 3x

DBSS* =1x

MOE  = 10	14-day dermal toxicity study in the mouse MRID 44389708

NOAEL (dermal) of 0.6 mg/animal, based on dermal irritation at 1.5
mg/animal (erythema, fissuring, eschar, thickening, and discoloration).
At 3.0 mg/animal, increased liver weight and centrilobular hepatocyte
hypertrophy was observed in both sexes.   



Dermal (intermediate term)	NOAEL = 40 mg/kg

	 Interspecies = 10x

Intraspecies = 10x

DBSS* =1x

MOE  = 100

	90-day Dermal Toxicity in Rats

MRID 43328001

LOAEL = 80 mg/kg/day, based on increased incidence occult blood in the
urine.

Dermal (long-term)	NOAEL = 40 mg/kg

	  Interspecies = 10x

Intraspecies = 10x

DBSS* =3x (lack of chronic dermal study)

MOE  = 300

	90-day Dermal Toxicity in Rats

MRID 43328001

LOAEL = 80 mg/kg/day, based on increased incidence occult blood in the
urine.

Inhalation (all durations)	LOAEL (males)= 50 mg/m3

(4.22 ppm; 3.21 mg/kg/day) 

 	 MOE = 1000	21-Day Inhalation Toxicity study in the rat

MRID 0087996

LOAEL = 3.21 mg/kg/day,  based on decreased thrombocytes, total derum
protein, and increased alkaline phospatase in males. 

Cancer (oral)	 In accordance with the EPA Final Guidelines for
Carcinogen Risk Assessment (March 29, 2005), the HED CARC classified
triclosan as “Not Likely to be Carcinogenic to Humans”.  



 a =  0.6 mg/animal x 1000 µg/mg  /  6cm2  =  100 µg/cm2

UF = uncertainty factor, DBSS = database uncertainty [special
sensitivity] factor, NOAEL = no observed adverse effect level, LOAEL =
lowest observed adverse effect level, PAD = population adjusted dose (a
= acute, c = chronic) RfD = reference dose, MOE = margin of exposure,
LOC = level of concern, NA = Not Applicable

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