Tammy E. Stoker, Ph.D.

Endocrine Toxicology Branch

Toxicity Assessment Division

NHEERL, ORD, U.S. EPA

Reproductive Toxicology Facility

Durham, NC NHEERL

National Health and

Environmental Effects

Research Laboratory

Triclosan Case Study: 

Experimental Studies Examining 

Effects on Thyroid Hormone.
Purpose 

 What is the dose-response relationship for the effect on T4?

 Are certain life stages more sensitive to the effects of triclosan

on the thyroid axis? 

 Since there is concern for the human fetus when the pregnant 

mother has hypothyroxinemia, what is the dose-response 

relationship in the developing fetus as compared to the dam?
Overview

 Focused Studies in Rats to examine effects of triclosan on 

serum thyroxine (sensitivity across different life-stage) 

 4 Day Juvenile Female Study 

 Pubertal Studies

 Male Pubertal Study (31 day oral exposure)

 Female Pubertal Study (21 day oral exposure)

 BMD Analysis 

 Gestational Study (Dam exposed GD6 to PND 21)

 3 DayWeanling Female Study 

 Conclusions
First study in weanling LE female rats

4 day exposure from PND 21 to 24

LE rat
Second study focused on the juvenile Wistar 

male rat.

 Triclosan exposure in the EPA Endocrine Disruptor’s 

Screening Program (EDSP) Tier 1 Pubertal Male 

Protocol (31 day oral exposure from PND 23 to 53) 

(Zorrilla et al., 2009, Toxicological Sciences).
Male Rat Pubertal Protocol

pups

21 25 30 35 40

Wean

4

3

or

Postnatal Day

Cull

45 50 53

Necropsy

Androgen 

dependent 

Tissue Wts.

Assign

 to Trt.

Males: Dose, Examine for PPS, Daily BW

Serum 

Hormones (T, 

T4, TSH).

Website for all EDSP Tier 1 Assays: http://www.epa.gov/scipoly/oscpendo/
Triclosan Effects on serum Thyroxine (T4) in Juvenile Male 

Wistar Rats

Thyroxine (T4)

C 3 30 300 C 100 200

0.00

0.75

1.50

2.25

3.00

3.75

4.50

5.25

*

 *

#

#

Triclosan (mg/kg)

ug/d l

Study run in two blocks. 

Suppression of serum T4 at 30 mg/kg (LOEL) and above, 

no difference in TSH.
Serum TSH in male rats.

TSH

C 3 30 100 200 300

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Triclosan (mg/kg)

ng/ml

No significant change in serum TSH on PND 53.
Triclosan Effects on serum T3 in males.

Triiodothyronine (T3)

C 3 30 100 200 300

0.00

0.25

0.50

0.75

1.00

1.25

1.50

*

Triclosan (mg/kg)

ng/ml

*
Thyroid Histology

Significant decrease in colloid area at only 300 mg/kg dose.
Organ Weights on PND 53.

L. Kidney (g) 1.23 0.02 1.20 0.03 1.16 0.03 1.28 0.04 1.26 0.04 1.28 0.04

L. Adrenal (mg) 25.05 0.56 25.67 1.11 23.68 2.04 23.71 1.08 22.97 3.24 23.97 1.66

Liver (g) 14.50 0.27 15.40 0.56 14.52 0.33 17.39 0.56* 17.18 0.79* 16.53 0.33*

Control 3 mg/kg 30 mg/kg 100 mg/kg 200 mg/kg 300 mg/kg

Increased liver weights at 100 mg/kg and higher.
Induction of hepatic enzyme activity on PND 53 (2 hr 

after last exposure).

Induction of UDGPT (1.5 fold) and PROD (Cyp2b, over 35 fold) activity at 

300 mg/kg .
No alterations in body weight following 31 day 

exposure. 

Mean Body Weight at

Puberty and Necropsy

0

50

100

150

200

250

300

350

30

Control

3

100

200

300

PND 44 PND 53

Triclosan (mg/kg)

Body Weight (g)
Third study focused on the juvenile Wistar 

female rat

 Triclosan evaluated in EDSP Tier 1 female pubertal 

protocol (21 day oral exposure from PND 23 to 53) 

(Stoker et al., 2010).
Female Pubertal Protocol

pups

21 25 30 35 40

Wean

4

3

or

Postnatal Day

Cull Necropsy

(second day 

diestrus in this 

study)

 Assign to Trt.

Females: Dose, Examine for VO, Daily BW

VO
Serum T4- Female Pubertal

Control

9.375 mg/kg

37.5 mg/kg 

75 mg/kg

150 mg/kg

0

1

2

3

4

5

37.5 mg/kg

9.375 mg/kg

Control

75 mg/kg

150 mg/kg

**

**

**

Treatment

Serum T4 (ug/dL)

Serum thyroxine (T4) on PND 42.
Serum TSH on PND 42.

Control

9.375 mg/kg

37.5 mg/kg

75 mg/kg

150 mg/kg

0.0

0.5

1.0

1.5

2.0 Control

9.375 mg/kg

37.5 mg/kg

75 mg/kg

150 mg/kg

Treatment

Serum TSH (ng/ml)
No changes observed in thyroid histology.

Normal colloid area and follicular cell height.
Body and Organ Weights

Control 9.375 mg/kg 37.5 mg/kg 75 mg/kg 150 mg/kg

PND 30 BW (g) 126.80 ± 4.43 124.52 ± 2.66 122.11 ± 3.39 121.86 ± 3.89 124.44 ± 1.73

Final BW (g) 187.24 ± 7.21 184.95 ± 4.37 180.19 ± 4.42 183.73 ± 6.62 185.69 ± 2.46

Liver (g) 8.988 ± 1.13 9.494 ± 1.01 9.031 ± 0.51 9.497 ± 1.11 9.582 ± 0.97

Relative Liver (g)

0.04756 ±

0.00140 

0.04971 ±

0.00116 

0.04914 ±

0.00066 

0.04998 ±

0.00070

0.04990 ±

0.00142

No statistical change in final body weight or liver weight following 21 day 

exposure to the female.
Summary of Effect of Triclosan on Thyroid hormones in male 

and female pubertal studies.

 There were no effects of triclosan on body weights, but an 

increase in liver weight was observed at higher doses in the 

male study, along with induction of hepatic enzyme activity. 

 Over 70% decrease in serum T4 two hours following the last 

dose of 150 mg/kg and higher in both the male and female 

studies. 
Are certain life stages more sensitive to the 

effects of triclosan on the thyroid axis? 
LE rat strain

Gestation/Lactation

Weanling
0

1

2

3

4

5

6

Control

1.18 mg/kg

2.35 mg/kg

4.69 mg/kg

9.38 mg/kg

18.75 mg/kg

37.5 mg/kg

75 mg/kg

* *

**

Treatment

Serum T4 (ug/dL)

Additional study in a Wistar weanling female rat: 3 day exposure 

(PND 19 to 21) with broad range of doses. Blood collected 6 h 

following third dose.
Wistar rat strain

Serum T4- Female Pubertal

Control

9.375 mg/kg

37.5 mg/kg 

75 mg/kg

150 mg/kg

0

1

2

3

4

5

37.5 mg/kg

9.375 mg/kg

Control

75 mg/kg

150 mg/kg

**

**

**

Treatment

Serum T4 (ug/dL)

0

1

2

3

4

5

6

Control

1.18 mg/kg

2.35 mg/kg

4.69 mg/kg

9.38 mg/kg

18.75 mg/kg

37.5 mg/kg

75 mg/kg

* *

**

Treatment

Serum T4 (ug/dL)

Male Pubertal- PND 53 

PND 19 to 21

PND 42 
What is the relevance of the change in T4 for 

adverse outcomes in humans?
Human Relevance and selection of BMR (10 or 20%) 

 Alterations in thyroid homeostasis is of particular concern as 

disruption of circulating concentrations of thyroid hormones have 

been associated with adverse outcomes in humans, including 

altered neurodevelopment. 

 Deficient thyroid hormones during development (e.g., severe 

iodine deficiency or congenital hypothyroidism) leads to severe 

adverse neurological outcomes (e.g., cretinism). 

 Only a few studies link mild to moderate perturbations in T4 to 

adverse neurological outcomes (Haddow et al., 1998 and Morreale

de Escobar, 2000) :

 Haddow et al., 1998 correlated a 25% decrease in maternal T4 

during the second trimester in women with 

neurodevelopmental and cognitive deficits in children (6-10 

point IQ drop). 
BMD analysis Results

 The T4 data in the two male pubertal blocks and the female 

pubertal block were pooled for analysis using EPA’s Bench Mark 

Dose Software (BMDS).

 This analysis was used to determine the benchmark dose estimate 

(BMD) and lower limit estimate (BMDL).

 The benchmark dose response (BMR) was set at both 10 and 20% 

using 

an unconstrained Hill estimate to fit the data.

 10% change or decrease in T4 is within normal limits of 

experiment to experiment variation in control values, but was 

calculated to estimate a significant change from controls. 

 20% change or decrease in T4 was calculated for BMD as a more 

biologically significant change. 
BMD analysis using 3 blocks of pubertal data
Results of benchmark analysis using pooled

blocks based on unrestrained Hill estimate.

 10% BMR --- BMD 10.37 mg/kg BMDL 3.47 mg/kg 

 20% BMR --- BMD 19.05 mg/kg BMDL 8.07 mg/kg 
Summary

 Studies provided dose-response data for suppression of T4

across several life stages and durations of exposure.

 A similar dose-response was observed between males and females 

given that the exposures were different (21 vs. 31 days). 

 Within rat strain, the fetus or weanling rat did not appear to be 

more sensitive to the effects of triclosan on serum thyroxine, as 

compared to the dam or older animals.

 With T4 data pooled from three blocks of pubertal studies, a BMR 

for 10 and 20% was determined to establish BMD and BMDL. 

 Suppression of thyroxine and increased liver weight correlate with 

proposed AOP.

Decreased

T4

Altered 

Development

Adverse

Neuro-Development

Hepatic

Phase I, II, III
Acknowledgements

 Kevin Crofton, Ph.D., NHEERL, ORD, EPA

 Woodrow Setzer, Ph.D., NCCT, ORD, EPA

 Tim McMahon, Ph.D., OPP, EPA

 Vicki Dellarco, Ph.D., OPP, EPA

 Leah Zorrilla, Ph.D., North Carolina State University/ U.S. 

EPA