## Tammy E. Stoker, Ph.D. Endocrine Toxicology Branch Toxicity Assessment Division NHEERL, ORD, U.S. EPA Reproductive Toxicology Facility Durham, NC 

# 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

Website for all EDSP Tier 1 Assays: http://www.epa.gov/scipoly/oscpendo/

## Triclosan Effects on serum Thyroxine (T4) in Juvenile Male Wistar Rats

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.

No significant change in serum TSH on PND 53.

# Triclosan Effects on serum T3 in males.

# 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 300 mg/kg 200 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.

# 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

# Serum thyroxine (T4) on PND 42.

# Serum TSH on PND 42.

## 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

## 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 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