# Presentation: 095277

##  Study of Phthalates in Pregnant Women and ChildrenShanna H. Swan, Ph.D.Center for Reproductive EpidemiologyUniversity of RochesterSTAR PROGRESS REVIEW WORKSHOPJuly 13, 2006

**Notes:**
Key: Thank you, for the opportunity to speak today about our phthalate study
Trans: I welcome the opportunity to share some recent results from  our Study for Future Families  These results, are consistent with a large toxicological literature and, though based on small numbers, suggest that humans males are undervirilized by prenatal phthalate exposure, an effect well established in rodents. 

## A large, coherent body of animal studies demonstrates reproductive toxicity of several phthalates
Phthalate exposure is prevalent in the US population (CDC 2002, 2003)
Urine assays are now sensitive, reliable and (relatively) inexpensive

**Phthalate exposure is prevalent in the US population (CDC 2002, 2003)**
**Urine assays are now sensitive, reliable and (relatively) inexpensive**

** *****No human studies of prenatal          phthalate exposure prior to ours***** **

## Phthalates and their metabolites

***Parent Compound***
**Dibutyl phthalate (DBP)**
- Benzyl butyl phthalate (BzBP)
- Di-isobutyl phthalate (DiBP)
- Diethyl phthalate (DEP)
- Di-n-octyl phthalate (DnOP)
- Dimethyl phthalate (DMP)
**Di-2-ethylhexyl phthalate (DEHP)**

***Primary Metabolites***
**Mono-n-butyl phthalate (MBP)**
- Mono-benzyl phthalate (MBzP)
- Mono-isobutyl phthalate  (MiBP)
- Mono-ethyl phthalate (MEP)
- Mono-3-carboxypropyl phthalate (MCPP)
- Mono-methyl phthalate (MMP)
**Mono-2-ethylhexyl phthalate (MEHP)**
**Mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP)**
**Mono-2-ethyl-5-oxohexyl phthalate (MEOHP)**

## Di-2-Ethylhexyl Phthalate (DEHP)

- US production>1 mill lbs/yr
- >95% used as a plasticizer in PVC
- Not chemically bound: migrates from plastic
- Multiple urinary metabolites
*MEHHP (52.7%)*
*MEOHP (31.8%)*
*MEHP (15.5%)*
- (Koch 2004)

**Notes:**
DEHP is thought to be most reproductively toxic, and of greatest concern. Concerns greatest for uses in PVC, particularly children’s toys and teethers, as well as medical products.

## Dibutyl Phthalate (DBP)

- US production:
- 	>1 million lbs/yr
- Occupational exposure: (e.g. nail salons)
- In:
- Cellulose acetate plastics
- Lacquer, varnish, adhesives
- Medical coatings and patches
- Cosmetics and nail polish

**Notes:**
DBP is ranked next in toxicity, concerns re use in  beauty products, (nail polish), medical uses. 

## Overview of Phthalate Toxicology

- Anti-androgenic
- Decrease AGD
- Phthalate Syndrome

## Phthalates decrease fetal testosterone

- (Parks et al, 2000)

- **

- NS

**Notes:**
And prepubertal testosterone is actually higher in exposed rodents

## Anogenital Distance (AGD)

**Sexually dimorphic: In rodents, AGD is about twice as long in males as in females**
**In rodents, male AGD is shortened by prenatal exposure to anti-androgens (including: phthalates, flutamide and vinclozilin)**

## DEHP and AGD (Moore, 2001)

- Male:female
- ratio = 2.0

- Male:female
- Ratio = 1.6

## “AGD is forever”

- “AGD is forever”
- (Gray 2006)

## Phthalate Syndrome

**Malformations of:**
- Perineum:
**Reduced AGD**
- Epididymis
- Vas deferens
- Seminal vesicles
- Prostate
- External genitalia:
- Hypospadias
- Cryptorchidism

**Downregulation of**
- Fetal testicular testosterone
- Insl-3

***Significant  ************ in testosterone ***
***and Leydig cell differentiation***
***@ doses below the  NOAEL for DBP***

## The question we addressed:

**Does prenatal phthalate exposure alter male sexual development in humans?**

## Study of Phthalatesin Women and Children  

**	****Designed to assess infant genital development ****in relation to prenatal phthalate exposure **

**Notes:**

Key:  In SFF I we had recruited pregnant women and their partners in four US cities. In SFFII we looked @ children and their development (motivated by studies of lab animals), 

## Study Population 

***Mothers who: ***
**Were recruited at a prenatal visit in:**
**    ****Columbia  MO, Minneapolis MN, and **
**    ****Los Angeles CA**
**Agreed to follow-up study**
**Provided a prenatal urine sample**
***Urine was not requested in first study year ***

## Study Population for this Analysis

**Boys: N=166**
**With two exams: N=68**
**With prenatal phthalate: N=106**
**Mean (SD) at first visit**
**Age:  12.6 (6.9) months**
**Weight:  9.7  (2.4) kg**

## Boys’ Physical Exam

**Anthropometry **
**Male genital exam**
***Anogenital distance***
***Testicular descent***
***Penile length and width***
***Scrotal size and condition***

## Measuring Anogenital Distance

***This is similar to toxicological measure ***
***AGD is repeatable (CV =7.2%)***

## AGD rarely measured in

**Callegari et al.  J of Pediatrics 111:240-243, 1987.**

- Humans
- In girls: AGD
-  is distance A to C

## AGD by Sex

- Male:female
- ratio = 1.5

- If male:female
-  ratio = 2.0

## Analysis of Male Anogenital Distance (AGD)

**AGD increases with both age and weight**
**These are strongly correlated (R****2**** = 0.88, p<0.0001)**
**We used standard growth curves to adjust for body size (CDC, 2000)  **
**Weight percentile (WT%) calculated for each boy at each visit**

## Slide 21

## Analysis of AGD (Continuous)

***Expected AGD*** modeled:
**Using all visits (mixed model)**
**WT% and age were the only significant predictors**
***AGD and phthalates***
**Using all visits (mixed model)**
**Log transformed phthalate metabolite concentration used (to normalize data)**

## Categorical Analysis of AGD

**Residual AGD** = Observed AGD - Expected AGD
- Categorized AGD by size of the residual:
**“****Shorter” **<25th Percentile
- 25th% <=**“Intermediate” < **75th%
- 75th percentile<= **“Longer” AGD: **

## Phthalate Exposure Assessment

**Samples were collected mid-pregnancy (median 28.6 weeks gestation) **
**Concentration of nine phthalate metabolites measured by CDC (blinded to identify of individuals or outcomes**
**Analyzed as continuous and categorical variables**
**low (<25****th****%), medium, high(>=75****th****%)**
**Creatinine (and square-root creatinine) not significant covariates **

## Results of regression analysis*: 

***Significant ***
***(p-value)***
**MBP** (0.048)
**MEP** (0.005)
**DEHP** metabolites
**MEHP** (0.017)
**MEOHP **(0.001)
**MEHHP **(0.002)

***Borderline ***
- MMP (0.053)
- MiBP (0.097)
***Not significant***
- MBzP (0.826)
- MCPP (0.591)

- * Mixed model including 106 boys and 165 visits

## Slide 26

## Odds ratio (95% CI) for Shorter AGD

| Exposure | Medium:low* | High:low* |  |  |
| --- | --- | --- | --- | --- |
| MBP | 5.7 (1.2, 27.3) | 9.2 (1.8, 46.2) |  |  |
| MEHP | 1.7 (0.5, 5.2) | 3.2 (0.9, 11.5) |  |  |
| MEOHP | 10.2 (1.3, 82.5) | 29.1 (3.4, 245.6) |  |  |
| MEHHP | 4.8 (1.0, 22.9) | 13.0 (2.6, 66.4) |  |  |
| MEP | 4.6 (1.0, 21.6) | 7.9 (1.5, 41.3) |  |  |

***Low < 25****th**** %, High >=75****th****%, Medium, other**

## Mean phthalate concentration by AGD category

## The problem of mixtures

- Until recently, toxicology examined one phthalate at a time
- But that is not how people are exposed: most people are exposed to multiple phthalates (CDC, 2005)
- New toxicology suggests:
***Dose Additivity***

## Gray, Personal communication

## Summary Phthalate Score

- Uses the five phthalates significantly associated with AGD
- Each phthalate contributes 1 point to score for each additional quartile >1st
- Distribution of score
- Low = 0-3 (25%)
- Medium = 4-11 (53%)
- High = 12-15 (22%)
- Min = 0, Max = 15, Median = 9

## * Fisher’s Exact test

| Phthalate Score | Shorter | Longer | P-value* |
| --- | --- | --- | --- |
| Low | 0 | 11
 | Referent |
| Medium | 16 | 14
 | 0.0014 |
| High | 13 | 1 | <0.0000 |

*** Fisher’s Exact test**

**AGD Category**

## 0 boys with

-  low Phthalate Score,
- 13 with high

- 1 boy with
-  high Phthalate Score
- 11 with low

- Shorter

- Longer

- Intermediate

## Other findings 

**Significantly correlated with AGD:**
***Degree of testicular descent ***
***Penile volume ***
***Scrotal size***
***This cluster of outcomes consistent with***
***“******phthalate syndrome” in rodents***

## Comparing animaland human studies

| Factor | Rodent | Human |
| --- | --- | --- |
| Route | Oral | Oral, inhalation, 
dermal, parenteral |
| Dose | High, medium | Low |
| Agent | Single | Mixture |

## Routes of Exposure

**Percutaneous **
**Ingestion**
**Dermal Absorption**
**Inhalation**
***Measurable in urine, serum, ***
***breast milk, amniotic fluid***

**Notes:**

Direct Transmission – IV tubing in NICU
Dermal Absorption – personal care products – lotions, aftershave, cosmetics
Ingestion – can be dietary or non-dietary - infants sucking on toys
Inhalation – can volatize

## Were these changes seen at very high phthalate levels?

- “High” level (>=75**th** percentile) was compared to levels in  NHANES samples
- “High” levels in our study seen in about 25% of adult US females
- How do these compare to EPA reference dose?

## Estimated exposure and EPA reference dose (μg/kg/day)

| Phthalate | Median | 95th% | Reference dose |
| --- | --- | --- | --- |
| DEHP | 1.32 | 9.32 | 20 |
| DEP | 6.64 | 112.3 | 800 |
| BBzP | 0.50 | 2.47 | 200 |
| DBP | 0.99 | 2.68 | 100 |

- (Marsee, et al, 2006)

## Clinical implications of the phthalate syndrome??

**In rodents:**
**At birth: Shorter AGD, impaired testicular descent, hypospadias**
**Later: Low sperm count, rarely  testicular tumors  **
**Our study of humans suggests:**
**At birth: Shorter AGD (some, but most NS, decrease in testicular descent, smaller penile volume)**
***Future studies needed to determine***
***clinical correlates in humans***

## U MO
Robin Kruse, 
Sara Stewart, Lynn Teague

UCLA
Christina Wang, Cathy Mao

U MN
Bruce Redmon, Chris Ternand 

U IA
Amy Sparks, Shannon Sullivan

- Robin Kruse,
- Sara Stewart, Lynn Teague
**UCLA**
- Christina Wang, Cathy Mao
**U MN**
- Bruce Redmon, Chris Ternand
**U IA**
- Amy Sparks, Shannon Sullivan

**CDC**
- Dana Barr, Antonia Calafat
**UC Davis**
- Jim Overstreet, Charlene Brazil
**U Copenhagen**
- Katharina Main
**U Rochester**
- Shanna Swan
- Fan Liu

*Funded by grants from the National Institute of Environmental Health Sciences and the US Environmental Protection Agency*