16.05.2013

Nancy B Hopf, Aurelie Berthet, David Vernez, Emilie Langard,

Philipp Spring, René Gaudin

Skin permeation and metabolism of

di(2-ethylhexyl) phthalate (DEHP)

Institut universitaire romand de Santé au Travail

Institut für Arbeit und Gesundheit

Institute for Work and Health

Background

• High-molecular-weight phthalates (HMWP) are used in plastic tubing, food packaging and processing materials, containers, vinyl toys, vinyl floor coverings, and building products

• Phthalate exposures have produced a variety of male reproductive effects

• Phthalates are assumed to have low dermal absorption however this is based on a few dermal absorption studies excluding the skin’s possibility to metabolize phthalates

• Skin permeation rates used in dermal exposure assessments might therefore underestimate the absorbed dose in health risk assessments.

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Biological monitoring

• Urinary metabolites MEHP, 5OH-MEHP, 5oxo-

MEHP, 5cx-MEPP, and 2cx-MMHP are collected

post shift

• 5cx-MEPP is the primary metabolite in humans1

• Half lives 2 - 24 hours2

1 Wittassek and Angerer, 2008

2 Koch and Angerer, 2007; Koch et al., 2005

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Workers’ exposures

• High exposures during manufacturing of products

containing phthalates1

• Urinary metabolite concentrations often exceed the 95th

percentile of the general population2.

• HMWPs are not very volatile, but readily form airborne

aerosols possibly inhaled by workers during work with

heated processes3

• Urinary HMWP metabolites are present after non-heated

processes. Can dermal exposures potentially play a role

in non-heated processes?

• 1 Gaudin et al., 2011; Gaudin et al., 2008; Hines et al., 2008; Hines et al., 2009; Koch et al., 2012

• 2 NRC 2008

• 3 Smith et al., 1980

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Dermal exposure during non-heated

processes

• workers preparing DEHP-containing plastisol at room

temperature and supervising the plastisol coating of glass

flasks automatically dipped into vats filled with the

plastisol - elevated 5cx-MEPP (median 107.5 μg/L)1.

• workers manufacturing vehicle filters with DEHP-based

plastisol - elevated 5OH-MEHP and 5oxo-MEHP (44.0

and 34.3 μl/L, respectively)2.

• workers refinishing sealants at room temperatures –

elevated DiNP/DiDP metabolite concentrations3.

1 Gaudin et al., 2011; Gaudin et al., 2008

2 Hines et al., 2008

3 Koch et al., 2012

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Experimental Design

Hypothesis

Dermal uptake is a significant contributor in overall DEHP exposure at room temperature

Aims

1. Determine DEHP lag time (τ), permeation rate (J) and permeation coefficient (Kp)

2. Assess extent of DEHP skin metabolism

3. Estimate skin absorption in a simple occupational exposure task using the obtained permeation parameters.

Materials and Methods

• Human skin preparation

• Flow-through diffusion cells

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Chemicals: DEHP, d4-

DEHP, MEHP,

Receptor fluid: cell culture

media (RPMI-1640)

Results: neat vs. aq DEHP

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Chemical

Applied

n Chemical

Measured

Dose

(mg)

Concentration

(µg/mL)

Kp

(10-5 cm/h)

J

(µg/h cm2)

AM±SD

Τlag (h)

d4-DEHP (aq) 4 d4-MEHP 50 166 15.1 0.025±0.021 8

DEHP (neat) 4 MEHP 980* Neat 0.13 0.0013±0.0017 30

Results: MEHP applied to skin

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Chemical

Applied

n Chemical

Measured

Dose

(mg)

Concentration

(µg/mL)

Kp

(10-5 cm/h)

J

(µg/h cm2)

AM±SD

Τlag

(h)

MEHP (aq) 6 MEHP 50 166 436.1 0.724±0.23 6

MEHP (aq) 5 5OH-MEHP 50 166 0.02 0.0004±0.0003 6

Dermally absorbed dose (DAD) mg/kg/day

• Workplace scenario: A vat with DEHP (166 μl/ml or neat), opened

once per day, 200 times per year, both hands immersed (800 cm2) for

3 min

• DEHP (aq): 2.85*10-2 mg/kg/day (Kp 15.1 * 10-5 cm/h)

• DEHP neat: 1.49*10-4 mg/kg/day (Kp 0.13 * 10-5 cm/h)

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𝐷𝐴𝐷 =𝐷𝐴𝑒𝑣𝑒𝑛𝑡 ∗ 𝐸𝑉 ∗ 𝐸𝐷 ∗ 𝐸𝐹 ∗ 𝐴

𝐵𝑊 ∗ 𝐴𝑇

DAevent absorbed dose per event (mg/cm/event) = 𝐾𝑝 ∗ 𝐶 ∗ 𝑡 where

Kp (cm/h) is the coefficient of percutaneous permeation obtained in our study

and by Ng et al., 1992,

C is the concentration of DEHP applied to the skin (mg/L)

t is the duration of exposure (h)

0.27*10-5

1.66

0.16

EV event frequency (events/day) 1

ED exposure duration (years) 1

EF exposure frequency (days/year) 200

A skin surface area (cm2) 800

BW body weight (kg) 70

AT average time in days for non-carcinogens AT = ED 1

Dermally absorbed dose (DAD)

• The dermally absorbed dose (DAD) estimated for the

maintenance task for

a) DEHP in aqueous solution was

I. 2.85*10-2 mg/kg/day using our Kp (15.1 * 10-5

cm/h) and

II. 1.02*10-2 mg/kg/day using the Kp (0.27 * 10-5

cm/h) obtained by Ng et al., 1992

b) DEHP neat was 1.49*10-4 mg/kg/day (Kp 0.13 * 10-5

cm/h)

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Discussion

Total dermal DEHP dose DAD was

• ~200 times greater for waterbased compared to neat solution

• ~ 3 times greater than using Kp from published papers (Ng et

al., 1992)

• > the daily intake calculated for the general population in

Germany (Wittassek et al., 2007)

• > the US EPA reference dose

• < the European Food Safety Authority (EFSA) Tolerable Daily

Intake (TDI)

• ~ estimated daily intake previously estimated for workers

manufacturing or using DEHP (Hines et al., 2008, 2011)

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Discussion

• Older studies concluded that DEHP permeation was

negligible; however, few used viable skin, mostly animal

skin, and did not measure metabolites

• In vitro skin permeation (Kp) previously reported for

DEHP ranged from 0.011 to 94 x 10-5 cm h-1 (ours 15 x

10-5 cm h-1 DEHP (aq))

• Recent phthalate skin permeation studies (DBP) have

shown similar skin metabolism results

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Discussion

• Other permeability determinants not explored were

– choice of reservoir fluid

– skin type (animal versus human, gender, age, type)

– storage (fresh and frozen)

– skin thickness

– SC separation methods (heat, NaBr, dermatomed)

• The vast number of permeability determinants

complicates comparisons with other studies.

• Human skin variability is large

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Discussion

• The rate was primarily determined by whether DEHP was applied

neat or in aqueous solution

– DEHP (neat) will reluctantly diffuse into the comparatively less

lipophilic SC (Tlag>30 h)

– DEHP (aq) have less affinity to water and prefer the lipophilic SC

as seen with the much higher permeation rate.

– Similar permeation rates DEHP (aq) and MEHP (aq) the rate

limiting step is the availability of DEHP in the dermis (aqueous

phase) and not the metabolism step.

– DEP skin permeation rates were almost double for DEP applied

as a saturated aqueous solution compared to neat.

– chemical enhancers commonly used to increase drug delivery are

alkyl esters, making phthalate diesters eloquent pro-drugs

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Conclusion

• Absorption of DEHP through the dermal route is

significant, and not as currently regarded: negligible or

low.

• Both DEHP and MEHP have shown low antiandrogenicity

effect and can bind to estrogen receptors in the skin (ERα

and ERβ). What endocrine effects this might exert is

currently unknown and needs further scrutiny.

Acknowledgement: We thank the Swiss Centre for Applied Human Toxicology

(SCAHT) for funding this study.

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Thank you for your attention

Nancy B. Hopf

Institute for Work and Health

University of Lausanne and Geneva