TO NON - PERSISTENT CHEMICALS

(1)

T ^HE ^QUEST ^FOR ^BIOMARKERS ^OF ^EXPOSURE

TO NON - ^PERSISTENT ^CHEMICALS

Holger M. Koch

koch@ipa-dguv.de

Institute for Prevention and Occupational Medicine of the German Social Accident Insurance

Institute of the Ruhr-University Bochum (IPA)

Bürkle-de-la-Camp-Platz 1 44789 Bochum, Germany www.ipa-dguv.de

(2)

2

Ambient Monitoring

external exposure

health effects chemicals:

in air, soil, water, diet, consumer products

Medical

Assessment

Biological Monitoring- Prospects in Occupational and Environmental Medicine Angerer and Weiss (eds.), Wiley-VCH, Weinheim, 1. Edition (2002)

adapted from:

model calculations, including

- various sources of exposure (known and unknown)

- routes of exposure (oral, inhalative, dermal)

Exposure and Risk Estimation

Exposure Assessment

(in the exposure-disease continuum)

(3)

3

Ambient

Monitoring Human Biomonitoring

health effects Biomarkers

of Exposure

Biochemical Effect Markers

Biological

Effect Markers chemicals:

parent chem., metabolites

protein adducts DNA adducts

cytogenetic parameters, enzymes

Medical

Assessment

adapted from:

Exposure Assessment

Exposure and Risk Estimation

(4)

4

Ambient

Monitoring Human Biomonitoring

health effects Biomarkers

of Exposure

Biochemical Effect Markers

Biological

Effect Markers chemicals:

protein adducts DNA adducts

cytogenetic parameters, enzymes

Medical

Assessment

adapted from:

Exposure Assessment

Exposure and Risk Estimation Specificity to helath effects

Specificity to health effects Specificity to chemical exposure

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5

Biomarkers of Exposure

- specific to target compound (exposure) ability to capture all:

- sources of exposure (known and unknown)

- routes of exposure (oral, inhalative, dermal)

Biomarkers of Exposure

… defined by:

• Toxikokinetics

• Metabolism

• Matrix (urine, blood, etc.)

• Specificity

• Contamination

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6 Blood

Toxicant/Metabolite

Urinary Metabolite

1 10 100 1000

Time (Days)

Henderson et al. Crit Rev Toxicol 20: 65-82 (1989) Needham and Sexton. JEAEE 10: 611-629 (2000)

taken from ISEA/ISES Wesolowski Award Presentation, Paris 2006:

Biomonitoring: An Integral Part of Exposure Analysis

Larry L. Needham, Ph.D., National Center for Environmental Health, Centers for Disease Control and Prevention

Toxikokinetics: persistent chemicals

• Long elimination half-times

• Higher blood levels relative to urine

Blood is preferred matix

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7 Blood

Toxicant/Metabolite

Urinary Metabolite

1 10 100 1000

Time (Days)

taken from ISEA/ISES Wesolowski Award Presentation, Paris 2006:

Biomonitoring: An Integral Part of Exposure Analysis

Larry L. Needham, Ph.D., National Center for Environmental Health, Centers for Disease Control and Prevention

Urinary Metabolite 3 Urinary Metabolite 2

Henderson et al. Crit Rev Toxicol 20: 65-82 (1989) Needham and Sexton. JEAEE 10: 611-629 (2000)

Toxikokinetics: non-persistent chemicals

• Quick metabolisation

• Short elimination half-times

• Higher urine levels relative to blood

Urine is preferred matrix

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8 1

10 100 1000 10000 100000

0 6 12 18 24

MnBP

3OH-MnBP

Toxikokinetics: n-butyl phthalate

Concentration (in µg/L)

time (in h)

5mg D4-DnBuP (oral, one human volunteer)

serum

Koch et al (2012) Arch Tox 86, 1829-1839.

Lorber and Koch (2013) Environ Int 59, 469–477.

Biomarkers: non-persistent chemicals

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9 1

10 100 1000 10000 100000

0 6 12 18 24

MnBP

3OH-MnBP MnBP

3OH-MnBP

Toxikokinetics: n-butyl phthalate

time (in h)

urine serum

(10)

10 0

2500 5000 7500 10000

0 6 12 18 24

MnBP

3OH-MnBP MnBP

3OH-MnBP

Toxikokinetics: n-butyl phthalate

time (in h)

urine serum

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11 1

10 100 1000 10000 100000

0 6 12 18 24

iso-BuP

2OH-iso-BuP

Toxikokinetics: iso-butyl paraben

time (in h)

5mg D4-iso-butyl paraben (oral, one human volunteer)

serum

Moos et al. (e-pub 11/25/2015) Arch Tox Serum data in preparation

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12 1

10 100 1000 10000 100000

0 6 12 18 24

iso-BuP

2OH-iso-BuP iso-BuP

2OH-iso-BuP

Toxikokinetics: iso-butyl paraben

time (in h) urine

serum

Urine is preferred matrix

Moos et al (e-pub 11/25/2015) Arch Tox Serum data in preparation

5mg D4-iso-butyl paraben (oral, one human volunteer)

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13

Parent chemical

Metabolism

Metabolite A Metabolite B

Parent chemical

-gluc

-sulf

Metabolite C

-gluc -sulf

-gluc -sulf -gluc -sulf

Parent chemical

Phase I Phase II

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14

Parent chemical

Metabolism

Parent chemical

-gluc

-sulf

Metabolite C

-gluc -sulf

-gluc -sulf -gluc -sulf

Parent chemical

Phase I Phase II

Enzymatic deconjugation

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15

Parent chemical

Metabolism

Metabolite C Parent

chemical

Phase I Phase II

Be careful!

• know, which conjugates you want to cleave

• (e.g. phthalates: gluc; parabens: gluc+sulfates …)

• check stability of target analytes

• (e.g. phthalates are broken down by aryl-sulfatases)

Partial deconjugation or analyte breakdown !

Enzymatic deconjugation

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16

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0-6 6-12 12-18 18-24 24-30

Methyl Paraben

free [%]

sul [%]

glu [%]

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0-6 6-12 12-18 18-24 24-30

n-Butyl Paraben

free [%]

sul [%]

glu [%]

Conjugate Distribution (Parabens)

to transform all conjugates into free phase I metabolites Gluc-sulf-enzyme (like HP2) is needed

Gluc-sulf-enzymes cleave ester-bonds of phthalates;

Phthalates/substitutes need pure Gluc-enzyme (like E.coli K12)

Moos et al (e-pub 11/25/2015) Arch Tox

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17

Metabolism

Parent chemical

Metabolite C Parent

chemical

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18

Parent chemical

Metabolism

Metabolite A

Metabolite B

Metabolite C

Parent chemical

chemical X chemical Y Biomarker Specificity ! Possible

Contamination !

5%

15%

Back-calculation of the dose

with metabolite conversion factors (F_ue)

3% 75%

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19

Metabolism (example parabens)

O H

O O

R

O H

OH O

PHBA

O H

NH O

O H PHHA O

Methyl Paraben

O H

O O

R

(free, glucuronide- and sulphate- conjugates) Methyl Paraben

17%

3%

60%

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20

Metabolism (example parabens)

O H

O O

R

O H

OH O

PHBA

O H

NH O

O H PHHA O

O H

O O

CH3 O

H

O O

CH₃ C

H₃

O H

O O

CH3

O H

O O

CH₃ C

H₃

iso-BuP n-BuP

MeP

O H

O O

R MeP

3-7%

57-64%

5.6%

6.8%

Other chemicals

Parent parabens as biomarkers:

• Different excretion fractions

• Rather low excretion fractions

• Contamination control!

• Preservative in biobanked samples*

* Longnecker et al. (2013) Environ Res 126, 211-214. (2013).

17%

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21

Metabolism (example parabens)

O H

O O

R

O H

O O

CH3 O

H

O O

CH₃ C

H₃

O H

O O

CH3

O H

O O

CH₃ C

H₃

iso-BuP n-BuP

MeP

O H

O O

R MeP

O H

O O

CH3 OH O

H

O O

CH₃ C

H₃ OH

2OH iso-BuP 3OH n-BuP

5.8%

15.8%

Moos et al (publication submitted)

5.6%

6.8%

17%

• dose extrapolation (daily intakes)

• risk assessment (comparison to

health benchmarks, HQ/HI)

Poster !

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22

Metabolism (example phthalates)

C

O

O ^C ^H ² ^C^C^C ^H ^H^H ²³ ⁽ ^C ^H ² ⁾ ³ ^C^H ³

C ^H ³

C ^H ² ⁽ ^C ^H ² ⁾ ³ ^C^H ³

C ^H

C ^H ²

O

C

Di(2-ethylhexyl)phthalate (DEHP)

C

O

O ^C^H ² ^C^C^C ^H ^H^H ²³ ⁽ ^C ^H ² ⁾³ ^C ^H ³

O ^H

O C

H ^O ^C ^H ² ^C^C^C ^H ^H ^H ² ³ ⁽ ^C^H ² ⁾³ ^C^H ³

H ^O ^C^H ² ^C^C^C^H^H^H ² ³ ⁽ ^C^H ² ⁾ ³ ^C^H ³

C

O O ^H

O ^H

O ^C

2-Ethylhexanol

Mono(2-ethylhexyl)phthalate (MEHP)

2-Ethylhexanol

Phthalic Acid

Koch et al. (2004) Arch Toxicol 78, 123-130.

Chain

length Phthalate Monoester-

Metabolite f_ue-pm reference

8 DEHP MEHP 6% Koch et al. (2005), Anderson et al. (2011), Kessler et al. (2012)

Chain

length Phthalate Monoester-

Metabolite f_ue-pm reference

2 DEP MEP ~80% ^estimated

4 DnBP MnBP 84% Koch et al. (2012), Anderson et al. (2001)

4 DiBP MiBP 71% Koch et al. (2012) Anderson et al. (2001)

6 BBzP MBzP 73% Anderson et al. (2001)

9 DiNP MiNP 1% Koch et al. (2007), Anderson et al. (2011)

10 DIDP/DPHP MiDP/MPHP <1% Wittassek et al. (2008), Schütze et al. (2014)

Pre-Analytical / Analytical

Contamination !

Plasticizers Personal

Care Products

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23

C

O

O ^C ^H ² ^C^C^C ^H ^H^H ²³ ⁽ ^C ^H ² ⁾ ³ ^C^H ³

C ^H ³

C ^H ² ⁽ ^C ^H ² ⁾ ³ ^C^H ³

C ^H

C ^H ²

O

C

Di(2-ethylhexyl)phthalate (DEHP)

C

O

O ^C^H ² ^C^C^C ^H ^H^H ²³ ⁽ ^C ^H ² ⁾³ ^C ^H ³

O ^H

O C

H ^O ^C ^H ² ^C^C^C ^H ^H ^H ² ³ ⁽ ^C^H ² ⁾³ ^C^H ³

H ^O ^C^H ² ^C^C^C^H^H^H ² ³ ⁽ ^C^H ² ⁾ ³ ^C^H ³

C

O O ^H

O ^H

O ^C

2-Ethylhexanol

Mono(2-ethylhexyl)phthalate (MEHP)

2-Ethylhexanol

Phthalic Acid

R ^C ^H ² ^C^C^C^H^H^H ²³ ⁽ ^C ^H ² ⁾ ³ ^C^O ^O ^H

R ^C^H ² ^C^C^C^H^H^H ²³ ⁽ ^C^H ² ⁾³ ^C^H ²

O ^H

R ^C ^H ² ^C^C^C ^H ^H^O ²^O ^H⁽^C ^H ² ⁾³ ^C ^H ³

R ^C^H ² ^C^C^C ^H ^H^H ²² ^O⁽^C ^H^H ² ⁾ ³ ^C ^H ³

R ^C^H ² ^C^C^C ^H ^H ^H ² ³ ^C ^H ² ^C

C ^H ²

O ^C ^H ³

R ^C^H ² ^C^C^C^H^H ³ ⁽ ^C^H ² ⁾³ ^C^H ³

O

R ^C ^H ² ^C^C^C ^H ^H ^H ² ³ ^C^H ² ^C^H

C ^H ²

O ^H ^C ^H ³

R ^C^H ² ^C^C^C ^H ^H^H ³^O⁽^H ^C ^H ² ⁾³ ^C ^H ³

2-(2-Hydroxyethyl)- hexylphthalate

2-Ethyl-6-hydroxy- hexylphthalate (<5%) 2-(1-Hydroxyethyl)-

hexylphthalate

2-Ethyl-4-hydroxy- hexylphthalate

2-Carboxymethyl- hexylphthalate

(2cx-MMHP)

2-(1-Oxyethyl)- hexylphthalate

2-Ethyl-5-carboxy pentylphthalate

(5cx-MEPP)

2-Ethyl-4-oxy- hexylphthalate

R ^C^H ² ^C^C^C^H^H^H ²³ ⁽^C ^H ² ⁾² ^C^H

C ^H ³

O ^H

R ^C^H ² ^C^C^C ^H ^H^H ²³ ⁽ ^C ^H ² ⁾² ^C

C ^H ³

O

2-Ethyl-5-hydroxy- Hexylphthalate

(5OH-MEHP)

2-Ethyl-5-oxy- Hexylphthalate

(5oxo-MEHP)

15.0%

23.3%

~4.2%

^t^1/2

18.5%

>24h

t_1/2 15h

t_1/2 10h

Metabolism (example DEHP)

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24 Chain

length Phthalate Monoester

Metabolite f_ue-pm Secondary

metabolite f_ue-pm reference

2 DEP MEP 80% ^estimated

4 DnBP MnBP 84% Koch et al. (2012), Anderson et al. (2001)

OH-MnBP 7%

4 DiBP MiBP 71% Koch et al. (2012) Anderson et al. (2001)

OH-MiBP 20%

6 BBzP MBzP 73% Anderson et al. (2001)

5OH-MEHP 23%

5oxo-MEHP 15%

5cx-MEPP 18%

9 DiNP MiNP 1% Koch et al. (2007), Anderson et al. (2011)

OH-MiNP 18%

oxo-MiNP 10%

cx-MiNP 9%

10 DPHP /DiDP

OH-MPHP 12% Schütze et al. (2014), Wittassek and Angerer (2008)

oxo-MPHP 14%

cx-MPHP 0.5%

Biomarkers of exposure for phthalates

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25

Metabolism (phthalate substitutes)

O

O O O

C₉H₁₉

O

O OH OH O

O OH

O

O OH O

O

O OH O

O

OH O

O OH O

DINCH MINCH

OH-MINCH

oxo-MINCH

cx-MINCH

CHDA

Hexamoll ® DINCH ®: market introduction in 2002

11% 2%

Schütze et al. (2012) J Chrom B. 895-896, 123-130 Koch et al. (2013) Arch Tox 87, 799–806

Schütze et al. (2015) Chemosphere 128, 216–224 Schütze et al. (e-pub) Arch Tox

2% 24%

<1%

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Hexamoll® DINCH®: Population Study

1999 2003 2006 2009 2012

Year

German Environmental Specimen Bank:

• 24 h urine samples

• with full info on 24 h urine volume, body weight, etc.

DINCH market introduction

26

(27)

Hexamoll® DINCH®: Population Study

1999 2003 2006 2009 2012

Year 0.1

1.0 10 100 1000

OH- + cx-MINCH in Urine (µg/L)

0.01

DINCH market introduction

Exposure Assessment:

• metabolite levels in urine

• daily intake calculation Risk Assessment:

• Comparison with TDI

• HBM values (UBA)

HBM I (OH-MINCH + cx-MINCH): adults 4500µg/L children 3000 μg/L

HBM Commission (2012)

Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz 57 (12), 1451-1461.

?

Schütze et al. (2014) Int J Hyg Environ Health 217, 421-426

27

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28

Metabolism (phthalate substitutes) DEHTP (Terephthalate)

O O

DEHTP ^O

O H

O O

OH O

O H

O O

O O H

O O

O OH O

O H

O O

O OH

MEHTP

5oxo-MEHTP 5OH-MEHTP

O O

O H

O O

5cx-MEPTP 2cx-MMHTP

0.3%

1.8% 13%

1.0%

Lessmann et al. (2016) J Chrom B. 1011, 196-203.

Lessmann et al. (accepted) Arch Tox.

O O

DEHP

Poster !

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29

Summary and conclusion

Biomarkers of exposure to non-persistent chemicals

• Urine is preferred matrix

• Toxikokinetics

• Metabolism

• Parent chemical – phase I met. – phase II met (conjugates)

• Metabolite conversion factors

• Specificity

• Contamination

• Exposure Assessment (comparisons; dose)

• Risk Assessment (TDI; HBM/BE)

• Risk Management

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Acknowledgement

Some of the work has been performed within the Cooperation for the enhancement of HBM between the German Ministry for the Environment, the German Environment Agency (UBA), and the German Chemical Industry Association (VCI)

30

IPA:

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T ^HE ^QUEST ^FOR ^BIOMARKERS ^OF ^EXPOSURE

TO NON - ^PERSISTENT ^CHEMICALS

Holger M. Koch

koch@ipa-dguv.de

Institute for Prevention and Occupational Medicine of the German Social Accident Insurance

Institute of the Ruhr-University Bochum (IPA)

Bürkle-de-la-Camp-Platz 1 44789 Bochum, Germany www.ipa-dguv.de

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low-molecular weight phthalates

high-molecular weight phthalates parent phthalate primary metabolite

(hydrolytic monoester)

secondary metabolite

(oxidised monoester) parent phthalate primary metabolite (hydrolytic monoester)

secondary metabolite (oxidised monoester)

Dimethyl phthalate (DMP) O

O OO

Mono-methyl phthalate (MMP) O

OH O O

n.a. Di(2-ethylhexyl) phthalate

(DEHP) O

O O O

Mono(2-ethylhexyl) phthalate (MEHP)

O

OH O O

5OH-Mono(2-ethylhexyl) phthalate (5OH-MEHP or MEHHP)

OH O

OH O O

Diethyl phthalate (DEP) O

O OO

Mono-ethyl phthalate (MEP) O

OH O O

n.a. 5oxo-Mono(2-ethylhexyl) phthalate

(5oxo-MEHP or MEOHP) O O

OH O O

Di-cyclohexyl phthalate (DCHP)

O

O O O

Mono-cyclohexyl phthalate (MCHP)

O

O OH O

n.a. 5carboxy- Mono(2-ethylhexyl)

phthalate (5cx-MEPP or MECPP) O

OH O

O OH

O

Di-n-penty lphthalate (DnPeP) O

O O O

Mono-n-pentyl phthalate (MnPeP)

O

O OH O

n.a. Di-iso-nonyl phthalate (DiNP)^*1

O

O O O

Mono-iso-nonyl phthalate (MiNP)^*1

O

O OH O

7OH-Mono-methyloctyl phthalate (OH-MiNP or MHiNP)

OH O

OH O O

Butyl-benzyl phthalate (BBzP) O

O O O

Mono-benzyl phthalate (MBzP) O

OH O O

n.a. 7oxo-Mono-methyloctyl phthalate

(oxo-MiNP or MOiNP) O O

OH O O

Di-iso-butyl phthalate (DiBP) O

O O O

Mono-iso-butyl phthalate (MiBP)

O

OH O O

3OH-Mono-methylpropyl phthalate (OH-MiBP)

O OH

OH O O

7carboxy-Mono-methylheptyl phthalate (cx-MiNP or MCiOP)

O OH O

OH O O

Di-n-butyl phthalate (DnBP) O

O O O

Mono-n-butyl phthalate (MnBP) O

OH O O

3OH-Mono-n-butyl phthalate (OH- MnBP)

OH O

OH O O

Di-iso-decyl phthalate (DiDP)^*1 and Dipropylheptyl phthalate (DPHP)

O

O O O

Mono-iso-decyl phthalate^*1 (MiDP or MPHP)

O

O OH O

6OH-Mono-propylheptyl phthalate (OH-MPHP, OH-MiDP or MHiDP)

OH O

O OH O

3carboxy-Mono-propyl phthalate (3cx-MiBP or MCPP)

O

OH O

O OH

O

6oxo-Mono-propylheptylphthalate (oxo-MPHP, oxo-MiDP or MOiDP)

O O

O OH O

Mono(2,7-methyl-7-carboxyheptyl) phthalate (cx-MiDP, MCiNP; mono- carboxyisooctyl phthalate)

OH O

OH O O

O

Koch and Calafat (2009):

Phil Trans R Soc B 364:2063-2078.

TO NON - PERSISTENT CHEMICALS

T HE QUEST FOR BIOMARKERS OF EXPOSURE

TO NON - PERSISTENT CHEMICALS

Holger M. Koch

Exposure Assessment

Exposure Assessment

Exposure Assessment

Biomarkers of Exposure

… defined by:

Toxikokinetics: persistent chemicals

Blood is preferred matix

Toxikokinetics: non-persistent chemicals

Urine is preferred matrix

Toxikokinetics: n-butyl phthalate

Toxikokinetics: n-butyl phthalate

Toxikokinetics: n-butyl phthalate

Toxikokinetics: iso-butyl paraben

Toxikokinetics: iso-butyl paraben

Metabolism

Phase I Phase II

Metabolism

Phase I Phase II

Metabolism

Phase I Phase II

Partial deconjugation or analyte breakdown !

Conjugate Distribution (Parabens)

Metabolism

Metabolism

5%

15%

3% 75%

Metabolism (example parabens)

Metabolism (example parabens)

Metabolism (example parabens)

Poster !

Metabolism (example phthalates)

15.0%

23.3%

~4.2%

18.5%

Metabolism (example DEHP)

Biomarkers of exposure for phthalates

Metabolism (phthalate substitutes)

Hexamoll ® DINCH ®: market introduction in 2002

Hexamoll® DINCH®: Population Study

Hexamoll® DINCH®: Population Study

?

Metabolism (phthalate substitutes) DEHTP (Terephthalate)

Poster !

Summary and conclusion

Acknowledgement

T HE QUEST FOR BIOMARKERS OF EXPOSURE

TO NON - PERSISTENT CHEMICALS

Holger M. Koch

T ^HE ^QUEST ^FOR ^BIOMARKERS ^OF ^EXPOSURE

TO NON - ^PERSISTENT ^CHEMICALS

T ^HE ^QUEST ^FOR ^BIOMARKERS ^OF ^EXPOSURE

TO NON - ^PERSISTENT ^CHEMICALS