ISBM_2013_P1.4 | page 1Manchester | 2013-09-09

A company of Bayer and LANXESS

Gabriele Leng and Wolfgang Gries

Institute of Biomonitoring, Leverkusen, Germany

Gabriele Leng and Wolfgang Gries

Institute of Biomonitoring, Leverkusen, Germany

Biomonitoring of 4,4´-methylenediphenyl diisocyanate(MDI) with new specific biomarker MDA-Val-Hyd

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MDI - AROMATIC ISOCYANATE Aromatic isocyanate: MDI

–N=C=O

substance for the production of polyurethanes, special plastics, lacquer, glues, paints, foams

Polymeric: foam for heat resistance materials, car seats Monomeric: shoe sole, coatings, composition leather

MDI (Diphenylmethan-4,4'-diisocyanat)

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ISOCYANATES – HEALTH ASPECTS

Acute• Irritations of skin and mucosa• Sensitization of respiratory tract

Chronic• Isocyanate asthma

Limiting values for MDI in Germany: • MAK: 50 µg/m³ air• BLW: 10 µg/L urine

Limiting values for MDI in UK: • Biological monitoring guidance value (BMGV) for isocyanates:

1 µmol/mol creatinine (ca. 4 µg/g creatinine)

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based on Sabbioni, 2000MDA in urine

albumin-adduct

N

HN

NH2

O

O

globin-adduct

METABOLISM IN RATS

Hydrolysis in vitro

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MDI – ANALYTICAL METHODS

1. Amines (urine):• Short term parameter, covers body dose of last 2.5 - 3.5 days• not isocyanate-specific as no differentiation between isocyanate and amine

2. Hemoglobin-adducts (blood): • Long term parameter, covers body dose of last 3 months• not isocyanate-specific as no differentiation between isocyanate and amine• bond at cystein

3. Albumin adducts (blood):• Long term parameter, covers body dose of last 3 weeks• Isocyanate specific• bond at lysin

4. Globin adducts (blood):• Long term parameter, covers body dose of last 3 months (cumulative)• Isocyanate specific• bond at valin; named: MDA-Val-Hyd• No human data available; knowledge derived from rats

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WHY IS SPECIFICITY SO IMPORTANT?

Fact: at many work places there is a potential simultaneous exposure tothe isocyanate MDI as well as to the aromatic amine MDA (Methylenedianiline)

Problem: Both substances are very different from toxicological point of view:MDA – Focus cancer (cat. 2)MDI – Focus sensitization / allergy

Urine marker shows sum of MDI + MDA body burdenGlobin-marker shows selectively MDI – body burden

Therefore: Globin-marker MDA-Val-Hyd is a specific marker for MDI!

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KNOWN FROM THR RAT

Sabbioni, Chem. Res. 2000

MAK: 0.050 mg/m³!

Hemoglobin-adduct (Cystein)

Globin-adduct (Valin)

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…AND HUMANS?

Questions to be answered:Is MDA-Val-Hyd formed in humans following MDI-exposure?In case it is, is it possible to measure it very sensitive?

Approach:1. Development of a sensitive as well as robust analytical method for the

determination of MDA-Val-Hyd

2. Applications:occupational medicineenvironmental medicine

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GLOBIN ADDUCT OF MDI

Synthesis of standard (Ehrenstorfer, Germany):5-Isopropyl-3[4-(4-aminobenzyl)phenyl]hydantoin

ABP-Val-Hyd = MDA-Val-Hyd

N

HN

NH2

O

O3

3

DCD

CD

CD

N

HN

NH2

O

O

Synthesis of internal standard (Ehrenstorfer, Germany): 5-Isopropyl-3[4-(4-aminobenzyl)phenyl]hydantoin-d8

ABP-Val-Hyd-d8 = MDA-Val-Hyd-d8

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METHOD DEVELOPMENT

Step DetailsErythrocytes –separation

Centrifugation of EDTA-blood sample, washing of erythrocytes with NaCl, water adding

Hemolysis of erythrocytes

- 20 °C

Globin-isolation Solution is denaturated first with HCl/isopropanol, thenwith ethylacetate

Acid hydrolysis + HCl, + ISTD (Aim: Cleavage of MDA-Val-Hyd fromterminal valin)

Extraction /derivatization

Chloroform / Heptafluoro butyric acid anhydrid (HFBA)

Gas-chromatography

Agilent GC HP 5890, Rtx 5 MS 20 m x 0.32 mm x 0.5 µm

Detection Waters AutoSpec Ultima; GC-HRMS-NCI

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DETECTION OF MDA-Val-HYD

LOQ: 0.05 ng/g MDA-Val-Hyd/l blood (0.155 pmol/g globin)

Substance m/z Rt (min)

MDA-Val-Hyd-HFBA 499.1331 ca. 8:50

MDA-Val-Hyd-HFBA-d8 507.1833 ca. 8:50

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PRECISION DATA OF MDA-HYD

56 spiked globin samples, analyte concentrations: 0.1, 0.5, 1.0 and 2.0 ng/g:

Precision in series: relative standard deviation between 3.0 and 9.3 %

Precision from day to day (on 5 days): relative standard deviation between 8.3 and 9.8 %

Fact: excellent precision data due to application of deuterated internalstandard (similar fragmentation, compensation of analytical variabilities)

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spiked globin,0.31 pmol/g globin

CHROMATOGRAM FROM SPIKED GLOBIN

MDA-Val-Hyd

ISTD

LOQ: 0.155 pmol/g

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CHROMATOGRAM FROM REAL SAMPLE

Real sample, 3.47 pmol/g globin Real sample, <0.155 pmol/g globin

MDA-Val-HydMDA-Val-Hyd

ISTD ISTD

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WORKERS FROM MDI-PRODUCTIONUrine Hb-Adduct Hb-Adduct Ratio

Worker MDA MDAMDA-Val-

HydMDA-Val-Hyd/

MDA

No.(pmol/g

creatinine) (pmol/g) (pmol/g)

17 20860 1.05 15.94 15.228 124490 0.63 9.63 15.329 18030 0.42 3.47 8.331 <500 <0.35 0.80 -255 <500 <0.35 1.89 -425 1480 0.41 9.82 24.0496 no sample 0.53 6.33 11.9851 4370 0.35 7.02 20.0897 no sample 1.09 15.26 14.0926 9820 1.12 16.24 14.5930 <500 <0.35 0.60 -

MDA in urine >>

globin-adduct >

Hb-adduct

Mean ratio:

MDA-Val-Hyd/MDA16.9 (± 4.8)

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40 persons without any known MDI-exposureBiomonitoring < LOQ

BACKGROUND LEVELS

Application with protection clothes of one-component-PU-foam (containing10 % free MDI) for 20 minutes; no dermal contact; only inhalative

2 - 5 µg MDI/m³ air (MAK: 50 µg/m³)Biomonitoring < LOQ

1. No known exposure:

2. Low inhalative exposure:

3. Provocation experiments: 3 - 20 µg MDI/m³ air Biomonitoring > LOQ

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SUMMARY MILESTONES

1. Development of a sensitive and robust analytical method (GC-HRMS) successfully managed

2. Specific MDI-marker in humans is MDA-Val-Hyd• Blood marker (adduct) covering body burden of the last 3 months• Differentiation between isocyanate and aromatic amine body burden

1. Application in MDI-exposed workers: MDA-Val-Hyd detected

2. Background level of MDI No

More information in our publication: Gries, Leng: Analytical determination of specific 4,4´-methylene diphenyl diisocyanate hemoglobin adducts in human blood. Analytical and Bioanalytical Chemistry (published online), 2013

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Synthesis of reference standards: International Isocyanate Institute

Method development: part of project on human biomonitoring as a cooperation betweenBMU (Federal Ministry for the Environment, Nature, Conservation and Nuclear Safety) andVCI (German Chemical Industry Association)

My biomonitoring team

ACKNOWLEDGEMENT