International Journal of Hygiene and Environmental Health 220 (2017) 103–112

Contents lists available at ScienceDirect

International Journal of Hygiene and

Environmental Health
journal homepage: www.elsevier.com/locate/ijheh

New human biomonitoring methods for chemicals of concern—the

German approach to enhance relevance
Marike Kolossa-Gehring a,∗,1 , Ulrike Fiddicke a,1 , Gabriele Leng b , Jürgen Angerer c ,
Birgit Wolz d
a
German Environment Agency (Umweltbundesamt), Dessau-Roßlau, Berlin, Germany
b
Currenta GmbH & Co. OHG, CUR-SI-GS-Biomonitoring, Leverkusen, Germany
c
Institute for Prevention and Occupational Medicine of the German Social Accident Insurance (IPA), Germany
d
Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety, Germany

a r t i c l e i n f o a b s t r a c t

Article history: In Germany strong efforts have been made within the last years to develop new methods for human
Received 9 September 2016 biomonitoring (HBM). The German Federal Ministry for the Environment, Nature Conservation, Building
Received in revised form 27 October 2016 and Nuclear Safety (BMUB) and the German Chemical Industry Association e. V. (VCI) cooperate since
Accepted 28 October 2016
2010 to increase the knowledge on the internal exposure of the general population to chemicals. The
projects aim is to promote human biomonitoring by developing new analytical methods Key partner of
Keywords:
the cooperation is the German Environment Agency (UBA) which has been entrusted with the scientific
Human biomonitoring
coordination. Another key partner is the “HBM Expert Panel” which each year puts together a list of
Method development
German environmental survey
chemicals of interest to the project from which the Steering Committee of the project choses up to five
Environmental specimen bank substances for which method development will be started. Emphasis is placed on substances with either
a potential health relevance or on substances to which the general population is potentially exposed to
a considerable extent.
The HBM Expert Panel also advises on method development. Once a method is developed, it is usually
first applied to about 40 non-occupationally exposed individuals. A next step is applying the methods to
different samples. Either, if the time trend is of major interest, to samples from the German Environmental
Specimen Bank, or, in case exposure sources and distribution of exposure levels in the general population
are the focus, the new methods are applied to samples from children and adolescents from the population
representative 5th German Environmental Survey (GerES V). Results are expected in late 2018.
This article describes the challenges faced during method development and solutions found. An
overview presents the 34 selected substances, the 14 methods developed and the 7 HBM-I values derived
in the period from 2010 to mid 2016.
© 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abbreviations: ASE/ASEP, Mesamoll®, C10-C20 Alkylsulfonic acid phenylester; BAuA, Federal Institute for Occupational Safety and Health; BfR, Federal Institute of Risk
Assessment; BHT, Butylated Hydroxy Toluene, 2,6-Di-tert-butyl-p-cresol; BMUB, German Ministry for the Environment, Nature Conservation, Building and Nuclear safety;
CIT/MIT (3:1), 5-Chloro-2-methyl-4-isothiazolin-3-one/2-Methylisothiazol-3(2H)-one; CMR, carcinogenic, mutagenic, reprotoxic; DEHTP, Di(2-ethylhexyl) terephthalate;
DINCH, 1,2-Cyclohexane dicarboxylic acid diisononyl ester; DPHP, Di(2-propylheptyl) phthalate or Bis(2-propylheptyl) benzene-1,2-dicarboxylate; ESB, German Environ-
mental Specimen Bank; GerES, German Environmental Survey; HBM, human biomonitoring; HBCDD, Hexabromocyclododecane; Hexamoll® DINCH®, 1, 2-Cyclohexane
dicarboxylic acid diisononyl ester or Diisononyl cyclohexane-1, 2-dicarboxylate; LOD, Limit of detection; LOQ, limit of quantification; 4-MBC, 3-(4-Methylbenzylidene)
camphor; 2-MBT, 2-Mercaptobenzothiazole; MDI, Methylenediphenyl-diisocyanate; NEP, N-ethyl-2-pyrrolidone; NMP, N-methyl-2-pyrrolidone; RKI, Robert Koch-Institute;
TDCP, Tris(2-chlor-1-(chlormethyl)ethyl)phosphat; UBA, German Environment Agency; VCI, German Chemical Industry Association.
∗ Corresponding author at: Umweltbundesamt, P.O. Box 33 00 22, 14191 Berlin, Germany.
E-mail addresses: [email protected] (M. Kolossa-Gehring), ulrike.fi[email protected] (U. Fiddicke).
1
Both authors contributed equally to this manuscript.

http://dx.doi.org/10.1016/j.ijheh.2016.10.012
1438-4639/© 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
104 M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112

1. Introduction and background 2007b; Angerer et al., 2011; Apel et al., 2017) which derives HBM
values from toxicological data. The HBM Commission was estab-
The exposure of the German population to chemicals is substan- lished in 1992 and has the mandate to support the UBA by giving
tially related to the use and production of chemicals, to products advice concerning HBM related issues and health-related environ-
and consumer goods, to the contamination of food as well as mental monitoring. The Commission members are appointed by the
todayı́s living conditions. Their safety for human beings and the President of the UBA. They are independent scientists and experts
environment has to be safeguarded by producers and the gov- from authorities at the Federal and Bundesländer (Federal States)
ernment. One important tool for identifying exposure levels and level, universities, public health institutes and clinical institutes.
sources, informing policy decisions and evaluating the success of In the following, the operation (modus operandi) and the
risk reduction strategies is human biomonitoring (HBM) (Angerer, achievements reached so far by the BMUB/VCI cooperation are
2012; Kolossa-Gehring et al., 2012). HBM is the most appropriate described.
scientific technique for assessing human exposure to environmen-
tal pollutants based usually on sampling and analysis of blood
or urine. By linking the biomonitoring results with environmen- 2. Methods
tal and health data or by a toxicologically/epidemiologically based
assessment of the exposure levels HBM can build bridges between 2.1. Identification of chemicals of relevance
exposure to pollutants and health effects and can give indication
for appropriate public health measures. Time trends of exposure The HBM Expert Panel consists of experts from the Federal Ger-
and different exposure levels of sub-groups of the population can man scientific agencies in charge of chemicals regulation, that is the
also be detected (Schulz et al., 2007a,b). Federal Institute for Risk Assessment (BfR), the Federal Institute for
Exposures of concern, such as to heavy metals like lead, to per- Occupational Safety and Health (BAuA) and the UBA as well as of
sistent organic pollutants like PCB and DDT, to PAHs taken up from experts from chemical industry enterprises and from universities
outdoor air pollution and to phthalates used e. g. as plasticizers and other scientific institutions. This panel provides advice on the
have been efficiently reduced by the introduction of risk reduc- selection of substances and the development of methods.
tion measures like restriction in use or complete bans of chemicals. A steering committee composed of representatives of the BMUB,
However, banned or restricted chemicals are often substituted by the UBA, the VCI and VCI member companies decides each year for
substances for which no data on internal exposure and/or no toxico- which substances new methods are to be developed.
logically derived guidance values for an assessment of their health The idea for the project was created in 2009 in a joint BMUB/VCI
relevance are available. Without data it is not possible to distin- meeting on HBM. An agreement was negotiated and the project
guish between recommendable and problematic substitutes or to then started in 2010. To get the work of the HBM Expert Panel
give advice to the general population on how to efficiently reduce started, a list of chemicals of interest was put together by the BfR,
an impact on health from the exposure to chemicals (Zota et al., the BAuA and the UBA. It was based on at that time available lists
2014; Schütze et al., 2014; Göen et al., 2011). HBM should thus also of nationally and internationally relevant substances already com-
be used to investigate substitutes. piled by other experts. The first focus was laid on the REACH-Annex
In the past HBM methods have primarily been developed for XIV-candidate list and the list of PBT/vPvB- substances. For the
chemicals relevant at the workplace. Later on, a set of methods following year a list of 120 substances of interest was set up.
for heavy metals, persistent compounds, biocides and industrial Table 1 gives an overview about the most relevant substance
chemicals relevant for the general population has been established. lists which were considered. Additionally the scientific Federal
Most of the HBM studies worldwide measure a similar set of chem- Agencies identified chemicals of interest from their respective
icals today (Kolossa-Gehring, 2012). In many cases the awareness fields of competence which had raised concern or were under dis-
of exposure and the subsequent development of appropriate risk cussion in science or in the public. Representatives from industry
reduction measures led to a reduction of exposure. Subsequent also proposed additional chemicals for method development.
studies observed and followed up these reductions. The above mentioned list is regularly reviewed in the course
In Germany, a cooperation was set up in 2010 to broaden the of the cooperation and supplemented as needed. Any expert, or
toolbox of HBM methods and thus enhance the use of HBM for institution, participating in the HBM Expert Panel, can suggest new
policy making. The German Ministry for the Environment, Nature substances. It is, however, required that any suggestion for a new
Conservation, Building and Nuclear safety (BMUB) and the Ger- substance is accompanied by a fact sheet with i.a. toxicological
man Chemical Industry Association (VCI), supported by the German information.
Environment Agency (UBA) and an “HBM Expert Panel”, identify In that list the substances are classified in 13 groups: 1)
chemicals with potential consumer exposure and potential health Phthalates and substitutes; 2) Flame retardants; 3) Per-and
relevance for the general population which up to now cannot be polyfluorinated alkyl substances (PFAS); 4) Chemicals used in
(reliably) measured by HBM. Each year up to five chemicals are cho- cosmetics; 5) (Musk) fragrances; 6) Allergenic substances; 7)
sen for the development of new HBM methods (Kolossa-Gehring (Phenol-)Benzothiazoles; 8) SVHC candidates (REACH Art. 57); 9)
et al., 2012; Kolossa-Gehring 2012). Aromatic amines; 10) Metals; 11) Nano particles; 12) Contami-
The VCI has taken on the responsibility of developing the new nants in food; 13) Others. Substance attribution to a group follows
detection methods. While advised by the HBM Expert Panel, it is either its chemical constitution or use (e.g. Phthalates, including
the task of the VCI to commission an analytical laboratory which it other plasticizers) or its potential health effect (Allergenic sub-
considers suitable for developing a valid analytical method. stances) depending on the main characteristics. The basic criteria
Applying the methods in suitable studies and interpreting their for selection of substances besides their toxicology are their good
results is the responsibility of the BMUB in close cooperation with or very good bioavailability (health relevance), a high likelihood
the UBA. Thus there is a clear division of responsibilities which is a of consumer exposure (consumer relevance) and non-existence or
core feature of the project. unsuitability of an existing HBM-method (for mother compound or
Reliable exposure data alone, however, does not allow to evalu- metabolites).
ate the health relevance of a given level of exposure. That evaluation Once a year, following a scientific discussion, the HBM Expert
is performed outside of the cooperation project. It is performed Panel releases a short list of proposed substances with sufficient
by the German Human Biomonitoring Commission (Schulz et al., data available to classify them as eligible for the cooperation
 M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112 105

Table 1 Table 2
Substance lists considered for the start of the BMUB/VCI cooperation in 2009. Characteristics of substances which hamper their use as a valid biomarker.

List of carcinogenic, mutagenic, reprotoxic (CMR) substances (as from January Substance characteristic Disadvantage
2009)a
Ubiquitously present in the Possibility of exogenous contamination
REACH-Annex XIV-candidate listb (as from March 2009)
environment (may lead to overestimation of
List of PBT/vPvB- substancesc (67/548/EECd and 793/93/EECe as from May
exposure)
2008):127 substances
Formed naturally in the body and No distinction between internal body
SVHC-candidate listf (34 substances, as from March 2009)
excreted in high concentrations dose and external supply possible (may
ICCAg HPV-list (International Council Chemical Associations, as from October
lead to overestimation of exposure)
2005)
Rapidly metabolized or eliminated Can easily result in an underestimation
OECD-HPV-Listh (as from 2004)
in humans (short half-live) of exposure
EDS-List (List of endocrine disrupting substance, Annex 1 of the EU-strategy) i
Formed or excreted in very small Can easily result in an underestimation
NORMAN-List of “Emerging Substances”j (as from March 2009)
amounts of exposure
NHANES-substancesk (National Health and Nutrition Examination Survey, USA)
Metabolite of several substances Specificity is insufficient (may lead to
HESIl -List (bioaccumulation, Health and Environmental Science Institute, USA).
overestimation of exposure)
a
Regulation (EC) No 1272/2008 of the European Parliament and of the Coun-
cil of 16 December 2008 on classification, labelling and packaging of substances
and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and human metabolism of the substance of concern is disclosed. Only
amending Regulation (EC) No 1907/2006 (Text with EEA relevance) http://eur-lex.
the appropriate biomarker (which might also be the mother sub-
europa.eu/eli/reg/2008/1272/oj.
b
echa.europa.eu/documents/10162/13585/pr 09 01 consultation substances
stance) provides a promising starting point for the development
authorisation 20090114 en.pdf. of an analytical method. To find this appropriate biomarker, i. e.
c
The TC NES sub-group on identification of PBT and vPvB substances that metabolite which reliably reflects internal human exposure to
assessed suspected PBTs (persistent, bioaccumulative and toxic) and vPvBs (very the substance in question, different actions are to be undertaken
persistent and very bioaccumulative) under the previous EU chemicals leg-
depending on the level of knowledge about the metabolism.
islation. https://echa.europa.eu/information-on-chemicals/pbt-vpvb-assessments-
under-the-previous-eu-chemicals-legislation. Each substance in question needs a separate, profound evalu-
d
Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, ation on the necessity to perform a short or extended study on
regulations and administrative provisions relating to the classification, packaging human metabolism depending on the existing knowledge. Table 3
and labeling of dangerous substances.
e
lists main aspects to consider for the decision of the necessity of
EC (European Commission), 1993. Council Regulation 793/93/EEC of March 1993
on the evaluation and control of risks of existing substances. Official Journal of the
a human metabolism study and examples within the BMUB/VCI
European Communities, L48/1. cooperation. Five levels of knowledge can be distinguished pro-
f
https://echa.europa.eu/candidate-list-table. voking different actions.
g
https://www.icca-chem.org/. The bigger the knowledge on human metabolism, the less com-
h
www.oecd.org/chemicalsafety/risk-assessment/33883530.pdf.
i plex is the preliminary work for method development.
Candidate list of 553 substances ec.europa.eu/environment/archives/docum/
pdf/bkh annex 01.pdf. Studies on human metabolism are also needed to derive toxi-
j
http://www.norman-network.net/?q=node/81. cology based guidance values like the HBM values of the German
k
Chemicals measured in selected participants for NHANES 2003–2004: http:// Human Biomonitoring Commission. Each human metabolism study
biomonitoring.ca.gov/downloads/chemicals-measured-selected-participants- has to have an authorization of an ethics commission.
nhanes-2003-2004-0.
l
http://hesiglobal.org/.
2.3. Method development

project. Then, the Steering Committee selects up to 5 substances After VCI has subcontracted a laboratory for method devel-
for that year for which HBM method development will be started. opment, the work can start. Besides the above mentioned
When deciding on the substances of choice, consideration is given recommendations for finding the appropriate biomarker, each
to whether the VCI is able to find industrial sponsors for the sub- method development should obey the following stipulations:
stance whose expertise is required for the effective development
of analytical methods. Therefore, it turned out to facilitate the pro- 1) The method should preferably be developed for urine (non-
cedures if the producer of a chosen chemical is located in Germany. invasive matrix) to facilitate the use in studies on children.
With the project running for 10 years, up to 50 substances shall Collection in conventional plastic vessels should be possible.
thus be selected until 2020. 2) The methods will afterwards be applied to large population stud-
ies, therefore the method should run in fast routine on devices
2.2. Biomarker identification which are regularly found in human biomonitoring laboratories.
The method should, if possible, not be cost-intensive.
The agreement between the BMUB and the VCI puts empha- 3) A low limit of quantification (LOQ) is necessary, suitable for
sis on developing a “valid method” in order to be able to properly environmental exposure of the general population, including
assess exposure levels. In the course of the project and discussion in children and adolescents. The LOQ must be well below the LOQ
the HBM Expert Panel, the conditions for an appropriate approach for occupational exposure, preferably be as low as 1 ␮g/l or less.
for method developing were established. One condition relates In general, a concentration of 0,1 ␮g/l urine or plasma should be
to the contracted laboratories: it is recommended that contracts achieved.
for method development should only be awarded to laboratories 4) Applying the new method to 30–40 occupationally non-exposed
which have sufficient experience in human biomonitoring. It is participants is also part of the method development.
of advantage if the laboratories are routinely involved in human
biomonitoring and do scientific work in this field. Fig. 1 provides a flow chart about the method development.
Another condition relates to the biomarkers: The HBM Expert Regularly, the first step of the method development is the syn-
Panel agreed that the key challenge in method development is to thesis of the analytical standards (substance which is supposed to
choose the right biomarker. Table 2 lists substance characteristics be the marker) and the internal standards (e.g. 13 C or deuterium
which are contradictory to a qualification as a biomarker. labeled standard). During the course of the cooperation project the
The use of an ill-suited biomarker may result in over- or under- development of these standard synthesis has shown to be some-
estimation of the exposure (Table 2). This can be circumvented if the times a time consuming factor.
106 M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112

Fig. 1. The flow chart shows the different steps of the development of an analytical method within the BMUB/VCI cooperation and depicts the involvement of the various
parties.
 M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112 107

Table 3
Necessity of a human metabolism study for detection of an appropriate biomarker.

Knowledge level Biomarker Human metabolism study Finalization of method Examples (full names see
development Table 4)

1: Human metabolism known identified Not necessary Directly possible HBCDD, 4-MBC
or results of animal studies
can be used
2: Metabolism of substances a relevant biomarker can be Recommended to validate the Possible after supporting DPHP, DEHTP (analogue to
with a similar structure are assumed on the basis of these assumed biomarker metabolism studies DEHP); DEHTP: also rested on
known (can be supported by findings in vitro studies;
results of in vitro studies) NEP (analogue to NMP)
3: Knowledge on human Not known; development can Necessary Only possible after metabolism CIT/MIT (3:1);
metabolism fragmentary or be complex, time-consuming is disclosed 4-tert-Nonylphenol branched;
lacking (without possibility and costly ASE/ASEP
to derive such findings)
4: No studies on human Possibly derived from in vitro Not allowed! Targeted Population background TDCP
metabolism exist or animal studies data administration of such exposure as basis for
(carcinogenic, mutagenic or substances is prohibited in metabolism extrapolations
reproduction-toxic humans.
substances)
5: Knowledge on metabolism Biomarkers from Maybe necessary after Identification of a suitable Chloroparaffins: consist of over
impeded due to complexity environmental monitoring complexity could be solved biomarker still requires major 10,000 individual compounds,
of the substances may not be suitable research difficult to separate
(multi-component isomer
mixtures)

Important steps and occurring problems during method devel- All five substances selected in 2010 have successfully passed
opment are addressed at the semiannual meetings of the HBM the validation process of the DFG (HexamollTM DINCHTM , DPHP,
Expert Panel. HBCDD, branched 4-Nonylphenol and 4-tert-Octylphenol). The
As part of the method validation, the procedure is cross- method developed for the simultaneous analysis of two alkylphe-
examined by a second laboratory from the ranks of the scientific nols (Nonyl- and Octylphenol) has the disadvantage that
working group “Analysis in Biological Materials” of the “Perma- preferentially glass vessels have to be used for urine collec-
nent Senate Commission for the Investigation of Health Hazards of tion to achieve a low LOQ (mother substance is subject to
Chemical Compounds in the Work Area” of the German Research contamination as alkylphenols occur ubiquitously). This is not man-
Foundation (DFG). The method including the examination results ageable in routine HBM-studies. First application results in 40
will be published online by the DFG in open access. This proce- not-occupationally exposed persons with 4-NP did not show any
dure turned out to be another time consuming factor. Therefore, a detectable amount. Additionally, the HBM-Commission of UBA had
method is referred to as “finalized” after a scientific paper has been doubts that the mother substance is an appropriate biomarker for
published in a peer-reviewed journal. 4-tert-Nonylphenol and favoured a human metabolism study to
search for other markers, i.e. metabolites to be detected in urine.
The metabolism study is ongoing – here the mother substance as
2.4. Substances selected and methods developed well as postulated metabolites are measured. The results will clar-
ify the best suitable marker and will be published peer reviewed.
From 2010 to 2016 the Steering Committee of the cooperation The correct marker can then be applied to human samples.
has selected 34 substances for method development. While aiming Method development for all substances selected in 2011 has
at 5 chemicals per year, in 2015 only four substances have been been completed.
selected (Table 4, also shows all full substance names). Eight of One substance selected in 2012 has gone through the develop-
the selected substances are phthalates and their substitutes (Group ment process, one other is still on its way. Method development
1). Two substances are flame retardants (Group 2); ten substances of three substances selected in 2012 did not lead to an acceptable
are chemicals used in cosmetics (Group 4); five substances belong result. Even though significant effort was spent, it was not possible
to Group 6 (allergenic substances), three substances to Group 7 to develop an appropriate and sufficiently sensitive HBM method
((Phenol-)Benzothiazoles) and six substances are summarized into for the simultaneous detection of Octamethylcyclotetrasiloxan,
Group 13 (Others). D4 (CAS 556-67-2), Decamethylcyclopentasiloxan, D5 (CAS 541-
The chemicals selected thus belong to five of the 13 groups 02-6) and Dodecamethylcyclohexasiloxan, D6 (CAS 540-97-6) in
from the priority substance list. Substances of other groups have urine. Method development failed because these cyclo-siloxanes
been discussed in the HBM Expert Panel but not proposed to the are metabolized to the same metabolite (dimethyl-siloxane), so
Steering Committee e.g. because of unclear consumer exposure, no separation between these three cyclo-siloxanes would be
very short half-lives (selected PFAS), already existing HBM methods possible which is important for taking substance specific mea-
(Limonene or environmental phenols like Triclosan or Parabenes), sures (e.g. regulation). Additionally there is a high background
or because method development for selected structural analogues contamination with dimethyl-siloxane which exacerbates analyt-
shall be bided (Homosalate, UV-327, OIT and BIT). Other substances ical and pre-analytical procedures (risk of contamination), and
proposed by the HBM Expert Panel have not yet been selected by the attainable limit of detection would be 10 fold higher than
the Steering Committee mostly because it is difficult to oblige an intended by the HBM Expert Panel. Thus, method development was
industrial sponsor especially for substances not produced or traded stopped.
in Germany (musk fragrances, some flame retardants). 3 out of 5 methods for substances selected in 2013 have been
Table 4 gives an overview of the substances selected and meth- developed, and 1 out of 5 for substances selected in 2014. Method
ods developed so far. Up to now a total of 14 new methods has been development for the substances selected in 2015 and 2016 is in
developed. Six of them have been published in peer-reviewed jour- process or has just begun.
nals (HexamollTM DINCHTM , DPHP, DEHTP, MDI, 2-MBT, Lysmeral).
 108
Table 4
BMUB/VCI-Cooperation: Prioritized chemicals and developed HBM methods 2010–2016 .

Year of selection Name (CAS-No.) Use Method development Biomarker Volume Method; Limit of
Matrix Quantification (LOQ)

2010 DINCH Plasticizer: phthalate Completed, published. OH-MINCH, 0,3 mL Urine HPLC–MS/MS
1,2-Cyclohexane substitute in toys, packing Validation by the German cx-MINCH, LOQ: all 0,05 ␮g/l
dicarboxylic acid material for food, medical Research Foundation (DFG) oxo-MINCH
diisononyl ester devices is completed.
(166412-78-8 Isomere
mixture)

M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112
DPHP Plasticizer: technologically Completed, published. oxo-MPHP (1), 1,0 mL Urine GC/HRMS
Di(2- replaces the phthalate Validation by DFG is in OH-MPHP (2), LOQ: (1) 0,25 ␮g/l;
propylheptyl)phthalate DIDP in PVC-products for process. cx-MPHxP (3) LOQ: (2) 0,3 ␮g/l;
(53306-54-0) technical applications LOQ:(3) 0,15 ␮g/l
HBCDD Flame retardant Completed. Validation by ␣-HBCDD; 1,0 mL Plasma LC–MS/MS
Hexabromocyclododecane DFG is completed. ß-HBCDD; LOQ:
(25637-99-4) ␥-HBCDD all 0,1 ␮g/l
4-Nonylphenol, branched Starting material for the Completed. Validation by 4-Nonylphenol, branched. 0,5 mL Urine HPLC–MS/MS
(84852-15-3) synthesis of nonylphenol DFG is completed. A metabolism study is One shot for LOQ:
ethoxylates, monomer for being performed for both all 1,0 ␮g/l
phenolic resins. validation substances
Antioxidant/Stabilizer in
technical oils
4-tert-Octylphenol Intermediate for the Completed. Validation by 4-tert-Octylphenol
(140-66-9) preparation of synthetic DFG is completed.
resins (used for rubber
production for tyres) and
non-ionic surfactants

2011 4-MBC UV-filter for cosmetics Completed. Submission to MBC-cx 0,5 mL Urine HPLC–MS/MS
3-(4-Methylbenzyliden)- DFG in 2016. (3-(4-Carboxy- MBC-cx:
camphor benzylidene) camphor) LOQ: 1,5 ␮g/l
(36861-47-9) and MBC-OH:
MBC-OH LOQ: 1,0 ␮g/l
(3-(4-Carboxy-
benzylidene)-6-
hydroxycamphor)
MDI Insulating foam Completed, published. 5-isopropyl-3-[4-(4- 5 mL EDTA-stabilized GC-HRMS-NCI
Methylenediphenyl- Validation by DFG is in aminobenzyl)phenyl]hydantoin whole blood LOQ:
diisocyanate process. ABP-Val-Hyd (Hemoglobin 0,05 ng ABP-Val-Hyd/g
(101-68-8 and adduct) Globin
26447-40-5)
2-MBT Vulcanization accelerator Completed, published. 2-MBT 0,5 mL Urine HPLC–MS/MS
(2-Mercaptobenzothiazole) Validation by DFG is LOQ: 1,0 ␮g/l
(149-30-4) completed.
NMP Solvent in many technical Completed. Validation by 5-Hydroxy-N-Methyl-2- 1,0 mL GC-EI-MS/MS
N-Methyl-2-pyrrolidone applications, ingredient in DFG is in process. pyrrolidone One shot for LOQ:
(872-50-4) paint, graffiti remover and (5-HNMP), NMP and NEP all 2,5 ␮g/l
carpeting 2-Hydroxy-N-methyl- analysis
succiminide
(2-HMSI)
NEP Polar aprotic solvent, Completed. Validation by 5-Hydroxy-N-
N-Ethyl-2-pyrrolidone substitute for NMP, e. g. for DFG is in process. Ethylpyrrolidone (5-HNEP),
(2687-91-4) surface coatings, in 2-Hydroxy-N-
cleaning agents and paint ethylsuccinimide
strippers (2-HESI)
2012 CIT/MIT (3:1) Preserving additive in Completed. Validation by NMMA
5-Chloro-2-methyl-4- cosmetics and industrial DFG is in process.

M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112
isothiazolin-3-one/2- products, biocides
Methylisothiazol-3(2H)-
one
(55965-84-9)
Geraniol Fragrance In process.
(106-24-1)
D4 Ingredient in Method
Octamethylcyclotetrasiloxane cosmetics development
(556-67-2) for urine not
D5 possible;
Decamethylcyclopentasiloxane finished
(541-02-6) without
D6 success.
Dodecamethylcyclohexa-
siloxane
(540-97-6)

2013 ASE/ASEP, Mesamoll® Plasticizer In process.

C10-C20 Alkylsulfonic acid
phenylester
(91082-17-6)
TOTM Plasticizer In process.
Tri(2-
ethylhexyl)trimellitate
(3319-31-1)
DEHTP Plasticizer Completed, published. 5cx-MEPTP (1), 0,3 mL Urine HPLC–MS/MS
Di(2- Validation by DFG is in 5OH-MEHTP (2), LOQ (1): 0,2 ␮L
ethylhexyl)terephthalate process. 2cx-MMHTP (3), LOQ (2): 0,3 ␮L
(DEHT, DOTP) 5oxo-MEHTP (4) LOQ (3): 0,4 ␮L
(6422-86-2) LOQ (4): 0,2 ␮L
OMC UV-filter for cosmetics In process.
Octylmethoxycinnamate
(5466-77-3)
BHT Antioxidant e.g. in food Completed. Validation by BHT acid LC–MS/MS
Butylated Hydroxy Toluene DFG is in process. LOQ: 0,1 ␮L
2,6-Di-tert-butyl-p-cresol
(128-37-0)

109
 110
Table 4 (Continued)

Year of selection Name (CAS-No.) Use Method development Biomarker Volume Method; Limit of
Matrix Quantification (LOQ)

2014 DINA, Diisononyladipate Plasticizer In process.

(33703-08-1)
DEHA Plasticizer In process.
Di-(2-ethylhexyl)adipate
103-23-1
OC, Octocrylene UV-filter for cosmetics In process.
(2-Ethylhexyl 2-cyano-3,3-
diphenyl-2-propenoate)

M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112
(6197-30-4)
TDI Foam and adhesives In process.
Tolylene-diisocyanate
2,4-TDI (584-84-9) and
2,6-TDI (91-08-7)
Lysmeral Fragrance Completed, published. TBBA (tert-butylbenzoic 1,0 mL Urine LC–MS/MS
2-(4-tert-Butylbenzyl) Validation by DFG is in acid) (1), Lysmerylic acid LOQ (1): 0,42 ␮g/l;
propionaldehyde process. (2), LOQ (2): 0,36 ␮g/l
(80-54-6) Lysmerol (3), LOQ (3): 0,10 ␮g/l
OH-Lysmerylic acid (4) LOQ (4): 0,45 ␮g/l

2015 EHS UV-filter for cosmetics In process.

2-Ethylhexylsalicylate
(Octisalate) (118-60-5)
Climbazole Cosmetics preservative
1-(4-Chlorophenoxy)-1-
(imidazol-1-yl)3,3-
dimethylbutan-2-one)
(38083-17-9)
UV 328 UV-absorber for plastics
P and paints
2-(2H-Benzotriazol-2-yl)-
4,6-di-tert.pentylphenol
(25973-55-1)
7-Hydroxycitronellal Fragrance
(107-75-5)

2016 TDCP Flame retardant Method

Tris(1,3-dichloroisopropyl) development
phosphate needs at least
(13674-87-8) two years.
Avobenzone (BMDBM) UV-filter in cosmetics
Butylmethoxydibenzoyl
methane
(70356-09-1)
Keromet MD Fuel additive
␣,␣‘-(1-methylethylene
diimino)di-o-cresole
(94-91-7)
DBA, Dibutyl adipate Plasticizer, Ingredient of
(105-99-7) cosmetics
Uvinul A Plus UV-filter in cosmetics
Hexyl 2-(1-(diethylamino
hydrox-
yphenyl)methanoyl)
benzoate
(302776-68-7)
 M. Kolossa-Gehring et al. / International Journal of Hygiene and Environmental Health 220 (2017) 103–112 111

Table 5
Human-Biomonitoring (HBM) values for blood and/or urine.

Analyte and sample material Population groups HBM I value HBM II value

Sum of Hexamoll® DINCH® metabolites Children 3,0 mg/l /

OH-MINCH and cx-MINCH in urine [2015] Adults 4,5 mg/l
Sum of DPHP metabolites Children 1,0 mg/l /
OH-MPHP and oxo-MPHP in urine [2015] Adults 1,5 mg/l
Hexabromcyclododecane (HBCDD) [2015] General population 0,3 ␮g/g lipid or /
1,6 ␮g/l blood
plasma
2-Mercaptobenzothiazole (2-MBT) in urine [2015] Children 4,5 mg/l /
Adults 7,0 mg/l
Sum of N-Methyl-2-pyrrolidone (NMP)-metabolites Children 10 mg/l 30 mg/l
5-Hydroxy-NMP and 2-Hydroxy-N-methylsuccinimid Adults 15 mg/l 50 mg/l
in urine [2015]
Sum of N-Ethyl-2-pyrrolidone (NEP)- metabolites Children 10 mg/l 25 mg/l
5-HNEP und 2-HESI in urine [2015] Adults 15 mg/l 40 mg/l
4-MBC metabolites 3–4CBC and 3–4CBHC Children 0,3 mg/l
in urine [2016] Adults 0,5 mg/l

This overview shows that method development is a time con- ing). After more than two decades of operation the ESB provides
suming process. Minimum time is two years from selection to a continuous historical record of the state of the environment in
method finalizing (without publishing) but this is only possible if Germany in this period. It allows the retrospective monitoring of
biomarker identification and biomarker standard synthesis don’t pollutants to identify temporal trends and spatial load differences.
face problems. Target compounds include those which had not yet been recog-
nized as hazardous when the specimens were archived (emerging
pollutants) or which could not be analyzed with the desirable pre-
2.5. HBM values derived
cision at that time. Thus, the ESB samples can be used to analyze
human exposure from the past using the analytical methods of the
The Human Biomonitoring Commission of the UBA derives
future.
toxicologically based HBM values to interpret the relevance
The newly developed methods for HexamollTM DINCHTM (Koch
of the exposure levels observed. Up to now HBM values
et al., 2013; Schütze et al., 2012, 2014, 2015a,b, 2016), DPHP (Gries
for seven substances of this cooperation have been derived
et al., 2012; Leng et al., 2014; Schütze et al., 2015b) and NMP/NEP
(HexamollTM DINCHTM , DPHP, NMP, NEP, HBCDD, 2-MBT, 4-MBC)
(Koch et al., 2014) have been applied in the ESB (Schütze et al.,
(Table 5). For CIT/MIT (3:1), Geraniol, DEHTP and Lysmeral discus-
2014, 2015b) and are being applied in GerES V samples. The 2-MBT-
sions are ongoing or in preparation. Derivation of HBM value for
method (Gries et al., 2015) is being applied in the current GerES
MDI failed because of missing metabolism data and the discussion
V, the HBCDD method is currently applied to ESB samples. Expo-
for HBM values for 4-tert-Nonylphenol and tert-Octylphenol will
sure levels in none of the samples analyzed up to now exceeded
go on after the metabolism study is finalized.
the respective HBM-I value. The method to detect MDI (Gries and
HBM values are regularly updated on the website of the
Leng, 2013) is based on the globin fracture found in whole blood.
HBM-Commission: https://www.umweltbundesamt.de/en/topics/
This procedure needs special attention and will be applied sepa-
health/commissions-working-groups/human-biomonitoring-
rately to human samples. All other methods are fairly new so they
commission-hbm-commission.
could not be integrated in ongoing studies and will be applied later
on (4-MBC, CIT/MIT, DEHTP (Lessmann et al., 2016a, 2016b), BHT,
2.6. HBM instruments Lysmeral (Pluym et al., 2016)).

On behalf of BMUB, the UBA employs two complementary HBM

tools – the German Environmental Survey (GerES) and the German Acknowledgements
Environmental Specimen Bank (ESB). The new methods, developed
within the cooperation to enhance HBM are applied to samples of Websites:
these studies. GerES: https://www.umweltbundesamt.de/en/topics/
GerES is a nationwide representative population study on health/assessing-environmentally-related-health-risks/german-
human and indoor air exposure and its sources which has repeat- environmental-survey-geres
edly been carried out in Germany since the mid-1980s. The three ESB: https://www.umweltprobenbank.de/en/documents
main instruments of investigation are human biomonitoring in BMUB/VCI cooperation: https://www.umweltbundesamt.de/
combination with indoor monitoring (air and tap water) as well as en/topics/health/assessing-environmentally-related-health-risks/
collecting information on possible exposure pathways and living human-biomonitoring/cooperation-for-the-promotion-of-human
conditions via questionnaires. GerES has always been conducted in
close co-operation with the National Health Interview and Exam-
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