Schweizerisches Zentrum für angewandte Ökotoxikologie | Eawag-EPFL CMEP Task 3.2 C- Effect Based...

Schweizerisches Zentrum für angewandteÖkotoxikologie | Eawag-EPFL

CMEP Task 3.2 C- Effect Based Monitoring Tools:

Effect based tools for hormonally active substances

Robert Kase Petra Kunz Cornelia Kienle Inge Werner

contact: [email protected] meeting, Praha the 30th June 2011

Structure

• Derivation of EQS and implementation in risk assessmentExample of EE2 a very potent estrogen receptor binding substance Overview of current EQS-proposals for waste water relevant micropollutants and EE2, E2, E1Detection possibilities

• How relevant are hormonally active substances and what are their effects?Environmentally relevant questions regarding fish toxicityResults of a Swiss wide risk assessment via biotests Options to reduce risks from estrogenic substances

• Which methods can be used to measure hormonally and reproduction toxic effects and what are the costs?Options to measure different mode of actionsSome cost and time factors for test procedures

• Overall summary and final conclusions

Seite 2 I Oekotoxzentrum | Eawag-EPFL

Derivation of EQS and Derivation of EQS and implementation in risk implementation in risk

assessmentassessment


Scope of Environmental Quality Standards (EQS)

EQS are based on reliable and relevant effect data.The critical step is to identify and to evaluate them.

– Goal: – protection against long-term exposure

– Comparison:– with the measured annual average

concentration (90th percentile)

– Data:– chronic effect data are preferred

• AA-EQS (Annual Average-EQS) must be derived as protection against the effects of long-term exposure

• MAC-EQS (Maximum acceptable concentration) against the effects of short-term exposure.

Because of the exposure scenario and the specific mode of action only an AA-EQS is proposed for

EE2 and E2


Risk Assessment = Exposure Assessement / Hazard Assessment

MEC= Measured environmental concentration (usually the 90th percentile)

QC= Quality criteria (usually the AA-EQS)

?QC

MEC (RQ)nt Riskquotie>1 intolerable risk

<1 tolerable risk

Source: Knacker 2007


Steps of the development of an EQS-proposal for the FOEN / BAFU

Source: Kase et al. 2011


http://www.oekotoxzentrum.ch/qualitaetskriterien

Coworking on the priority substances in the (Sub)Working Group E

Substances proposed for EQS derivationLeads and associated MS/stakeholders at 28th June 2010

Substance Lead for EQS derivation Associated MS/stakeholders

Methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate (Bifenox)

COM BE, ECPA

Terbutryn NL BE, DE, PT, ECPA

Cybutryne (Irgarol®) SE BE, CH, DE, (NL), ECPA

Cypermethrin NL BE, UK, PT, ECPA

Dichlorvos COM BE, (DE), ECPA

Heptachlor/Heptachlor epoxide COM BE

Polychlorinated biphenyls (PCBs) Multi-MS / COM BE, (FR), (IT), NL, PT, SE

Dioxin (2,3,7,8 - Tetrachlorodibenzo-p dioxin,TCDD) IT BE, SE, (FI)

Perfluorooctane sulfonic acid and its salts (PFOS) and perfluorooctane sulfonyl fluoride

UK BE, NL

1,2,5,6,9,10-Hexabromo-cyclododecane (HBCDD)/ 1,3,5,7,9,11-Hexabromo-cyclododecane (HBCDD)

SE BE, (AT), CEFIC

Quinoxyfen COM BE, PT, ECPA

Dicofol COM BE, ECPA

Cyanides COM BE, (NL), (UK), PT, CEFIC, CONCAWE, EUROFER, EUROMINES

Aclonifen COM BE, ECPA, PT

Diclofenac DE BE, CH, NL, PT, AESGP, EEB, EFPIA

17alpha-ethinylestradiol (EE2) UK BE, CH, DK, (NL), PT, EEB, EFPIA

Zinc UK BE, (FR), NL, CEFIC, EUROFER, Eurometaux

Ibuprofen DE BE, CH, EEB, AESGP, EFPIA

17 beta-estradiol (E2) UK BE, CH, DK, (NL), EEB, EFPIA

Aim: To develop harmonizedEnvironmental Quality Standards(EQS) and allow a border crossing risk management

In the future a regulation of EE2 and E2 is expected.

Prioritized wastewater relevant micropollutants from over 250 candidate substances

Contact persons: pharmaceuticals, industrial chemicals and hormone active substances / biocides, plant protection products and chelating agents

Robert Kase ([email protected]) Marion Junghans ([email protected])

Composition of 47 waste water relevant micropollutants

Pharmaceuticals; 21

Steroidal hormonactive substances; 3

Industrial chemicals; 6

Sweetener ; 2

Chelating agents; 2

Biocides and Plant Protection Products; 13

43% 27%


mailto:[email protected]

mailto:[email protected]

Hormone active and „endocrine disrupting“ effectsestrogenic effect androgenic effect

In both cases, the possible effects are:

• changes in the behaviour• changes in the fertility• intersex• population relevant effects

The steroidal hormone system is highly conserved among different taxa, so there is a widespread effect in animals and humans.

Most effects are considered relevant according to the TGD for EQS


Example EE2: SSD approach in the EU dossier

The EU EQS guidance notes than an assessment factor in the range of 1-5 should be applied to the HC5 derived. Based on the available dataset it is considered that an assessment factor of 2 is appropriate based on the data for EE2.

This AF is warranted because:

•The mode of toxic action is well understood. The HC5 has been derived based on data for two of the most sensitive taxonomic groups, i.e. fish and amphibians.

•In addition where several studies were available for a species the lowest reliable effect concentration has been used adding a further level of conservatism.

The HC5 from this distribution is 0.07 ng/l. Applying an assessment factor of 2 to this HC5 gives an AA-QSfreshwater,eco of 0.035 ng/l.


List of existing Swiss EQS-proposals and proposed EU EQS 33 EQS derivations were derived according to the Technical Guidance Document for EQS (TGD for EQS). The dossiers were sent to external reviewers for a plausibilty and quality check.

Substances

MAC-EQS-proposal

AA-EQS proposal

tentative AA-EQS-proposal

of the WG E

risk of secondary poisoning

Pharmaceuticals and steroidal hormonactive substances 17-alpha-ethinylestradiol no proposal 0.037 ng/l 0.035 ng/l not at this

concentration

17-beta-estradiol no proposal 0.400 ng/l 0.400 ng/l not at this concentration

Atenolol 330 µg/l 150 µg/l / Azithromycin 0.09 µg/l 0.09 µg/l / x Bezafibrat 76 µg/l 0.46 µg/l / x Carbamazepin 2550 µg/l 0.5 µg/l 0.5 µg/l Clarithromycin 0.11 µg/l 0.06 µg/l 0.1 µg/l x

Diclofenac 700 µg/l 0.05 µg/l 0.1 µg/l x would lead to 7ng/ L

Estron no proposal 3.6 ng/l / not at this concentration

Ibuprofen 23 µg/l 0.3 µg/l 0.01 µg/l x Mefenamic Acid 40 µg/l 4 µg/l / x Metoprolol 76 µg/l 64 µg/l / Naproxen 370 µg/l 1.7 µg/l / (x) Sulfamethoxazol 2.7 µg/l 0.6 µg/l 0.12 µg/l Trimethoprim 1100 µg/l 60 µg/l / x for these substances a risk of secondary poisoning exists, which is not numerically included yet

Estrogenic potency:

EE2

E2

E1

At least for some of the steroidal hormonactive substances (EE2 and E2) problems with the analytical LOQ are expected in routine methods.


Bioanalytical detection limits for hormone active substances

FSA

XE

MA

Pa-

Rep

ro 1

Pa-

Rep

ro 2

Dm

-Rep

ro

Chi

rote

st

MV

LN

YE

S M

cDon

.

YE

S S

umpt

er

YA

S S

umpt

er

H29

5R

ER

Cal

ux

AR

Cal

ux

E-S

cree

n

ELR

A

AW

AC

CS

Sam

ple

conc

entra

tion

in n

g/L

0.01

0.1

1

10

100

1000

10000

min. LOEC oder ECx max. LOEC oder ECx min. EC50 max. EC50

EE2

kein

e A

ngab

e

kein

e A

ngab

e

kein

e A

ngab

e

kein

e A

ngab

e

kein

e A

ngab

e

EE2

T

E2

EE2

E2

T

E2

DHT

E2

E2

Even bioanalytical methods reach detection limits below 1 ng/L. Moreover they can measure estrogenic effects in an integrative way, so they could be suitable as screening methods.

With these methods it is possible to measure integrative E2-equivalents (EEQ) with different cell based bioassays

Comparison of 5 in vivo and 10 in vitro test procedures. More information on the compared test-procedures can be found in (Kase et al. 2009)

Abbreviations: T= testosteroneDHT= dihydrotestosteroneE2=17-β-estradiolEE2= 17-α-ethinylestradiol


How relevant are How relevant are hormonally active hormonally active

substances and what are substances and what are their effects?their effects?


Environmentally relevant questions

Environmental Science and Technology 2005


Situation analysis 2005 and theories 2010

John Sumpter 2010, one Sunday morning in May at the Pharmaceutical Advisory Group in Sevilla:

•Presentation regarding “Pharmaceuticals in the Environment: What are the key unresolved issues?”

•STP effluents entering rivers are the main route of entry of pharmaceuticals into the environment.

•So, aquatic organisms, particularly fish, may be at greatest risk.”

Fish seem to be exposed at Diclofenac and EE2 concentrations above their effect levels, for many other substances it remains unclear.

2005


Estrogenic impacts on fish populations ?

• The SSD based EQS for E2 will be at 0.4 ng/L (derived from 11 chronic NOECs of different fish species)

• Fish species are the most sensitive taxonomic groups.

• The most sensitive study is done with rainbow trout (Lahnsteiner et. al. 2006) NOEC of 0.5 ng/L (for endpoints: fertilization success, sperm density and volume).

• E2, the metabolite estrone (E1) and the pharmaceutical 17-alpha ethinylestradiol (EE2) contribute additionally to the estrogen receptor mediated estrogenicity there is a cumulative risk.

• Additionally the ER receptor binding for several non steroidal substances (Dang 2011) indicate a need of integrative measurement methods to identify estrogenic potentials.


Waste-water

Primary clarifierMechanical treatment

PAC-UF treatment

Ozonation Sand filtration

Secondary clarifier

LF

EN

OZ SF

PAC-UF

Moving Bed biology

Results of the pilot study at the STEP de Vidy Lausanne with the YES and ER-Calux assays in 17β-estradiol-equivalents (EEQ) (4th measurement campaign):

Influent37.15 ng/L 9.8 ng/L

Biological Treatment3.03 ng/L; CILF/EN = 120.90 ng/L; CILF/EN = 11

PAC-UF Treatment0.29 ng/L; CIPAC/EN= 1260.16 ng/L; CIPAC/EN= 61

Ozonation + SF0.23 ng/L; CIOZ+SF/EN= 1600.15 ng/L; CIOZ+SF/EN= 65

Legend:Blue: YES-EEQ [ng/L]Red: ER-Calux-EEQ [ng/L]CITEQ = Change Index = (EEQ Treatment Step / EEQ Influent) -1

Conclusions: •Compared to the conventional biological treatment a 6-13 times lowered receptor binding estrogenicity results via additional wastewater treatment steps.•Without bioanalytical tests like YES- and ER-Calux a performance control of WWTP regarding the elimination of estrogenicity is hardly or not possible.

Example: Estrogenicity during wastewater treatment steps

AA-EQS proposal for E2 = 0.4 ng/L

Water quality during wastewater treatment

Main conclusions for bioassays applied in the FOEN-Project “Strategy Micropoll” :•A broad range of micropollutants and their effects were eliminated by more than 80% (by PAC-UF and Ozonation AF).•There was no evidence for a toxicity increase due to constant formation of stable toxic ozonation by-products.•An ozonation should be followed by a final filtration step with biological activity.•Quality of treated effluent was significantly improved, leading to improved surface water quality.

Report in prep.: Cornelia Kienle, Robert Kase, Inge Werner 2011

PAC-UF and Ozonation-SF can lead to 6-13 times lowered estrogenicity compared to the conventional biological treatment. Ozonation-SF can lead to more than 73 times lowered risk quotients for Diclofenac.

Conclusion: There are options developed and available to reduce ecotoxicological and fishtoxic effects of wastewater in surface waters.


Conclusions for steroidal hormone active substances

In conclusion it is very likely that different steroidal estrogens (EE2, E2 and E1) have an impact on fish populations at environmental relevant concentrations (see map before), also at median flow conditions close to WWTPs.

But there are limitations:-to detect the different steroidal estrogens due to analytical problems at these low effect levels. -to detect several mode of actions for endocrine disruptors.

Fischnetz: - Downstream SWTP: up to 5 ng/L EEQ

NRP50: - Surface Waters: 0.5 - 4 ng/L EEQ

Strategie Micropoll: - Effluent Vidy (upgraded): 0.15 - 0.29 ng/L EEQ

- Effluent Würi (upgraded): 0.05 - 0.33 ng/L EEQ

Source: Kunz et al. 2011


AA-EQS proposal for E2 = 0.4 ng/L

Which methods can be Which methods can be used to measure used to measure hormonally and hormonally and

reproduction toxic effects reproduction toxic effects and what are the costs?and what are the costs?


in vivo in vitro

Embr

yo-to

xicity

e.g. fish egg test or a non acute test with invertebrates

Reproduction-toxicity

Thyroidal-

modulation

Test with invertebrates, e.g. reproduction test with

Potamopyrgus antipodarumor Daphnia magna

H295R-Steroido-genesis Assay

ER/AR receptorbinding

YES/YAS Sumpter and modified to Schultis/Metzger or

ER/AR-Calux

Receptor binding-assays,

e.g. ELRA

Receptor-binding if

you have strong

(cyto)toxic effects

optional biotests

Xenopus laevis metamorphosis

assay

Ster

oidg

enes

is- a

nd

Arom

atas

emod

ulat

ionMode Of Action (MOA)

Is it possible to detect different modes of action with a modular test battery?

The modular system presented here allows the switching between test modules according to the continuously developing state-of-the-art of science and technology as well as the incorporation of novel developments.

Or short: In principle yes, but far too expensive for a routine monitoring

Not applicable for all environmental samples!

Source: Kase et al. 2009

How much time do you have to invest?

In general: in vivo-tests consume more time than in vitro-tests, scaling days vs. hours

Specific remarks:

in vivo: the reproduction test with Potamorpyrgus antipodarum needs most overall time but minimum working time

in vitro: MVLN and E-Screen have the most overall and working time need

0

30

60

90

120

150

180

Gesamtzeitbedarf Arbeitszeit

Zeit

in S

tund

en [h

]

MVLN YES McDon. YES Sumpter YAS Sumpter H295RER Calux AR Calux E-Screen ELRA AWACCS

overall time working time

Tim

e in

hou

rs [h

]

0

10

20

30

40

50

60

70

Gesamtzeitbedarf Arbeitszeit

Zeit

in T

agen

[d]

FSA XEMA Pa-Repro Dm-Repro Chirotest

overall time working time

Tim

e in

day

s [d

]


How expensive are the materials for one test run?In vivo and in vitro:

0

1

2

3

4

5

Materialkosten

FSAXEMA

Pa-Repro

Dm-Repro

Chirotest

MVLNYES McDon.

YES Sumpter

YAS Sumpter

H295R

ER Calux

AR Calux

E-Screen

ELRAAW

ACCS

FSAXEMAPa-ReproDm-ReproChirotestMVLNYES McDon.YES SumpterYAS SumpterH295RER CaluxAR CaluxE-ScreenELRAAWACCS

keine Angabe

<50 Euro

<150 Euro

<450 Euro

<1350 Euro

>1350 Euro

# #

# comment: these are commercial tests, so additional costs will occure

In general the in vivo tests are more expensive than in vitro tests regarding materials needed and in addition more time consuming.

Since November 2010 a DIN/ISO Standardization for different cell based ER receptor binding assays is ongoing, but there is still a long way to go in order to have standardized methods for regulative purposes available.


Overall summary and Overall summary and final conclusionsfinal conclusions


Thus, it is safe to say that…..

•for environmental samples with an unknown composition (unknown mixtures) only integrative bioanalytical tools, used as screening methods, are able to detect specific hormone active effects•Specific hormone active effects are resulting from an exposure to a mixture of known and unknown substances with endocrine disrupting properties.

These tools are available and have been proposed in the past, for example within:

•the international validation efforts by the OECD and the US EPA EDTA for methods to detect endocrine disruptive effects•the Global Water Research Coalition (GWRC) report "Tools to detect estrogenic activity in environmental waters" (Leusch 2008). •an extensive literature search and evaluation in which 15 biological test methods (5 in vivo and 10 in vitro) were evaluated (Kase et al. 2009), comprising 8 OECD methods and 3 out of five in vitro methods mentioned in Leusch (2008 and 2010).

Summary


These tools are able to measure the total receptor binding potential of an environmental sample by expressing its potency in EEQ (or EE2Q). These estrogenic potencies are then directly comparable with proposed EQS for E2 or EE2 for a decision for which samples additional analytical measurements are necessary.

Accordingly with effect based tools there is a potential:

To reduce the high costs for specific analytical measurementsTo provide reliable information for endocrine disrupting potentials covering several modes of action

However, for regulatory purposes the concept of dose addition is a sufficiently accurate model to predict combination effects of groups of endocrine disruptors with similar effects (Kortenkamp, 2007), but in order to do so an extensive data set of analytically measured EDCs has to be available.

In most cases this analytical data set is not available, therefore we recommend cost efficient and sufficiently sensitive effect based tools on a cellular basis to detect hormone active potentials in environmental samples in a first step.

Final conclusions


But, definitely without risks

Do not hesitate to contact us for further information:

[email protected](risk-assessor)[email protected](multiple effect assessment)[email protected](DIN/ISO standardization)[email protected](Head of Ecotox centre)

Thank you for your attention

Remember What’s Good for the Fish is Good for Us Also

Source: US-EPA, Lazorchak J 2010


Abegglen C et al. (2009): Ozonung von gereinigtem Abwasser. Schlussbericht Pilotversuch Regensdorf. Eawag Bericht Dübendorf, 16. Juni 2009

Burkhardt-Holm P, Giger W, Güttinger H, Ochsenbein U, Peter A, Scheurer A, Segner A, Staub E, Suter M J F (2005): Where have all the fish gone? The reasons why fish catches in Swiss rivers are declining. Environmental Science and Technology 39 (21) pp 441-447.

Dang Z C, Rub S, Wang W, Rorije E, Hakkert B, Vermeire T (2011): Comparison of chemical-induced transcriptional activation of fish and human estrogen receptors: Regulatory implications. Toxicology Letters 201:152–175.

EC (European Commission) (1997): (DG XII – Environment and Climate Research Programme). European Workshop on the Impact of Endocrine Disruptors on human Health and Wildlife. Report of Proceedings of a Workshop 2-4 December 1996, Weybridge, UK. Commission of the European Community, Brussels; Report EUR 17549

FNSNF (2008): Konsensplattform “Hormonaktive Stoffe in Abwassern und Gewässern” Schlussdokument. Nationales Forschungsprogramm “Hormonaktive Stoffe“. http://www.nrp50.ch/final-products/final-reports-consensus-plattforms.html

Götz C W, Kase R, Hollender J (2011): Mikroverunreinigungen - Beurteilungskonzept für organische Spurenstoffe aus kommunalem Abwasser. Studie im Auftrag des BAFU. Eawag, Dübendorf.

Götz C W, Kase R, Kienle C, Hollender J (2010): Mikroverunreinigungen aus kommunalem Abwasser - Kombination von Expositions- und ökotoxikologischen Effektdaten. Gas Wasser Abwasser, 7/2010.

More supporting information and references


Götz C W, Stamm C, Fenner K, Singer H, Schärer M, Hollender J (2009): Targeting aquatic microcontaminants for exposure categorization and application to the Swiss situation. Environ Sci Pollut Res. DOI 10.1007/s 1356-009-0167-8

Hutchinson T H, Ankley G T, Segner H, Tyler C R (2006): Screening an Testing for Endocrine Disruption in Fish-Biomarkers As „Signposts“, Not “Traffic Lights” in Risk Assessment. Environmental Health Perspectives 114:106-114

Kase R, Eggen R I L, Junghans M, Götz C, Hollender J (2011): Assessment of Micropollutants from Municipal Wastewater- Combination of Exposure and Ecotoxicological Effect Data for Switzerland, Waste Water - Evaluation and Management, Fernando Sebastián García Einschlag (Ed.), ISBN: 978-953-307-233-3, InTech

Kase R, Kunz P, Gerhardt A (2009): Identifikation geeigneter Nachweismöglichkeiten von hormonaktiven und reproduktionstoxischen Wirkungen in aquatischen Ökosystemen. Umweltwiss Schadst Forsch 21(4). DOI :10.1007/s12302 009 0072 2.

Kienle C, Kase R, Werner I (2011 in prep.): Evaluation of bioassays and wastewater quality. In vitro and in vivo bioassays for the performance review in the Project "Strategy MicroPoll". Summary. Oekotoxzentrum Eawag/EPFL, Dübendorf.

Knacker T (2007): Möglichkeiten der ökotoxikologischen Bewertung für Einzelstoffe und Gemische. Presentation at the IKSR-Workshop Mikroverunreinigung aus der Siedlungswasserwirtschaft 23. Mai 2007, Bonn, Germany.

Kortenkamp, A (2007): Ten years of mixing cocktails: a review of combination effects of endocrine disrupting chemicals. Environ. Health Persp 115, Suppl. 1: 98-105.



Kunz P, Kienle C, Homazava N (2011): Endocrine Effects in the Aquatic Environment – The Situation in Switzerland. Presentation at the Endocrine Disruptors Workshop 8. Februar 2011, Zürich, Switzerland.Lahnsteiner F, Berger B, Kletzl M, Weismann T (2006): Effect of 17β-estradiol on gamete quality and maturation in two salmonid species. Aquatic Toxicology 79:124–131.

Leusch F (2008): Tools to Detect Estrogenic Activity in Environmental Waters. Global Water Research Coalition 2008

Maack G, Adler N, Bachmann J, Ebert I, Hickmann S, Küster A, Rechenberg B (2010):Environmental Risk Assessment of medicinal products for human use: Does the risk assessment reflect the reality? Presentation May 2010; SETAC Sevilla.

TGD for EQS 2009/2010: CHEMICALS AND THE WATER FRAMEWORK DIRECTIVE: TECHNICAL GUIDANCE FOR DERIVING ENVIRONMENTAL QUALITY STANDARDS. Draft 2010. Draft version 6.0, 23 February 2010.

US-EPA, Lazorchak J (2010): Results of a 21-Day Fathead Minnow (Pimephales promelas) Fecundity Study Following Exposure to Ethinylestradiol (EE2). Presentation at the SETAC Annual Meeting Portland, OR November 11, 2010.

Vermeirssen E L M, Suter M J –F, Burkhardt-Holm P (2006): Estrogenicity Patterns in the Swiss Midland River Lützelmurg in relation to treated domestic sewage effluent discharges and hydrology. Environmental Toxicology and Chemistry 25(9):2413-2422



Annex: Additional information for the proposed test procedures 1

Organismus/ System Name Endpunkte Organisationslevel Versuchdauer Guideline/

Validierungsstatus Aquatische Vertebratentests

Fisch Zebrabärbling, Danio rerio

Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages,

Fischeitest Embryotoxizität, Fischeier werden exponiert und nach 48 h wird das Überleben

erfasst, keine endokrine Spezifität organismisch 48 h OECD 212 und DINENISO 15088

Amphibien Südafrikanischer Krallenfrosch Xenopus laevis

Amphibian Metamorphosis Assay (AMA) oder (XEMA)

Verzögerung oder Beschleunigung der Entwicklung , (NF-Stadien, Hinterbeinlänge, Körperlänge, Gewicht), abhängig von thyroidaler Modulation

Schilddrüsenhistologie (Genexpression möglich), organismisch 21 d

OECD draft TG in abschließender Kommentierung, Verabschiedung

voraussichtlich 2.Q 2009

Invertebratentests Mollusken

Zwergdeckelschnecke,Potamopyrgus antipodarum Reproduktionstest mit

Potamopyrgus antipodarum Embryonenzahl in der Bruttasche, leicht zu bestimmender Endpunkt der sehr

empfindlich auf estrogene und androgene Subsatnzen reagiert organismisch / Population 4-8 Wochen

in Prävalidierung, auf OECD-Ebene entsteht z.Zt. ein Detailed Review Paper zu

Molluskentests,

Crustaceen Daphnia magna Daphnienreproduktionstest

Überleben und Reproduktionsfähigkeit , Erweiterung für Geschlechterverhältnisse geplant organismisch / Population 21 d OECD 211

zelluläre Reportergenassays ER-Calux humane Brustkrebszellen ER-Calux

Messung einer Luziferaseaktivität als Maß für die agonistische und antagonistische Induktion eines estrogen- abhängigen Reportergens für hER alpha zellulär ca. 54 h

Validierung in 2008, Präsentation der Daten im 1. Quartal 2009 durch EC/ECVAM

AR-Calux humane Brustkrebszellen AR-Calux Messung einer Luziferaseaktivität als Maß für die agonistische und antagonistische

Induktion eines androgen- abhängigen Reportergens für hAR zellulär ca. 54 h Validierung in 2008, durch EC/ECVAM

YES nach Sumpter/Routledge und Schultis/Metzger modifiziert

rekombinant veränderte Hefezellen

Messung einer Galaktosidaseaktivität als Maß für die Induktion eines androgennabhängigen Reportergens für hER zellulär ca 30 h

mehrfach für Umwelprobentestungen eingesetzt

YAS nach Sumpter/Routledge und Schultis/Metzger

modifiziert rekombinant veränderte

Hefezellen Messung einer Galaktosidaseaktivität als Maß für die Induktion eines

androgennabhängigen Reportergens für hAR zellulär ca. 30 h mehrfach für Umwelprobentestungen

eingesetzt

Steroidgenese und Aromatase-Assays

H295R-Adrenokarzinomsäugerzellen H 295 R Steroidgenesis Assay

Modulation der Steroidhormongenese für 17-beta- Estradiol und Testosteron, Aromatasemodulation möglich zellulär 48-72 h

bereits validiert, Peer Review wir für das 2. Quartal 2009 erwartet

Rezeptorbindungsassays

ELRA Enzyme-Linked-Receptor-

Assay Agonistische und antagonistische Bindung an dem humanen Estrogenrezeptor alpha,

funktioniert auch bei zytotoxischen Ueberlagerungen molekular 4 h

mehrfach für Umwelprobentestungen eingesetzt und derzeit in einer

Weiterentwicklung der Virginia University in den USA

A full summary of available and OECD tests is available in Kase et al. 2009


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