1. No category

March 18, 2015
Endocrine Disruptors in the Drinking Water
Current Progress of Testing Methods for Assessment of
Potential Endocrine Disrupting Chemicals in Japan
１．Endocrine Disruptors
2． Laws and regulations for water quality in Japan
3. Framework for assessment of potential Endocrine Disrutpros
4. Tier 1 assays (screening) and methods
5. Tier 2 assays (definitive testing) and methods
6. Future needs
Taisen Iguchi
National Institute for Basic Biology
Okazaki Institute for Integrative Bioscience
1
Earthquake
March 11, 2011
Tsunami
March 11, 2011
Mount Otake erupted in Central Japan
September 27, 2014
4
1．Endocrine Disruptors
1962
1996
1975
Abnormalities Induced by Chemicals (WHO/IPCS 2002)
Imposex: vas deferens, spermatogensis
in females
Organotins: TBT, TPT
Sex reversal-intersex-ovotestis-abnormal
steroidogenesis–reduction of sperm EE2,
nonylphenol, equiline, UV absorbers
Human
RXR
ER
AR
Sex reversal-intersex-ovotestis
Atrazine ?
Malformation
ER
Arom
Sex reversal-abnormal penis-abnormal
steroidogenesis dicofol, DDT-related
pesticides
ER
AR
TR
Egg shell thinning, crossed bill, reproductive
abnormalities PCBs, dioxins, pesticides
ER
AhR
Abnormal reproductive tracts, abnormal
steroidogenesis PCBs?
?
Reduction of sperm counts and qualities
Increase in hypospadias, etc.
?
Human Disease Trends: Endocrine Cancers
Increases in:
endometrial, ovarian, breast,
prostate, thyroid and
testicular cancers.
Testicular
Prostate
Breast
EDCs may contribute substantially to male reproductive
disorders and diseases, with nearly €15 billion annual
associated costs in the EU. These estimates represent only a
few EDCs for which there were sufficient epidemiological
studies and those with the highest probability of causation.
These public health costs should be considered as the EU
contemplates regulatory action on EDCs.
J. Clin. Endocr. Metab., 2015.
WHO/UNEP
2012
DDT Associations with Breast Cancer
Odds Ratio
Critical Windows:
Age in 1945 (proxy for age at 1st exposure)
Fetal Origin of Adult Disease: Barker Hypothesis
WWII Dutch Famine – Complexity of Biology
First Trimester
 CV Disease
 Hypertension
 Dyslipidemia
 Obesity
Second Trimester
 Pulmonary Disease
 Renal Disease
Third Trimester
 Diabetes
 Depression
 Schizophrenia
 Anti-Social Personality
Disorder
Developmental Origins of Adult Disease
Contaminant Exposure and Women's Health
Crain et al. 2008 Fertility & Sterility
The New Disease Paradigm:
Developmental Origins of Disease



The environment during
development..
 stress, nutrition, environmental
exposures, infections and drugs
Functional changes
 aberrant developmental
programming, permanently
A bad
start…lasts
alters gland,
organ
or system a lifetime!
potential
• Alter gene And
expression
and/or
maybe…several
lifetimes!
protein regulation
• Persist throughout life
Developmental changes lead to
increased susceptibility to disease
Epigenetic/Environmental Basis of Disease
Normal Stem Cell
CH3
Hormones
CG CG
CG CG
Normal Growth
and Development
CG CG
CG CG
Disease/Dysfunction
EDCs
CH3
CG CG
Altered Gene
Expression persists
CH3
Changes in DNA
methylation pattern
CG CG
Abnormal Growth &
Development
Endocrine Disrupting Chemicals
HERBICIDES
2,4,-D
Pesticides
2,4,5,-T
Alachlor
Amitrole
Atrazine
Linuron
Metribuzin
Herbicides
Nitrofen
Trifluralin
FUNGICIDES
Benomyl
Ethylene thiourea
Fungicides
Fenarimol
Hexachlorobenzene
Mancozeb
Maneb
Metiram - complex
Solvents
Tri-butyl-tin
Vinclozolin
Zineb
METALS
INSECTICIDES
Aldicarb
Flame
beta-HCHRetardants
Carbaryl
Chlordane
Chlordecone
DBCP
Dicofol
Industrial
byproducts
Dieldrin
DDT and metabolites
Endosulfan
Heptachlor / H-epoxide
Lindane (gamma-HCH)
Malathion protectors
Surface
Methomyl
Methoxychlor
Oxychlordane
Parathion
Synthetic pyrethroids
Sunscreens
Transnonachlor
Toxaphene
Testosterone synthesis inhibitor
Thyroid hormone disruptor
INDUSTRIAL CHEMICALS
Bisphenol - A
Plastics
Polycarbonates
Butylhydroxyanisole (BHA)
Cadmium
Chloro- & Bromo-diphenyl
Dioxins
FuransPlasticizers
Lead
Manganese
Methyl mercury
Nonylphenol
Octylphenol
PBDEs Cosmetics
PCBs
Pentachlorophenol
Penta- to Nonylphenols
Perchlorate
PFOA
Over 1,000 EDCs
p-tert-Pentylphenol
Phthalates
Styrene
Estrogen receptor agonist
Androgen receptor antagonist
Diet
Nutrients &
Contaminants
Stress
Behavioral &
Environmental
(pesticides, metals,
plasticizers, phytoestrogens)
Body Burden
Drugs &
Supplements
Stored Contaminants –
Fat & Bone
Legal & Illegal
(smoking, alcohol, vitamins)
Personal Care
Products
(phthalates, musk xylene)
Genetics
Parental Genetics &
Epigenetics
Predisposition: Health or Disease
Genome - Epigenome Influences
The {(gene X environment) X environment} interaction……or
{(genomeM+P X epigenomeM+P) x epigenomeE)} X (genomeE X
epigenomeE')
Thus: not just gene mutation leads to disease
Rather… gene regulation probably more critical.
Guillette and Iguchi (2012) Science 337:1614
２. Laws and Regulations in Japan

Basic Environment Law
→Environmental water quality standards
 Standards for protecting human health
 Standards related to conservation of living environment
• Water Pollution Control Law
•

•
Effluent standards
Sewerage Law
Effluent water quality standards for wastewater treatment
plant
 Waterworks
•
law
Water quality standards for drinking water
EQS and Effluent Standards for Water
Environmental Quality Standard (EQS)
 Established as part of the government’s objectives (standards
that are to be followed) to prevent health hazards and conserve
the living environment” by the Environment Basic Law
 “Effluent Standards” are applied on
factories and establishments in order to
satisfy “EQS”
 In consideration of dilution effect by river
water, an effluent standard value for a
certain item is decided as 10 times as an
EQS for water
environment quality standard for the
same time.
Effluent
Standard
（Source: MLIT)
17
Environmental Water Quality Standards
 Standards

for protecting human health
Defined uniformly for all public water bodies
• Heavy metals, Toxic chemicals
 Standards
related to conservation of living
environment

Defined according to water body classification
and water quality characteristics
• pH, BOD, SS, DO, Coliform, Nitrogen, Phosphorus
• Zinc, Nonylphenol and LAS (Linear Alkylbenzen
Sulfonate) -- surfactant
Environment Quality Standards (EQSs)
for Human Health
 Uniform
Standards
1,4-Dioxane
(Source: http://www.env.go.jp/en/standards/)
≦0.05mg/L
Environment Quality Standards for Human Health
» Monitored substances and guideline values
(Source: http://www.env.go.jp/en/standards/)
Environmental Water Quality Standards
 Standards

for protecting human health
Defined uniformly for all public water bodies
• Heavy metals, Toxic chemicals
 Standards
related to conservation of living
environment

Defined according to water body classification
and water quality characteristics
• pH, BOD, SS, DO, Coliform, Nitrogen, Phosphorus
• Zinc, Nonylphenol and LAS (Linear Alkylbenzen
Sulfonate) -- surfactant
EQSs for Conservation of the Living Environ.
 Example

of the river water’s EQSs
Category A : Water use
(Source: http://www.env.go.jp/en/standards/)
EQSs for Conservation of the Living Environ.

Category B : Aquatic life habitat
Item
class
Aquatic life A
Aquatic life
Special A
Aquatic life B
Aquatic life
Special B
Adaptability to aquatic life
habitat conditions
Water bodied inhabited by aquatic
organisms such as char, salmon and
trout, and also their prey, which favor
relatively low-temp. ranges.
Water bodies categorized in “Aquatic life
A” need to be conserved in particular as
breeding or nursery grounds for the
aquatic life categorized in “Aquatic life
A”.
Water bodies inhabited by aquatic
organisms such as carp and crucian,
and also their prey, which favor relatively
high-temp. ranges.
Water bodies categorized in “Aquatic life
B” need to be conserved in particular as
breeding or nursery grounds for the
aquatic life categorized in “Aquatic life
B”.
Standard value
Total zinc
Nonyl phenol
LAS
≦0.03mg/L
≦0.001mg/L
≦0.03mg/L
≦0.03mg/L
≦0.0006mg/L
≦0.02mg/L
≦0.03mg/L
≦0.002mg/L
≦0.05mg/L
≦0.03mg/L
≦0.002mg/L
≦0.04mg/L
Remarks: Standard value are based on annual average values
(Source: http://www.env.go.jp/en/standards/)
Standards for Water Pollution Control

Water Pollution Control Law
Effluent standards
※direct application of penalty provisions for
exceeding effluent standards
•
 Sewerage Law
• Effluent water quality standards for wastewater treatment
plant
Uniform effluent standards 【Health item】
Kinds of harmful substances
Cadmium and its compounds
Cyanide compounds
Organic compound (limited to parathion, methyl parathion, methyl
demeton and EPN (ethyl p-nitrophenyl phenylphosphorothioate))
Lead and its compounds
Hexavalent chromium compounds
Arsenics and its compounds
Mercury and alkyl mercury, and other mercury compounds
Alkyl mercury compounds
Polychlorinated biphenyl
Trichloroethylene
Tetrachloroethylene
Dichloromethane
Carbon tetrachloride
1,2-dicholoroethane
1,1-dichloroethylene
cis-1,2-dichloroethylene
Tolerable limit
0.1 mg/L
1 mg/L
1 mg/L
0.1 mg/L
0.5 mg/L
0.1 mg/L
0.005 mg/L
Not detected
0.003 mg/L
0.3 mg/L
0.1 mg/L
0.2 mg/L
0.02 mg/L
0.04 mg/L
0.2 mg/L
0.4 mg/L
Note
The effluent standard shown in this table is applicable to the effluent water discharged by a plant, factory, or
business establishment which discharges 50m3/day or more of effluent water on daily average.
（Source: MoE)
Uniform effluent standards 【Health item】(cont’d)
Kinds of harmful substances
1,1,1-trichloroethane
1,1,2-trichloroethane
1,3-dichloropropene
Thiram
Simazine
Thiobencarb
Benzene
Selenium and its compounds
Boron and its compounds
Fluorine and its compounds
Ammonia, ammonium compounds, nitrite compounds and
nitrate compounds
1,4-dioxane
Tolerable limit
3 mg/L
0.06 mg/L
0.02 mg/L
0.06 mg/L
0.03 mg/L
0.2 mg/L
0.1 mg/L
0.1 mg/L
Other than sea area: 10
mg/L
Sea area: 230 mg/L
Other than sea area: 8
mg/L
Sea area: 1 mg/L
(*) 100 mg/L
0.5mg/L
(*) 0.4 times the ammonia nitrogen compound, and the total of nitrite nitrogen and nitrate nitrogen
Note
The effluent standard shown in this table is applicable to the effluent water discharged by a plant, factory, or business
establishment which discharges 50m3/day or more of effluent water on daily average.
（Source: MoE)
Uniform Effluent Standards 【Living environment item】
Kinds of harmful substances
Hydrogen ion concentration (pH)
Tolerable limit
Other than sea area: 5.8 – 8.6
Sea area: 5.0 – 9.0.
Biochemical oxygen demand (BOD)
160 mg/L (Daily mean value: 120 mg/L)
Chemical oxygen demand (COD)
160 mg/L (Daily mean value: 120 mg/L)
Suspended solids (SS)
200 mg/L (Daily mean value: 150 mg/L)
Normal-hexane extracts content (mineral oils
content)
Normal-hexane extracts content (animal and plant
fats content)
Phenols content
Copper content
Zinc content
Soluble iron content
Soluble manganese content
Chromium content
Coliform group number
Nitrogen content
Phosphorus content
5 mg/L
30 mg/L
5 mg/L
3 mg/L
2 mg/L
10 mg/L
10 mg/L
2 mg/L
Daily mean value: 3,000/cm3
120 mg/L (Daily mean value: 60 mg/L)
16 mg/L (Daily mean value: 8 mg/L)
Note
The effluent standard shown in this table is applicable to the effluent water discharged by a plant, factory, or
business establishment which discharges 50m3/day or more of effluent water on daily average.
（Source: MoE)
The Relevant Government Ministry
Ministry of the Environment
Environmental Management Bureau
Water Environment Management Division
29
Standards for Water Pollution Control

Water Pollution Control Law
Effluent standards
※direct application of penalty provisions for
exceeding effluent standards
•
 Sewerage Law
• Effluent water quality standards for wastewater treatment
plant
Effluent Water Quality Standard for WWTP in Japan
The Relevant Government Ministry
Ministry of Land, Infrastructure, Transport and
Tourism (MLIT)
Water and Disaster Management Bureau
Wastewater Management Department
Sewerage Planning Division
Sewerage Works Division
Director for Watershed Management
Standards for Drinking Water Quality
 Waterworks
•
law
Water quality standards for
drinking water
Water Quality Standards for Drinking Water
Detection of Potential Endocrine Disrupting Chemicals
In Raw Water for Supply and Tap Water at 10 Cities in
Tokyo in 2013
Potential endocrine
disrupting chemicals
and natural and
synthetic estrogens
Detection limit Raw water Clean water
(mg/L)
in
(Tap water)
10 cities
in
10 cities
Nonylphenol
0.1
ND
ND
Bisphenol-A
0.01
2/10
(0.03, 0.11)
ND
Butyl benzyl phthalate
0.05
ND
ND
Di-n-butyl phthalate
0.1
ND
ND
17b-Estradiol
0.002
ND
ND
17a-Ethynylestradiol
0.002
ND
ND
35
The Relevant Government Ministry
Ministry of Health, Labour and
Welfare
Health Service Bureau
Water Supply Division
How to Control Chemicals in Water
Environment --- PRTR System
•
Identify the hazardous Chemicals released
to water
⇒Pollutant Release and Transfer Register
(PRTR)
•
•
A PRTR is a catalogue or database of releases and
transfers of potentially harmful chemicals including
information on the nature and quantity of such
releases and transfers.
A typical PRTR covers releases to air, water and land
as well as wastes transported to treatment and
disposal sites.
http://www.unitar.org/cwm/prtr/what-is
Pollutant Release and Transfer
Register (PRTR) System
（Source: http://www.env.go.jp/en/chemi/prtr/about/overview.html）
38
PRTR System
462
Evaluate the
management
conditions
Improve public’s
understanding of
chemical
Report
theand
estimation
METI
MoE
of the amounts
of
estimate
the amount
chemical
substances
of chemicals
released
released
and
from
other sources
transferred
to the
(households,
Government
farmlands,
automobiles, etc) that
are not subject to
report
39
PRTR System
Targeted business operators are
required to confirm the
released/transferred amount of
class 1 chemicals, and report them
to the government every year.
http://www.meti.go.jp/policy/chemical_management/law/msds/sin1shueng.pdf
40
PRTR System and Sewage Works
Top 10 chemical substances transferred to sewerage
No.
（PRTR）
9999
232
71
407
411
13
277
405
68
20
56
Substance
物質名称
(TOTAL)
N,N-Dimethylformamide
Ferric chloride
Poly(oxyethylene)
alkylether
Formaldehyde
acetonitrile
Triethylamine
Boron compounds
1,2-Epoxypropane
2-aminoethanol
ethylene oxide
[合計]
Ｎ，Ｎ－ジメチルホルムアミド
塩化第二鉄
ポリ（オキシエチレン）＝アルキルエー
テル（アルキル基の炭素数が１２から１
５までのもの及びその混合物に限る。）
Transfer to
Sewerage（kg/year）
1,367,575
180,133
160,617
138,360
ほう素化合物
92,109
61,210
59,019
56,105
１，２－エポキシプロパン（別名酸化プ
ロピレン）
50,601
ホルムアルデヒド
アセトニトリル
トリエチルアミン
２－アミノエタノール
エチレンオキシド
43,905
37,697
PRTR System and Sewage Works
Top 10 chemical substances
released from sewerage to public water bodies
No.（PRTR）
9999
405
374
412
1
272
144
48
87
150
332
Substance
物質名称
[合計]
(TOTAL)
ほう素化合物
Boron compounds
hydrogen fluoride and its waterふっ化水素及びその水溶性塩
soluble salts
manganese and its compounds マンガン及びその化合物
zinc compounds(water-soluble） 亜鉛の水溶性化合物
copper salts (water-soluble,
銅水溶性塩（錯塩を除く。）
except complex salts)
inorganic cyanide compounds
無機シアン化合物（錯塩及びシアン酸
(except complex salts and
塩を除く。）
cyanates)
ＥＰＮ
EPN
chrome and chromic
クロム及び三価クロム化合物
compounds
１，４－ジオキサン
1,4-Dioxane
arsenic and its inorganic
砒素及びその無機化合物
compounds
Release from
Sewerage（kg/year）
4,073,401
1,623,784
1,268,962
506,796
467,979
57,957
24,818
21,656
19,544
17,683
11,180
42
Pharmaceuticals
Nanomaterials
95 organic wastewater contaminants
139 streams across 30 states
1999-2000
SETAC Pellston meeting
Salt Lake, in 2003.
Environ. Sci. Technol., 36: 1202-1211, 2002
Tokyo
Nagoya
Osaka
800
Concentration (ng/L)
600
400
300
200
Population density
Crotamiton
Ibuprofen
Naproxen
Fenoprofen
Mefenamic acid
Ketoprofen
Propyphenazone
Ethenzamide
Triclosan
Thymol
Diethyltoluamide
Carbamazepine
100
8.0
6.0
4.0
2.0
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R13
R13’
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
R35
R36
R37
0
10
River no.
(Nakada et al., ES&T, 2008)
0
Population density (x103 capita/km2)
44/34
Distributions of PPCPs in 37 Japanese Major Rivers
Concentration of Detected PPCPs in Influent
Outline of Survey
 Conventional Activated Sludge Process (8 Plant): 5,000-120,00m3/d
 Oxidation Ditch Process (4 Plant): 220-1,000m3/d
 Selected PPCPs: 92 PPCPs
 Spot Sampling: Influent and Final Effluent
We detected 86 PPCPs at WWTPs’ influent. 35 substances was detected over 10ng/L.
Ketoprofen
Enrofloxacin
Ciprofloxacin
Norfloxacin
Furosemide
Ceftiofur
Dipyridamole
Levofloxacin
Chloramphenicol
Tylosin
Diclofenac
Oxytetracycline
Tetracycline
Naproxen
Nalidixicacid
Sulfapyridine
Ifenprodil
Propranolol
Sulfamerazine
Indomethacin
Roxithromycin
Sulfathiazole
Sulfadimizine
Clenbuterol
Griseofulvin
Azithromycin
Mefenamicacid
Fenoprofen
photolysis rate constant [hr⁻¹]
Photolysis
10
1
Bar；average
Error bar；SD
quinolone antibiotics
tetracycline antibiotics
0,1
・ 18 out of 57 PPCPs degraded more than 20% in average
・ The quinolone antibiotics, tetracycline antibiotics, ketoprofen and
furosemide, showed higher photodegradability
Number of PPCPs
* determined in the
Yodo river system
High
Photolabile
Persistent
19
ketoprofen furosemide
9
diclofenac etc.
carbamazepine
crotamiton
quinolones
etc.
tetracycline
2
1
6
acetaminophen
6
ifenprodil
dypiridamol
e
Ibuprofen
caffeine etc.
High
biodegradability*
0
sulpiride etc.
9
diltiazem
disopyramide
Macrolides etc.
High
adsoptivity*
48
49
Occurrence of Anti-influenza Drug during
Influenza Out Bread
50
Time-dependent Dynamics of OC
51
Feminization of Wild Fish and its Causation in UK Rivers
Sexual Disruption in Wild Roach (Rutilus rutilus) in English Rivers
Synopsis of findings ………
Percentage of sexually disrupted
males at survey sites
Intersex present at 44 (86%)
of 51 sites
0%
1–20%
21 – 40%
Overall incidence of intersex in ‘males’ of 23%
41 – 60%
61 – 80%
81–100%
Feminized reproductive duct
North
East
Oocytes in the testis
Jobling,S., Williams, R., Johnson, A., Taylor, A., Gross-Sorokin,
M., Nolan, M., Tyler, C.R., van Aerle, R.,Santos, E.M.,and
Brighty, G. (2006). Environ Health Perspectives 114: 32-29
Midlands
Anglian
Thames
Southern
Induction of Feminizing Effects by Controlled Effluent Exposures
(UK-Japan)
Identification of Estrogenic Compounds in Sewage Effluent
IO N T R AC E . M a x.S c a n = 1 3 0 9 # 3 0 :0 2 .4 2 .
T o ta l Io n C u rre n t. M a x.In t.= 1 8 6 .5 9 8 8 5 . 1 0 0 % In t.= 1 8 6 .5 9 8 8 5 .
100
In te n s ity (% a g e )
80
Bile
NP
NP1EO NP2EO
60
NP3EO
40
NP4EO
20
0
Determination of the
estrogenic activity of
the test effluents
4 :0 0
6 :0 0
8 :0 0
1 0 :0 0
1 2 :0 0
1 4 :0 0
1 6 :0 0
1 8 :0 0
2 0 :0 0
R e te n tio n T im e
Identification of
(anti)estrogenic
compounds and
their metabolites
(enzymatic
hydrolysis–RPHPLC, yeast Ty
screens, LC-NMRMS/GC-MSMS
HO
DHQ E2 EE2 E1
3.0
NP +
NPnEO
(n=1-4)
2.5
Absorbance (ABS)
Exposure of
fish to effluent
OH
2.0
1.5
1.0
0.5
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
45
47
49
51
53
55
57
59
T im e (m inutes)
E2
2.5
E1
Absorbance (ABS)
E2
2.5
Absorbance (ABS)
2.0
1.5
E1
Control
2.0
1.5
1.0
0.5
1.0
0.0
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
45
47
49
51
53
55
57
T im e (m inutes)
0.5
Gibson etal., 2005
Tyler et al., Environ. Sci. Technol., 2009.
Tyler et al., 2005
EE2: 17a-ethinylestradiol, NP: nonylphenol, 17b-Equ: 17b-dihydroequilenine
0.0
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
T im e (m inutes)
39
41
43
45
47
49
51
53
55
57
59
59
Long-term Effects of EE2 on Gonadal Development
Exposure of roach from fertilization until two years of age to
environmental concentrations of EE2

EE2 exposure
0
100
200 dph
Depuration Phase
120
200
300
400
500 dph
 Proportion of males and females
collected between 250 – 720 dph
120
100
ovary
n = 54
testes
n = 54
ovotestes
n = 59
n = 58
n = 47
33 %
of males
80
percentage
n = 96
100
60
percentage
120
80
60
40
40
20
20
0
control
0
control
0.3 ng/L
4.0 ng/L
exposure concentration during early life
 Exposure to 4 ng/L EE2 for resulted in an ALL-FEMALE population
Lange et al., Environ. Sci. Technol., 2009.
ovary
0.3 ng/L
4.0 ng/L
testes
ovotestes
Does Life Long Exposure to Estrogenic Effluent
Compromise Breeding Capabilities of Male Roach?
100 %
Effluent exposure
0
0.5
1.0
control fish
Scenario 1
1.5
2.0
2.5
3.5 years
Breeding
100 % effluent fish
Scenario 2
3.0
Scenario 3
Scenario 4
3 males
X
X
3 females
Breeding
6 males
3 females
3 males
X
X
6 males
No Breeding
3 females
3 males
3 females
Breeding
3 males
Breeding

Breeding groups of 9 fish (3 females & 6 males)

4 different combinations of control and effluent-exposed fish (each in duplicate)

All fish were fin-clipped for parentage analysis

Fish were allowed to breed naturally

Tanks were checked once a weeks and fertilised eggs transported to Exeter

Sampling of fry at 4 dph for parentage assignment  Microsatellites
Lange et al., Environ. Sci. Technol., 2011.
Population Effects of Ethinylestradiol on Fathead
Minnow in Lake 260 in Canada


Whole lake exposure of environmentally relevant
concentration of EE2 (5 ng/L)
 Fish population effects
Estimate estrogen effects on
fresh water fish
Close to UK river situation
3
Depth 14 m
Area– 34 ha
3
In flow
Fish species
Lake trout
White sucker
Fathead minnow
Pearl dace
Lake chub
Finescale dace
Slimy sculpin
Out flow
•Exposure 2001-2003
•EE2 exposure: 3 times/week, 20-21 weeks
•EE2 measurement: 5 points/week
(RIA and GC/MS/MS)
• Concentration: 5 ng/L
• Mean concentrations: 4.8 to 6.1 ng/L
(2001 – 2003）
Experimental Plan
Recovery?
Effects on individual and population
EE2 exposure
Basic data
1999
2000
2001
2002
2003
2004
Data from Control Lake
2005
2006
Fathead minnow
Lake 260 - Fall
+EE2
+EE2
+EE2
・Population decrease by
・Reduction of spermatogenesis
and testis-ova
・undeveloped ovaries
・Predator fish also declined
・Population has been recovered
when stopped EE2 exposure
Effects on Human?
May, 2001
June, 2008
３. Framework for Assessment of Potential EDCs
SPEED ’98 (1998-2005), ExTEND2005 (2005-2010), EXTEND2010 (2010-2015)
Framework has been established for assessment of the
environmental risk of endocrine disrupting effects of chemicals
and to take appropriate management measures.
→ Needs establishment of assessment methodologies and
implementation of assessment.
• It gives priority to ecological effects, and also consider risks to
human health caused by chemicals in the environment.
• It further enhances international collaboration.
• Conceptual Framework for Testing and Assessment of EDCs
• Framework for Assessment of Potential EDCs in EXTEND2010
• Developments of these Test Methods have been supported by
MOE through, Basic Study, UK-Japan Research Collaboration,
US-Japan Bilateral Cooperation, LRI etc.
Collaboration with NIES (Dr. Tatarazako), WatchFrog, Idea Co. (Dr.
62
Onishi) and JANUS (Dr. Kawashima).
Conceptual Overview of Actions in EXTEND2010
1) Promotion of Research for
the Biological Observation of Wildlife
and Fundamental Studies
Grasping of changes and
mechanisms in wildlife
2) Development of
Test Methods and
Establishment of
Assessment Framework
4) Implementation of
Actions and Effects Assessment
・Selection of chemicals to be considered
・Reliability evaluation of literature
・Test implementation
7) Promotion of
International Cooperation
・OECD
・Japan-UK joint research
・Japan-US partnership
・Regions including Asia
Acceleration
Utilization for selection of test chemicals
3) Survey on Environmental
Concentrations and Exposure Assessment
・Utilization of Environmental Survey
and Monitoring of Chemicals
4) Hazard
Assessment
×
3) Exposure
Assessment
Acceleration
5) Risk Assessment
5) Risk Management
6) Promotion of Information Sharing
・ Websites
・ Opportunities including research presentation
OECD Conceptual Framework for Testing and Assessment of EDCs
Mammalian and
Non-mammalian toxicology
Level 1
Existing data and non-test
information
Physical & chemical properties
Available
dataAvailable
Chemical
categories, QSAR,
in silico
Physical &(eco)toxicological
chemical properties,
(eco)toxicological
data, Read
across
Read
across
Chemical
categories, QSAR, in silico,ADME
ADMEmodel
modelpredictions
predictions
Level 2
In vitro assays: selected
endocrine
mechanisms/pathways
Estrogen, androgen
receptor binding
Estrogen
&
androgen
receptor
binding,
ER transactivation,
ER transactivation
OECD
TG455-TG457AR, THR,
Steroidogenesis
OECD TG455-TG457
AR,
THR
Transactivation
Transactivation OECD TG456
Steroidogenesis
OECD TG456
Level 3
In vivo assays: selected
endocrine
mechanisms/pathways
Uterotrophic (TG 440)
Hershberger (TG 441)
Amphibian metamorphosis assay (OECD TG 231)
Xenopus embryonic thyroid signaling assay (XETA)
Fish reproductive screening assay (OECD TG 229)
Fish screening assay (OECD TG 230)
Androgenized female stickleback screen (GD 140)
Level 4
In vivo assays: adverse
effects on endocrine
relevant endpoints
OECD TG407, 408, 415, 414, 451,
426 etc.
Fish sexual development test (OECD TG234)
Larval amphibian growth & development assay
(LAGDA)
Daphnia reproduction test (with male induction)
(OECD TG 211)
Chironomid toxicity test, Mollusc partial lifecycle assay,
Earthworm reproduction test, etc.
Avian reproduction assay (OECD TG 206)
Level 5
In vivo assays: more
comprehensive data on
endocrine relevant
endpoints, life cycle of the
organisms
Extended one-generation
reproductive toxicity study (TG
443)
2-Generation reproduction toxicity
study (TG 416)
Medaka extended one generation reproduction test
Daphnia multigeneration assay
Avian 2-generation reproductive toxicity assay
Mysid life cycle toxicity test
Copepod reproduction and development test
Mollusc full life cycle assays
64
Current Status of Testing Methods in EXTEND2010
Effects
OECD Level 2
Tier 1 In Vitro
Screening Assay
Level 3
Tier 1 In Vivo Screening
Assay
Level 4, 5
Tier 2 Definitive
Testing
Estrogenic effects
Anti-estrogenic
effects
Medaka estrogen receptor
a transactivation assay
Medaka reproductive screening
assay (OECD TG 229)
Fish screening assay (OECD TG
230)
○Medaka extended
one generation
reproduction test
Androgenic effects
Medaka androgen
receptor b transactivation
assay
Medaka reproductive screening
assay (OECD TG 229)
Fish screening assay (OECD TG
230)
○Medaka extended
one generation
reproduction test
Anti-androgenic
effects
Medaka androgen
receptor b
transactivation assay
○Juvenile medaka anti-androgen
assay
○Medaka extended
one generation
reproduction test
Thyroid hormone-like
effects
Anti-thyroid hormonelike effects
Xenopus tropicalis
thyroid hormone receptor
b transactivation assay
△Xenopus embryo thyroid
signaling assay (XETA)
Amphibian
metamorphosis assay
(TG 231)
○Larval amphibian
growth and
development assay
(LAGDA)
Juvenile hormone-like
effects
△Daphnia juvenile
hormone receptor
transactivation assay
○Daphnia juvenile
hormone receptor twohybirid assay
○Daphnia short-term juvenile
hormone-like effect screening
assay
Daphnia reproduction
test (with male
induction) (OECD TG
211)
○Daphnia
multigeneration test
Ecdysone-like effects
○Daphnia ecdyone
receptor transactivaion
assay
△Daphnia short-term ecdysonelike effect screening assay
○Daphnia
multigeneration test
65
Flowchart of Tiered Assessment in Japan
Tier 1 (to assess actions to endocrine systems)
OECD
Level
Candidates for testing on endocrine disruption
Re-evaluation of knowledge obtained
through the reliability evaluation
1
In vitro assay
2, 3
Monitoring Data:
identification in the
Japanese Environment
10 or more literatures
Literature evaluations
Prioritization for in vivo testing
Reporter gene assays
(receptor-based)
ER, AR, TR, EcR, JHR
In vivo tests
Tier 1 assessment
TG230 (VTG) or TG229?
Transgenic frog (XETA)
Short-term anti-andorngen
Daphnia TG211
pending
Tier 2 (to characterize adverse effects)
In vivo tests
4, 5
Hazard Assessment
Daphnia
EcR, JHR
Medaka
Medaka Extended One generation T
LAGDA or Metamorphosis TG231
Daphnia Multigeneration test?
pending
To risk assessment framework
African
clawed frog
ER, AR
TR
Reliability Evaluation of the Existing Knowledge
Obtained from the Literature
• Chemicals detected in the aquatic environment in the national
monitoring programs have been nominated for the testing and
assessment under the EXTEND2010.
• Reliability evaluation of existing knowledge obtained from the
literature is conducted focusing on the following information.
– Results of in vitro assays which suggest chemical binding to
hormone receptors (such as estrogen, androgen, aromatase or
thyroid hormone)
– Results of in vivo assays or epidemiological survey which suggest
direct effects to endocrine system (such as genital organs, thyroid
or pituitary gland), reproduction or development, or effects to
immune or nervous system via endocrine system
• Identification of “candidate chemicals for testing” has been
carried out based on:
– whether results mentioned in the literature is evaluated as reliable
or not, and
– whether the positive results could be caused by endocrine
disruption or not.
• Selected chemicals have been named as “chemicals that can be
67
subjected to tests for endocrine disrupting effects”.
Evaluation of Literature Reliability
Chemicals detected by the Environmental Monitoring: 328
Literature survey >10 publications:
114
Evaluation of literatures:
79
Selected for in vitro testing:
40 (18)
• Assessment of ecotoxicological effects should be conducted, focusing
on organisms whose test methods have already been developed.
• The following three effects caused by endocrine disruption will be
targeted.
– Reproduction – (anti)estrogen, (anti)androgen (Oryzias latipes)
– Development (metamorphosis) – (anti)thyroid hormone (Xenopus laevis)
– Growth – ecdysone, juvenile hormone (Daphnia magna)
• Framework for conducting ecotoxicity testing should be developed for
ecological risk assessment caused by endocrine disruptors.
Following two actions/effects should be evaluated.
– Identification of actions in the endocrine system
– Characterization of adverse effects on organisms
• For conducting assessment efficiently,
– In vitro assays should be conducted to prioritize candidate chemicals68for
in vivo assays.
Development of Two-Tier Framework for
Testing and Assessment
• Tier 1
– Chemical actions on endocrine system should be identified.
– “Chemicals that can be subjected to tests for endocrine
disrupting effects” identified through the reliability
evaluation of existing knowledge should be examined.
– Tier 1 assays should be comprised of in vitro assays and
short-term in vivo assays.
– Tier 1 assessment should be conducted based on the
existing knowledge and test results.
• Tier 2
– Adverse effects caused by endocrine disrupting effects
should be characterized.
– Chemicals whose actions to endocrine system were
recognized in the Tier 1 assessment should be candidates
for Tier 2 in vivo testing.
– Ecological risk assessment should be conducted.
69
４. Tier 1 In Vitro Assays
Dual-Luciferase® Reporter Assay System
Test Chemicals
Hormone
receptor
vector
Transiently co-transfected
Mammalian cell
(HEK293, HepG2)
Hormone
receptor
binding
cofactor
Hormone
response-element
Firefly luciferase gene
Experimental reporter vector
Transcription
Firefly
luciferase
beetle luciferin
(substrate)
Translation
Luminescence
the effect of specific
experimental conditions
Renilla luciferase gene
Transcription
Control reporter vector
Translation
Renilla
luciferase
Luminescence
coelenterate-luciferin
(substrate)
internal control that serves as
the baseline response 70
Development of Test Methods
Developments of Tier 1 In Vitro Assays
• Estrogenic and anti-estrogenic activities
Medaka (Oryzias latipes) estrogen receptor a (ERa) reporter
gene assay.
• Androgenic and anti-androgenic activity
Medaka androgen receptor b (ARb) reporter gene assay.
(Validity for detection of anti-androgen activity should be
examined.)
• Thyroid and anti-thyroid activities
Xenopus tropicalis thyroid hormone receptor b (TRb) reporter
gene assay.
• Juvenile hormone activity
Daphna magna juvenile hormone receptor (JHR) reporter
gene assay (under development)
• Ecdysone activity
Daphnia magna ecdysone receptor (EcR) reporter gene
71
assay
Evolution of Estrogen Receptors
ERa, ERb1, ERb2
3 subtypes of ERs
in teleost fish after two
genome duplications
Roach
Zebrafish
Carp
Gar (a, b)
Bowfin
Rainbow trout
(a1, a2, b1, b2)
Arowana
Stickleback
Eel
Sturgeon (a1, a2, b, ...)
Bichir
X
Ascidian
ER/PR/CR
Lamprey
Shark (b)
Rayfish
WGD (whole genome duplication)
ER/SR
Amphioxus
ER→ERa, ERb
AcsER→ER, SR
Medaka
ERa→ERa1, ERa2?
ERb→ERb1, ERb2
ERa, ERb
Lungfish (a, b), Coelacanth
Amphibians, Amniota
Evolution of Estrogen Receptors
ERa, ERb1, ERb2
Roach
Zebrafish
Carp
Gar (a, b)
Bowfin
Arowana
Rainbow trout
(a1, a2, b1, b2)
Medaka
Stickleback
Eel
ERa
ERb1
Sturgeon (a1, a2, b, )
ERb2
Bichir
X
Ascidian
ER/PR/CR
Lamprey
Shark (b)
Rayfish
ER/SR
Amphioxus
ER→ERa, ERb
AcsER→ER, SR
WGD
ERa→ERa1, ERa2?
ERb→ERb1, ERb2
ERa, ERb
Lungfish (a, b), Coelacanth
Amphibians, Amniota
Responses to chemicals of each ER subtype differed between fish species.
Miyagawa et al. Environ. Sci. Technol., 48, 5254-5236, 2014.
Evolution of Androgen Receptors
Androgen receptor (AR)
AR duplication
NTD
hinge
LBD
DBD
ARα, ARβ
Appearance
of AR
AR
Ogino et al. Endocrinology, 150, 5415-5427, 2009.
Ogino et al. Evol. Dev., 13, 315-324, 2011.
Priority-setting for Tier 1 In Vivo Testing
• Provisional priority-setting for Tier 1 in vivo assays based on
results of Tier 1 in vitro assays
– Priority 1: Substances whose activities were detected in the in
vitro assays conducted in this program
– Priority 2: Substances whose activities were suggested in the
existing literature but were NOT detected in the in vitro assays
conducted in this program
– Priority 3: Substances whose activities were NOT suggested
in the existing literature and were NOT detected in the in vitro
assays in this program
• Remaining issue for the priority-setting
– How to deal with substances whose actions and effects
cannot or will not be detected by the reporter gene assays.
75
Characteristics of Daphnia magna as
an Experimental Animal
1 mm
(Daphnia magna)
OECD TG211
21 day Reproduction test
(Fecundity)
Acute Toxicity test
Water quality, Chemical toxicity
1. Easy to breed
2. High breeder (mature ca. 5 days
after birth and lay eggs every 3
days)
3. Parthenogenesis (easy to obtain
genetically clone individuals)
4. Translucent (easy to observe
internal organs without dissection)
5. In vitro development is possible
(developmental process can be
observed under the microscope)
6. ESTs and Genome database can
be used
7. Established RNAi and TALEN
8. Cloned ecdysone receptor and
juvenile hormone receptor
Ecdysteroids and Ecdysone Receptor (EcR)
・Major hormone
・development
・molting
・metamorphosis
・Reproduction
20-hydroxyecdysone (20E)
Ponasterone A (PoA)
・Act through EcR
・Nuclear receptor superfamily
・Heterodimer with ultraspiracle (USP)
← essential for ligand-dependent
gene activation
Kato et al., J. Endocrinol., 193, 183-194, 2007.
Sex Differentiation Model in Daphnids
Maturation
Oocytes
Growth
♀
- JH
?
+ JH
dsx1↑
JHR
In the ovary
♂
Critical Period:
Develop in the brood chamber
7-8 h before ovulation
PLoS Genet , 7, e1001345, 2011.
4, 1856, 2013.
Juvenile Hormone Receptor
Met
SRC
Miyakawa et al., Nature Communications, 2013.
JH synthesizing cells are unknown
Farnesoic acid
Both Met and SRC are grouped in the family of bHLH-PAS
nuclear transcription factors.
Target cells
OH
JHAMT
MF
MF
MF
JH epoxide hydrolase
Met
or
JH III
JH III
?
Met
SRC SRC
Met
MF
SRC Met
MF: methyl farnesoate
JHIII: juvenile hormone III
Met: methoprene-tolerant
SRC: steroid receptor coactivator
MF
MF
SRC Met
JHRE
?
?
Juvenile Hormone Receptor Two-hybrid Assay
Daphnia pulex
Daphnia magna
Juvenoids bind to Met and form heterodimer of Met and SRC
RNAi ：Dapma-Met と Dapma-SRC
Experimental Design of TG229
Pre-exposure
1 2
Chemical exposure period (days)
3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Checking fecundity
Selection of fish
Daily observation (general endopoints, fecundity)
Checking test conditions (Water temp, chemical conc., etc.)
Initiation of exposure
Termination of exposure
Endpoint measurements
Daily observation
► Survival
► Abnormal response (appearance, behavior, feeding)
► Egg production and Fertility
Endpoints at exposure termination
► VTG (hepatic)
► Secondary sex characteristics
(Anal fin papillae)
► Gonad-histology (option)
• 2014
4-hydroxybenzoic acid,
4-nonylphenol,
4-t-octylphenol,
bisphenol-A
81
Establishment of Anti-Androgen Detection Test
using Juvenile Medaka
83
Secondary Sex Characters (Papillary Processes)
in the Anal Fin of Male Medaka
♀
♂
amplexus
Papillary processes
Androgen Induces Papillary Processes
in the Anal Fin of Female Medaka
Anal fin 10 days
Control ♀
Androgen (MT)
Papillary processes
Androgen activates the Lef1/b-catenin signaling, which might
contribute to the proliferation of mesenchymal cells surrounding
the bone nodule.
Outgrowth
Lef1/b-catenin
Cell
proliferation
ARs
Androgen
Bone
Bmp7
deposition
Osteoblast
Differentiation
Androgen-induced Bmp7 expression
promotes the bone deposition.
Ogino et al. Endocrinology, 159, 449-462, 2014.
Transgenesis Spiggin 1 (4.2Kb)-eGFP
Medaka
Oryzias latipes
Anthony Sébillot
1200 injected eggs with Spg1
300 eggs GFP positive on
mesoderm and yolk
Three-spined stickleback
Gasterosteus aculeatus
73 ALIVE FOR SCREENING
GFP expression in Kidneys with MT 10-6 M
Sébillot et al., Environ. Sci. Technol.,
48, 10919-10928, 2014.
88
Exemples of Anti-androgenic pollutants
Fungicide
Procymidon
Fenitrothion
Insecticide
Herbicide
Vinclozolin
Linuron
Flutamide
p,p’-DDE
Pharmaceutical
molecule
Spg1.22-gfp transgenic medaka
Spiggin
Fry exposed to Evian
head
kidneys
tail
GFP
Fry exposed to the reference
hormone : 17α-methyltestosterone
head
kidneys
tail
Androgenic specificity of the
spiggin1.22-gfp line
DHT 29 mg/L
Anastorozole
DHT + Flutamide
17 MT 3 mg/L
Anti-androgen detection assays
with 17MT
Estradiol
MT + Flutamide
MT + Fenitrothion
MT + Limuron
MT + Vinclozolin
Human and Amphibian Thyroid System
Hypothalamus
CRH
Pituitary
SYNTHESIS
-
TSH
IThyroid Gland
Blood Vessels
Tg
TPO
HT-TT
DEIODINASE METABOLISM
OF THYROID HORMONES
TRANSPORT
Free TH
TH
Target Cells
TH
TR
TR
RECEPTORS
INTERACTIONS
MODULATED GENE EXPRESSION
GFP
TH/bZIP
ISAREN 2014
OECD RING TEST : XETA
92
Human and Amphibian Thyroid System
Hypothalamus
CRH
Pituitary
SYNTHESIS
-
TSH
IThyroid Gland
Blood Vessels
Tg
TPO
HT-TT
TRANSPORT
Thyroid
Disruption
DEIODINASE METABOLISM
OF THYROID HORMONES
Free TH
TH
Target Cells
TH
TR
TR
RECEPTORS
INTERACTIONS
MODULATE GENE EXPRESSION
GFP
TH/bZIP
ISAREN 2014
OECD RING TEST : XETA
93
XETA : an Ethical alternative to AMA test
ENDOGENEOUS FEEDING
(autotrophic)
EXOGENEOUS FEEDING
(heterotrophic)
Maturation
resorption of yolk sac
Fertilisation
Hatching
Egg
(unfertilised
Oocyte)
Embryo
Metamorphosis
Eleutheroembryo
EMBRYONIC STAGES
LARVA
JUVENILE
ADULT
Laboratory animal-Protected
animal
Non laboratory animal-Not protected
stage
XETA
Ethical Alternative
Animal Welfare
Secondary
sexual
characteristics
free swimming
and feeding larva
AMA
To be replaced/ reduced/ refined
94
Xenopus Embryonic Thyroid Assay (XETA)
With thyroid hormone activity, tadpole show fluorescence.
The transgenic Xenopus can detect (anti-)thyroid hormone activity
In 5 days.
Selection of the biomarker : THbZIP
100
Arbituary Units
80
60
40
20
T3
0
55
60
63
THbZIP :
•Protein  transcription factor.
•Gene expression directly modulated by thyroid
hormones during metamorphosis.
66
stages
Leloup and Buscaglia, 1977
The Xenopus transgenic model THbZIP-GFP
Tadpole exposed to
reference hormone
Triiodothyronine (T3)
Tadpole exposed to
FETAX medium
head
head
tail
tail
OECD RING TEST : XETA
97
Normalised mean fluorescence
(Arbituary units)
T3 Dose Response
OECD RING TEST : XETA
100
An OECD Ring test for the validation of the XETA
OECD RING TEST : XETA
101
OECD TG211
Daphnia magna Reproduction Test
Endpoint: number of offspring in 21 days
Male offspring production
M4 medium
21±1℃
16L：8D
Detection Method for Chemicals with Male Induction Activity
In A Week
Abe et al., J. Appl. Toxicol., 35, 75-82, 2015.
104
105
5. Developments of Tier 2 In Vivo Assays
• Estrogenic, anti-estrogenic, androgenic and anti-androgenic
activities
Medaka Extended One Generation Reproduction Test
(MEOGRT) (sent to OECD)
• Thyroid hormone and anti-thyroid hormone activities
Amphibian Metamorphosis Assay (TG231) or
Larval Amphibian Growth and Development Assay (LAGDA)
(under development).
• Juvenile hormone activity
Daphnia magna reproduction test (OECD TG211) ANNEX 7;
Multi-generation assay using daphnids. (under development)
• Ecdysone activity
Multi-generation assay using daphnids. (under development)
106
Exposure and Measurement Endpoint Timelines for the MEOGRT
(Medaka Extended One Generation Reproduction Test)
MEOGRT Exposure and Endpoint Timeline
F0
1 2 3 4
F1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 5 16
Study
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Week
Lifestage Key
Embryo
Larvae
Juvenile
Subadult
Adult
Endpoints

Experimental design has 7
groups of replicates
Fertility
F1
F0
o
5 for test substance
treatments
Hatch
F0
F1
o
2 for control treatments (4 if
Survival
F1
F1
F1
solvent is used)
 Within-group design
Growth
F1
F1
F0
o
12 replicates for reproduction,
Vitellogeni
F1
adult pathology and SSC
n
(Wks 10 thru 18)
SSC
F1
F1
o
6 replicates for hatch, survival,
Histopathol
Vtg; and - subadult SSC and
F1
ogy
growth (Wks 1 thru 9)
SSC: secondary sex characters; Wks:
Study
weeks;
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Week
Vtg: vitellogenin
Fecundity
F0
F1
107
Nakamura et al., J. Appl. Toxicol., 35, 11-23, 2015.
108
Amphibian Metamorphosis Assay OECD TG231
• Exposure of X. laevis tadpoles (stage 51 to 66) for total 21 days
• 6 replicates per test group (20 tadpoles)
• Exposure in a flow-through system
• Multiple Endpoints
Hind Limb
Morphological criterias
Whole Body
Weight
Snout-Vent
2 mm
Lengths
Developmental Stage
Histological criteria
2.0 mm
Thyroid Gland Histology / diameter
109
Life-cycle Test for Amphibian in Japan
Mar. 2009
ADGRA SPSF (OECD WNT)
Jan. 2010
6th bilateral meeting
Initiation of ADGRA (EE2) in Japan
Apr. 2010
Nov. 2010
Feb. 2011
Apr. 2011
Sep. 2011
Jul. 2011
Feb. 2012
Jul. 2012
May. 2013
Revised protocol (LAGDA) from US
End of ADGRA (40 weeks)
7th bilateral meeting
Latest Revised protocol, LAGDA, from US
MED meeting in Duluth
Benzophenone-2 (BP2) and EE2 LAGDA
8th bilateral meeting
Tamoxifen citrate (TC) LAGDA
Integrated Summary Report (ISR) from US
110
Larval Amphibian Growth and Development Assay
(LAGDA)
Experimental Design
• Test species: Xenopus laevis
• Test duration: ~16-20 weeks
– NF8 embryo to 10 weeks post-NF62 in
control
• Dosing: 4 concentrations and control
• Replication: 8 control and 4 for each conc.
– Total test vessels: 24
111
LAGDA Experimental Design
N.F. stage
8
Life cycle
LAGDA
Gonadal
differentiation
Male, female maturation
66
6-7 w
0d
NF 8
exposure
NF 62
Cull after NF66
5-6 w
10 w
Test termination
16 w (10 weeks post metamorphosis)
Larval sub-sampling st66 culling Juvenile sampling
X. laevis
Endpoints
- Mortality
- Body Mass/Length
- Vitellogenin
-T4, TSH
-Time to NF stage 62
-Thyroid Histology
- Liver Somatic Index
- Nuptial Pads
- Gonad Histology
- Sex Phenotype/Genotype
-Liver histology
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Test timeline
NF62 sub-sample
•HPT axis assessment
•n = 5 (randomized block)
•1 of every 4 that reach NF62
Exposure
start
NF8 embryo
Gonadal
differentiation
Cull to 10 per tank
~6 weeks
~8 weeks ~10 weeks
Test
termination
~16 weeks
n = 20/tank
NF66: completion of
metamorphosis
HPG axis assessment
3/20/2015
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Overt toxicity endpoints
Apical Endpoints
Mortality
Clinical signs of disease and/or toxicities
Growth (weight and snout-vent length)
Liver-somatic index (LSI)
Liver histology
Kidney histology
Daily
Stage 62
Test
Termination
x
x
x
x
x
x
x
Thyroid (HPT) endpoints
Endpoint
Daily
Time to NF stage 62
Thyroid histopathology
Stage 62
Test
Termination
x
x
Reproduction (HPG) endpoints
Endpoints
Daily
Stage 62
Genetic/histological sex comparison
Gonad histopathology
Reproductive duct histopathology
Endpoints removed: Plasma T4, vitellogenin, nuptual pads
Test
Termination
x
x
x
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Inter-laboratory validation studies
• Prochloraz (aromatase inhibitor;
AR antagonist)
– 4 labs (EPA/MED + 3 other U.S. labs)
• 4-tert-octylphenol (ER agonist)
– 3 labs (EPA/MED + 2 other U.S. labs)
• 17-β trenbolone (AR agonist)
– 1 lab (EPA/MED)
• Benzophenone-2 (ER agonist; TPO inhibitor)
– 1 lab (Japan)
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How to design the test procedure ?
• Just repeat the general reproduction test?
– Most of multi-generation experiments were conducted by repetition of
the general reproduction test (OECD TG 212) (Sánchez et al., 2004;
Brennan et al, 2006; Péry et al., 2008).
• How to ensure enough number of neonates for the second
generation test?
• How to compare the results from 1st generation and 2nd
generation?
– Need to consider the variation in test organisms used in 1st generation
and 2nd generation.
F1
Control
Exposed
F2
?
?
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Summary
• Two-tier framework of testing and assessment of EDCs on aquatic
organisms.
• Progress in development of testing protocols
– Reporter gene assays using receptors of fish, amphibian and daphnids.
– Medaka Short-term Reproduction Assay (OECD TG229)
– Medaka Extended One Generation Reproduction Test (MEOGRT) and
Larval Amphibian Growth and Development Assay (LAGDA) : developed
in cooperation with US EPA have submitted to submission to OECD Test
Guideline Programme.
– Medaka short term assay for detection of (anti)androgenic activities is
now under development, and establishing transgenic medaka and
Xenopus for (anti) androgenic activities and (anti) thyroid hormone
activities
• Progress of testing and assessment (number of substances) total: 328
– Selection of chemical substances for literature review: 114
– Reliability evaluation of existing literature is finished:
79
(candidate substances for testing:
51)
– Tier 1 in vitro assays:
40 (18)
118
– Tier 1 in vivo assays:
10 (6)
6. Future Needs
• Establishment of testing protocols to be used in two-tier framework of
testing and assessment.
– Tier 1 in vitro assays
– In vivo assays using medaka, amphibians and daphnids
• Implementation of two-tier framework for testing and assessment
– Tier 1 assessment for selecting candidates for Tier 2 testing
– Implementation of Tier 2 in vivo tests as definitive ones
– Tier 2 assessment
• Re-evaluation of the substances identified as endocrine disrupting
substances under the previous program: SPEED’98
– Nonylphenol, octylphenol and bisphenol-A
• Effective selection of target substances [bilateral collaboration]
– New tools such as high-throughput screening methods
• Advanced studies [bilateral collaboration]
– Adverse Outcome Pathways (AOP)
• Accelerated implementation of testing and assessment
119
• Regulatory actions for risk management of endocrine disruptors
Maternal Prenatal Exposure to Phthalates on Child IQ
December 10, 2014
intelligence quotient (IQ)
Maternal prenatal urinary metabolite concentrations measured
in late pregnancy of DnBP (di-n-butyl phthalate) and DiBP (diisobutyl phthalate) are associated with deficits in children’s
intellectual development at age 7 years.
Eco & Child 2011-2032
2014: ca US$ 51 million
Over 100,000 pregnant women (103,106) from 2011-2013.
In pregnancy: blood, urine
At parturition: umbilical cord blood, blood from parents,
dried blood spot
One month: breast milk, hair of baby
Six months: questionnaire, interview, up to 12 years old
Chemical measurements
Analyses
Evaluation
121