1

Which information identifies a chemical as endocrine disrupting?

Poul BjerregaardInstitute of Biology

University of Southern DenmarkOdense

and Danish Centre on Endocrine Disrupters

UNEP EDC Advisory Group meeting, Geneva , September 25-26, 2015

2

WHO EDC definition

1. Endocrine mechanism

2. Adversity

3. Plausible link between the two

3

Adversity according to WHO/IPCS 2004

• A change in morphology, physiology, growth, reproduction, development or lifespan of an organism which results in impairment of functional capacity – or

• impairment of capacity to compensate for additional stress or increased susceptibility to the harmful effects of other environmental influences

4

Adversity?

• Risk assessment of chemicals– What do we want to protect?

• Human risk assessment:– Every individual

• Environmental risk assessment:– The structure and function of the ecosystem

5

Environmental risk assessment

• Protection of ecosystem structure and function

• The aim is to protect 95% of the species• ‘Adversity ’defined from population effects

– Not effects on the individual

6

Environmental risk assessment

• Protection of ecosystem structure and function

• The aim is to protect 95% of the species

• ‘Adversity’ defined from population effects– Not effects on the individual

7

Relevant OECD test guidelines

• Human risk assessment– TG 416 or 433 (mammals)

• E.g. malformed male genitals, altered AGD

• Environmental risk assessment– TG 234

• Fish Sexual Development Test

8

Fish Sexual Development Test

• Altered content of yolk proteins– Controlled by oestrogen

• Indicates endocrine mechanism - not adversity

• Altered sex ratio – Indicates endocrine mechanism and adversity

– OECD Guidance Document 150

9

Danish suggestion for EDC-criteria

• Category 1 – Endocrine Disrupter– Adverse in vivo effects– ED mode of action in vivo clearly linked to adverse in vivo effects

• Category 2a – Suspected Endocrine Disrupter– Adverse effects in vivo where an ED MoA is suspected – ED MoA in vitro combined with toxicokinetic in vivo data– ED MoA in vivo suspected to be linked to adverse effects in vivo

• Category 2b – Indicated Endocrine Disrupter– in vitro/in silico evidence indicating potential for ED in vivo

10

Exemplified by chemical UV

filters

O

OH

OHBenzophenone-1

O

OH OH

OH OHBenzophenone-2

OBenzophenone

O

O

OHBenzophenone-3

O

OH OH

4,4'-dihydroxybenzophenone

• Danish 2012-assessment

Category 2a

Category 2a

Category 2a

Category 1

BP-3: Indications of oestrogenic and anti-androgenic effects.Not consistent

TG234 needed

11

Sexual development in zebrafish

Hatch

♀

♂20 days 40 days 60 days

12

Sex ratio altered – more ♀

Control Solventcontrol

191±5 388±23 470±30

Benzophenone-3 concentration (g L-1

)

0

50

100

Perc

ent d

istr

ibutio

n

Females Undiff.Males

*

Fig. 2

102 101 88 80 95

*

Intersex

Kinnberg et al. 2015. Environ.Toxicol.Chem. In press.

0 100 200 300 400 500

Benzophenone-3 concentration (g L-1

)

20

30

40

50

60

70

Pe

rce

nt

dis

trib

uti

on

Fig. 3

% =54.8 - 0.056*[BP-3]p = 0.003r2 = 0.965

% =38.9 + 0.047*[BP-3]p < 0.001r2 = 0.9993

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No consistent effect on yolk proteins

Control

Solvent

control 191±5

388±23470±30

Benzophenone-3 concentration (g L-1)

510

102

103

101

102

103

104

Vite

lloge

nin

le

vel (

ng g

-1 w

et w

eig

ht)

101

102

103

104

105

106

107

Fig. 5

72(13) 1177(8) 6

Male fish

Female fish

Undifferentiated and intersex fish

39 4240 47 56

5455 41 28

5

24

4 106 3

A

B

C

Fig. 5

Adult male zebrafish vitellogenin

Benzophenone-3 exposure concentrations

ControlSolvent control

63 µg/L268 µg/L

437 µg/L17 ng/L E2

ng vite

llog

enin/m

L ho

mo

ge

nate

0

20

40

60

80

100

120

140

160

180

200

3 3 3 3 3

*

*

3

12 d exposure

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Conclusion

• Exposure to benzophenone 3 skews sex ratio– so

• Benzophenone 3 is an EDC

15

Is BP-3 oestrogenic or anti-androgenic?• Does it matter ?• Depends on the requirement for detailed

knowledge about the mechanism of action• Detailed knowledge about ‘Adverse

Outcome Pathways’ is desirable• But not always obtainable

16

A chemical may have multiple AOPs

Cl

Cl

ClO

N

O

N

N

ProchlorazFungicide

17

Prochloraz: More male zebrafish

%

0

20

40

60

80

100 Female

Male

Intersex

Undifferentiated

Control 16 mg/lProchloraz

64 mg/lProchloraz

202 mg/lProchloraz

73 72 72 69

*

Kinnberg et al. 2007. Comp. Biochem. Physiol.145C, 165-170

18

Prochloraz is anti-androgenic in rats

Laier et al. 2006. Toxicol.Appl.Pharmacol. 213, 160-171

19

Effect of prochloraz

OH

O

OH

Cholesterol

Testosterone

Oestrogen

ProchlorazInhibits aromatasein zebrafish (?)

OH

OH

Prochloraz Anti-androgenic in rats

20

Conclusion

• Detailed knowledge on ‘Adverse Outcome Pathways’ is not necessarily needed to reach conclusions on endocrine disrupting activity