Combined low-dose exposures to anti-androgenic substancescefic-lri.org/wp-content/uploads/2014/03/12.JUNKER... · 2018. 12. 12. · LRI Presentation 151112.pptx 1 Combined low-dose

LRI Presentation 151112.pptx 1

Combined low-dose exposures to anti-androgenic substances:

Steffen Schneider, November 15, 2012

Reproductive toxicity studies in rats with three compounds (and their combinations)

First Results of the LRI EMSG 56 project


There may be effects at low doses of individual substances, which might be systematically overlooked (Bisphenol A, hormesis)

Knowledge gaps in single substance assessments (old 2-Gen studies)

Effects are seen at mixtures of substances combined at their individual NOAELs

More-than additive mixture toxicity

No clear evidence, but debate is ongoing

Optimize single substance evaluation

Only in case of common toxicity No human-relevant exposure situation

No evidence (except specific combinations)

Public concerns


Concept of the project Assess combination effects at different doses

ADINOAEL

LOAEL

ADINOAEL

LOAEL

ADINOAEL

LOAEL

?

Androgen ReceptorAntagonist 1


Inhibitor of Steroidogenesis

ADINOAEL

LOAEL

ADINOAEL

LOAEL

ADINOAEL

LOAEL

ADINOAEL

LOAEL

ADINOAEL

LOAEL

ADINOAEL

LOAEL

?



Inhibitor of Steroidogenesis

2 AndrogenReceptor

Antagonists

Molecular Effects Downstream Effectsand Homeostasis

EndocrineDisruption

No Adverse

EffectWP1In vivo Testing of the EndocrineActivity of Single Compounds

and Complex Mixtures

WP2Analyses of the Transcriptome and miRnome

1 Inhibitor of Steroido-

genesis

WP3Overall assessment and publication

2 AndrogenReceptor

Antagonists

Molecular Effects Downstream Effectsand Homeostasis

EndocrineDisruption

No Adverse

EffectWP1In vivo Testing of the EndocrineActivity of Single Compounds

and Complex Mixtures

WP2Analyses of the Transcriptome and miRnome

1 Inhibitor of Steroido-

genesis

WP3Overall assessment and publication

Project „Combined low dose exposure to antiandrogenic substances“


Reference value Expected NOAEL Effect level

BAS 352 F Vinclozolin

0.05 mg/kg bw/day 4 mg/kg bw/day 20 mg/kg bw/day

Flutamide NOAEL / 100 = 0.0025 mg/kg bw/day

0.25 mg/kg bw/day 2.5 mg/kg bw/day

BAS 590 F Prochloraz


Doses used in the study


Exposure duration and endpoints

Dams

GD 6 PND 0 PND 21 PND 28-49 PND 83

F1

Subset 1 OW, Histo RNA, µRNA Metabol, H Subset 2

10 m+f sexual maturity OW, Histo RNA, µRNA, Metabol, H

Subset 3 10 m+f OW, Histo RNA, µRNA Metabol, H spermatology

PND 0

PND 21 OW, Necropsy Metabol, H

GD 18 Plasma kinetics in dams and fetuses at Tmax

GD 6

GD – gestation day PND – postnatal day OW – organ weights Histo – full histopathology Metabol – Metabolome H - Hormones

LRI Presentation 151112.pptx

F1 Ano-genital Distances PND 1

6


F1 Male Nipple Retention

7


F1 Male Sexual Maturation

VIN FLT

PRO FLT


Male Sexual Maturation

9


Histology - Subgroup 2

10

Control animal Flutamide-treated, high dose

Epididymis:


F1 Physical Development

Female sexual development occurred normally

F1 males exposed to 2.5 mg/kg bw/d flutamide (putative LOAEL) had increased incidences of developmental defects

Subgroup 2 (day of sexual maturity)

– Hypospadia clinically observed in 2/10

– Small Penis clinically observed in 2/10

Subgroup 3 (PND 83±2)

– Hypospadia clinically observed in 2/10

– Small Penis clinically observed in 2/10

– The testes of one animal (No. 767) were not palpable, and found to be histopathologically abnormal

11


Histology - Subgroup 3

12

Control animal Flutamide-treated, high dose, animal No 767

Testes (higher mag):


F1 Organ Weights

No changes were observed in female sex organ weights

Male sex organ weights were significantly reduced in animals exposed to 20 mg/kg/d Vinclozolin (LOAEL), 0.25 and 2.5 mg/kg/d Flutamide (putative NOAEL and LOAEL), and 30 mg/kg/d Prochloraz (LOAEL) across a variety of timepoints

– Bulbo-urethral gland

– Cauda epididymis

– Epididymides

– Glans penis

– Bulbo-cavernous and Levator ani muscles

– Prostate

– Ventral Prostate

– Seminal Vesicle

No change was observed in testes weight


Conclusions

Dose Group 1 2 3 4 5 6 7 8 9

Dose (mg/kg bw/day)

VIN 0.005 4 20 FLT 0.0025 0.25 2.5 PRO 0.01 5 30

Ano-genital Distance/Index + + +++

Nipple Retention PND 12 +++ ++ +++ +++ +++ PND 21 +++

Developmental Abnormalities +++

Male Pup Sexual Maturation Age + + +++ + Weight +++

Organ Weights

PND 21 ++ ++ +

Puberty + ++ +++ +

PND 83 ++ +++ +

Histopathology

PND 21

Puberty ++

PND 83 +++

Putative NOAEL (0.25 mg/kg/d) for Flutamide is no NOAEL


Dose Group 1 2 3 4 4b 5 6 7 8 9

Dose (mg/kg bw/day)

VIN 0.005 4 20 FLT 0.0025 0.025 0.25 2.5 PRO 0.01 5 30

Ano-genital Distance/Index + + +++

Nipple Retention PND 12 +++ ++ +++ +++ +++ PND 21 +++

Developmental Abnormalities +++

Male Pup Sexual Maturation Age + + +++ + Weight +++

Organ Weights

PND 21 ++ n/a ++ +

Puberty + n/a ++ +++ +

PND 83 ++ n/a +++ +

Histopathology

PND 21 n/a

Puberty n/a ++

PND 83 n/a +++

Ongoing current study to establish Flutamide NOAEL


Future outlook

Reference value Expected NOAEL Effect level

BAS 352 F Vinclozolin

0.005 mg/kg bw/day

4 mg/kg bw/day 20 mg/kg bw/day

Flutamide NOAEL / 100 = 0.00025 mg/kg bw/day

0.25 mg/kg bw/day

0.025 mg/kg bw/day

2.5 mg/kg bw/day

0.25 mg/kg bw/day

BAS 590 F Prochloraz


In-life phase of the mixtures study begins January 2013

Excise mammary glands for whole-mount mammary analysis

Hormone measurement in juvenile testes

16


Brian, J.V.1, Beresford, N.1, Smith, A.2, White, S.2, Bean, T. 3, Katsiadaki, I.3, Sebire, M3 & Hutchinson, T.H.3. (contact [email protected])

HIGHLIGHTS The goal of this research is to characterize the in vitro and in vivo activities of plant-derived endocrine active compounds (EACs) and to evaluate potential effects of relevance to fish reproductive health Fallen leaves (beech, oak & reed) were collected from south west England and processed for screening using a battery of (anti)estrogenic, (anti)androgenic & (anti)gestagenic in vitro screens (the YAS, YES and YPS assays, respectively). This initial work addressed the impact of temperature and solvent on the extraction of dissolved organic carbon (DOC) and consequent responses in the YAS, YES and YPS assays (as both agonism and antagonism) Aqueous extracts (10 µl volumes) were directly screened in the YAS, YES and YPS assays. Aqueous extract subsamples (10 ml) were concentrated by solid phase extraction (SPE) onto a Sep-Pak C18 cartridge & the concentrated ethanol extracts were then also screened in the three yeast assays. Extraction temperature had a marked proportionate effect on aqueous DOC values. For the reed aqueous extracts, there was a DOC-related response in YES assay responses, while oak aqueous extracts gave DOC-related anti-YAS activity. Impacts of leaf extracts on fish reproduction will be addressed.

1Institute for the Environment, Brunel University, Middlesex, UK; 2Centre for Environment, Fisheries & Aquaculture Science (CEFAS) Lowestoft Laboratory, Suffolk, UK & 3CEFAS Weymouth Laboratory, Dorset, UK.

EMSG55: Evaluating Plant Leaf Extracts for Endocrine Activity & Reproductive Toxicity in Fish

mailto:[email protected]

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Combined low-dose exposures to anti-androgenic substancescefic-lri.org/wp-content/uploads/2014/03/12.JUNKER... · 2018. 12. 12. · LRI Presentation 151112.pptx 1 Combined low-dose