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The science and methodologies required to perform the Endocrine Disruptor Screening Program’s Tier 1 In Vivo Mammalian Assays will be discussed including the nature of the reproductive and general toxicity endpoints and the alterations that may signal endocrine disruption. As the Male and Female Pubertal studies can be performed as a Combined Male and Female Pubertal Study, the advantages and disadvantages of the combined assay will also be discussed. Regulatory agencies have consistently agreed that endocrine disruption must be evaluated by “Weight of Evidence” (WoE) procedures. A discussion of the relative weight for each endpoint and the use of the hypothesis-based “WoE” approach for determination a positive response in this assay will be discussed. More info at http://www.huntingdon.com/Chemical/Endocrinedisruptorscreeningprogram/Webinars
Citation preview
www.huntingdon.com
Endocrine Disruptor
Screening Program
Webinar week
20-23 January 2014
www.huntingdon.com
Science and methodologies behind
performance and interpretation
EDSP in vivo mammalian assays
Bob Parker PhD, Diplomate ABT
Director, Safety Assessment
Reproductive and Developmental
Toxicology
www.huntingdon.com
Robert M. Parker, PhD, DABT
Colin Williams
Huntingdon Life Sciences
Science and Methodologies behind the Performance
and Interpretation of Endocrine Disruptor Screening
Program’s Tier 1 In Vivo Mammalian Assays:
Hershberger, Uterotropic, Male Pubertal and Female
Pubertal Studies –
A Study Director’s Perspective
www.huntingdon.com
EDSP Tier 1 Assays Estrogen Receptor Binding
Estrogenic Non-estrogenic
Androgen Receptor Binding
Androgenic Non-androgenic
Uterotrophic Hershberger
Female Pubertal
HPG/HPT
Male Pubertal
HPG/HPT Thyroid
Function
Amphibian Metamorphosis (HPT)
Aromatase
Steroidogenesis Estrogen Testosterone
Fish Short-term Reproduction (HPG)
In v
itro
In
viv
o
Ec
oTo
x
ER
Activation
From: Marty MS, Carney EW and Rowlands JC.
Toxicol Sci 120(S1), S93–S108 , 2011.
www.huntingdon.com
The EDSP Tier 1 screening assays encompass key endpoints within a Mode of
Action (e.g., receptor binding) and along endocrine pathways (e.g., effects on
HPG and HPT axes, steroidogenesis) through which a chemical has the
potential to interact with the Estrogen, Androgen, or Thyroid hormonal
pathways.*
Estr
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Estr
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Ag
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Estr
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Study Design
Uterotrophic g
Hershberger g g g1
Male Pubertal Study g g g g g
Female Pubertal Study g g g g g
15-reductase inhibition only
Receptor BindingSteroidogenesis
Inhibitor
HPG
Axis
HPT
AxisMode of Action
*Complementary endpoints across assays are indicated (solid red box) within each column.
U.S. EPA 2011a. EPA-HQ-OPPT-2010-0877-0021
www.huntingdon.com
Examples of Neuro-Endocrine Pathways that are
affected by Endocrine Disrupting Compounds
DETAILED REVIEW PAPER ON THE STATE OF THE SCIENCE ON NOVEL IN VITRO AND IN
VIVO SCREENING AND TESTING METHODS AND ENDPOINTS FOR EVALUATING ENDOCRINE
DISRUPTORS. Series on Testing & Assessment, No. 178; ENV/JM/MONO(2012)23
Black arrows denote contiguous pathways. Red arrows highlight examples of cross-talk between pathways
Hypothalamic-Pituitary
-Adrenal Axis Hypothalamic-Pituitary-
Thyroid Axis
Somatotropin Axis
RETINOID
SIGNALING
PATHWAY
VITAMIN D
SIGNALING PATHWAY
Hypothalamic-Pituitary-
Gonadal Axis
www.huntingdon.com
Reproductive Physiology: Rat and Human
Similarities
• Steroid hormone control of reproductive function relies on testosterone,
estradiol, dihydrotestosterone and progesterone.
• CNS-hypothalamic secretion of gonatropin-releasing hormone controls
pituitary synthesis and release of follicle-stimulating hormone (FSH) and
luteinizing hormone (LH) that regulate germ cell development after
puberty. LH surges induce spontaneous ovulation in the female, LH
regulates testis Leydig cell testosterone production.
• Hormonal regulation of uterine function and onset of delivery.
• Androgens are required to maintain male spermatogenesis and secondary
sex characteristics.
• Dramatic endocrine changes resulting from CNS-hypothalamic-pituitary-
gonadal maturation responsible for puberty in males and females. Females
generally attain puberty at an earlier age than males of the same species.
Gray et al, 2004 ILAR Journal, 45:4, 425
www.huntingdon.com
Reproductive Physiology: Rat and Human
Differences
• The rat placenta lacks aromatase; estrogen is produced during pregnancy by the
ovary. Human placental tissue expresses high levels of aromatase.
• Rat sexual differentiation is perinatal, whereas CNS sexual differentiation is
postnatal, regulated to a great degree by aromatization of testosterone to
estradiol. In nonhuman primates and presumably humans, more CNS events are
prenatal, and androgens are more important than in rats .
• The rat has a 4- to 5-day estrous cycle, with no functional corpora luteum. The
estrous cycle can be monitored easily by examining daily cytology. The female rat
displays sexual receptivity only during estrus after ―lights out‖ after a proestrus
vaginal smear. This behavior is exquisitely dependent on estrogen followed by
progesterone. Humans have a menstrual cycle approximately 28 days in duration
and do not display periods of peak behavioral ―estrus‖ during the cycle.
• Puberty in the rat (as measured by the age at vaginal opening and the onset of
estrous cyclicity) occurs at about 32 days of age in females and 42 days of age
(as measured by preputial separation an androgen-dependent event) in male
Sprague-Dawley and Long-Evans rat strains. In humans, puberty occurs at 9 to
12 years of age in girls, and 10 to 14 years of age in boys.
Gray et al, 2004 ILAR Journal, 45:4, 425
www.huntingdon.com
OPPTS 890.1500:
PUBERTAL DEVELOPMENT AND
THYROID FUNCTION IN INTACT
JUVENILE/PERIPUBERTAL MALE RATS
Body Weights checked daily; Dose daily adjusting for body weight
Daily examination for PS
Wean &
Group
Assign
21 23 25 30 35 40 45
BW
Blood Collection
Necropsy
Dosing Period Cull to
8 – 10
pups
4 Postnatal Day
50 53
www.huntingdon.com
MALE PUBERTAL ASSAY
ENDPOINTS Estr
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Ag
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ist *
Estr
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An
tag
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An
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Ag
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An
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An
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Th
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Th
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Growth
Age and Weight at Preputial Separation
Hormones
Thyroxine (T4)
Testosterone
Thyroid Stimulating Hormone (TSH)
Organ weightsA
Testis (separately) B
Epididymides (separately) B
Ventral Prostate B
Dorsolateral Prostate
SV (with CG) with Fluid B
SV (with CG) without Fluid B
Levator ani/Bulbocavernosus muscles
Thyroid
Liver
Pituitary
Histopathology: `
Testis C D E F
Epididymus C G C
Thyroid follicular epithelial height
Thyroid colloid area
* Not designed to detect this modality however effects do occur A. Androgen agonist - A statistically significant increase
in any two or more of the five required androgen-dependent tissue weights is considered positive; B. Paradoxical weight
decrease; C. Atrophy; D. Aspermia and ductal atrophy; E. Variable progression to tubular atrophy; F. Hyperplasia/
hypertrophy of the interstitial cells of the testis; G. Hypospermatogenesis and interstitial cellular atrophy
www.huntingdon.com
OPPTS 890.1450:
PUBERTAL DEVELOPMENT AND
THYROID FUNCTION IN INTACT
JUVENILE/PERIPUBERTAL FEMALE RATS
Body Weights checked daily; Dose daily adjusting for body weight
Daily examination for VO; then daily vaginal lavage for cyclicity
Wean &
Group
Assign
21 22 25 30 35 40 42
BW
Blood Collection
Necropsy
Dosing Period Cull to
8 – 10
pups
4
Postnatal Day
www.huntingdon.com
FEMALE PUBERTAL ASSAY
ENDPOINTS Estr
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Ag
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Estr
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An
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Ag
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An
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An
tag
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Th
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Ag
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Th
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Ste
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ogenesis
Inhib
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Growth
Age and Weight at Vaginal Opening
Hormones
Thyroxine (T4)
Thyroid Stimulating Hormone (TSH)
Estrous Cyclicity
Age at first estrus
Organ weights:
Ovaries
Uterus
Thyroid
Liver
Adrenals (paired)
Histopathology:
Ovarian A B A
Uterus A B A
Thyroid follicular epithelial height
Thyroid colloid area
* Not designed to detect this modality however effects do occur A. Androgen agonist - A statistically significant increase
in any two or more of the five required androgen-dependent tissue weights is considered positive; B. Paradoxical weight
decrease; C. Atrophy; D. Aspermia and ductal atrophy; E. Variable progression to tubular atrophy; F. Hyperplasia/
hypertrophy of the interstitial cells of the testis; G. Hypospermatogenesis and interstitial cellular atrophy
www.huntingdon.com
Combined Male and Female
Pubertal Assay Schematic
Male Body Weights checked daily; Dose daily adjusting for body weight
Daily examination for PS Wean &
Group
Assign
23 25 30 35 40 45
•Male
•BW
•Blood
Collection
•Necropsy
Cull to
8 – 10
pups
4
Postnatal Day
50 53
Female Dosing Period
Female Body Weights checked daily; Dose daily adjusting
for body weight
Daily examination for VO; vaginal lavage for cyclicity
42
Male Dosing Period
•Female
•BW
•Blood Collection
•Necropsy
www.huntingdon.com
Combined Pubertal Study versus Individual
Male and Female Pubertal Studies It is acceptable to run a combined male and female pubertal study. (OPPTS
890.1500 and 890.1450).
The combined study duration is the same as the Male Pubertal Assay.
Separately conducted studies require 50 time-mated F0 animals.
In contrast, a combined Male and Female Pubertal Assay design requires 26 time-
mated F0 animals.
This 48% reduction in the number of F0 animals meets the spirit of the three Rs
espoused by ECVAM.
The other major advantages of a combined pubertal study design are:
1) most pups are evaluated rather than having one sex discarded per single
gender study designs;
2) reduced reporting time; and
3) decreased costs (e.g., fewer animals, rooms; cages, technical evaluations,
technician time).
The only minor disadvantage is concentration of technician time during the VO and
PPS evaluations.
www.huntingdon.com
Concerns and Potential Pitfalls for
Pubertal Studies Scheduling
Parturition is often over two days causing a staggered start
Stagger may be required based on necropsy technical staff
Litter sizes may be unbalanced
Method of animal allocation
Very detailed (performed on PND 21; tight schedule)
Ranked bodyweight and distributing litters across groups
No placing of same-sex litter mates in the same group
(controls Litter Effect)
EPA Required Spreadsheets and Statistical Analysis
Requires manual entry and therefore another QC/QA effort
www.huntingdon.com
Concerns and Potential Pitfalls for
Pubertal Studies Caging and Bedding Requirements
Feed and Water Requirements
Treatment (between 7 and 9 am)
Oral gavage recommended
Stainless steel catheter with ball
Dose administration (Day of Necropsy)
Transfer from dosing room to holding room
Transfer from holding room to Necropsy Lab to minimize stress
effects
Initiation of necropsy two hours after dosing
Euthanasia – decapitation (alternatives now acceptable)
Completion of Necropsy by 1:00 pm
www.huntingdon.com
Problems with Interpreting Pubertal Assays Inherent variability in (apical endpoints) Significant inherent biological variability
in the endpoints (puberty onset (age and weight at VO and PPS), estrous cycle,
organ weights) complicates interpretation. Endpoints can be altered by either
endocrine or non-endocrine modes of action or by non-specific, systemic
toxicity, or by impairment in growth (such as reduced food consumption).
Assay Specificity
Female pubertal assay:
Insufficient monitoring period for estrous cycling
Ovarian and uterine weights complicated by estrous cycling
Male pubertal assay:
False negative: Phenobarbital (< 100 mg/kg/day) thyroid effects not detected
Male and Female Pubertal Assays:
―SAP: “…that a negative control substance has not been identified (in the pubertal assays)…is a major limitation to the Tier I battery. Lacking demonstration of expected negative results remains an issue for the validity of these assays”.
EPA is in the process of conducting negative control studies
www.huntingdon.com
Using Body Weight for MTD and
Pubertal Assay Specificity A 10% decrement in final body weight has been established as a criterion
for establishment of a Maximum Tolerated Dose, but subsequent studies (Marty et al., 2003; Laws, et al., 2007) suggest more than 6% decrement in final body weight of males may result in thyroid perturbations. The possibility of body weight-associated changes rather than direct endocrine disruption should be considered when there is >6% change in body weight in male rats.
Feed restriction studies performed with the pubertal assay designs:
9-12% change in terminal body weight
Decreased absolute Adrenal, Pituitary (♀♂) and Ovarian weights
Decreased absolute Epididymal, Ventral Prostate and Seminal Vesicles weights
T3 and T4 are sensitive to body weight changes 9% body weight change altered thyroid endpoints (♂)
SAP: ―Body weight reductions were closely associated with perturbations in the onset of puberty and/or normal cycling. Therefore the specificity of the pubertal assays for detecting alterations in the HPG axis due to purely endocrine-related disruption is currently unclear‖.
www.huntingdon.com
OPPTS 890.1400:
HERSHBERGER BIOASSAY STUDY
DESIGN
Acclimatisation
0 42 49 59
Dosing period
Postnatal Day 60
Post-surgical care
continued
acclimatisation
Necropsy Castration
Related timings must remain the same but the plan may be shifted provided dosing
commences no earlier than Day 49 and no later than Day 60
www.huntingdon.com
HERSHBERGER ASSAY
ENDPOINTS Estr
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Ste
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ogenesis
Inhib
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GrowthWeight at NecropsyClinical SignsHormones (Optional)
TestosteroneFollicle Stimulating HormoneLeutinizing Hormone
Organ weightsA,B
Cowper's gland
Glans Penis
Ventral Prostate
SV (with CG) with Fluid
Levator ani/Bulbocavernosus
Liver - optionalKidneys (paired) - optionalAdrenals (paired) - optional
A. Androgen agonist - A statistically significant increase in all five androgen-dependent organs is a clear indication of
potential androgenic activity while any two or more of the five required androgen-dependent tissue weights should be
considered a positive androgen agonist result.
B. Androgen antagonist - A statistically significant decrease in all five androgen-dependent organs is a clear indication of
potential androgenic activity while any two or more of the five required androgen-dependent tissue weights should be
considered a positive androgen agonist result.
www.huntingdon.com
Issues with Hershberger Assay
1.EPA and OECD test guidelines require a castrated male model.
2.Weights of the target tissues may be altered by agents other than
androgen agonists or antagonists, therefore significant alterations in two
or more target organ weights are required for a positive assay outcome.
3.Glans penis weights can only be collected from animals that have
completed preputial separation, yet there is wide inter-laboratory
variation in the mean age at which preputial separation occurs.
4.For optional endpoints, ease of measurement and ability to interpret
results need to be fully considered to avoid potential ambiguities. If
optional measurements are included, it is advisable to develop a priori a
set of internal interpretive criteria specified in the study protocol before
conducting the assays.
5.If optional steroid hormone levels are measured, the anesthetic agent
and euthanizing method should be chosen carefully to avoid artifacts.
Borgert et al. 2011. Regulatory Toxicology and Pharmacology 59: 397–411
www.huntingdon.com
OPPTS 890.1600:
UTEROTROPHIC ASSAY STUDY
DESIGN: OVERIECTOMIZED RAT
Related timings must remain the same but the plan may be shifted provided ovariectory
is conducted in animals between 6 and 8 weeks of age
59
Acclimatisation
35 42 49 56
Vaginal
Swabbing
Postnatal Day
Post-surgical care
continued
acclimatisation
Necropsy Ovariectomy Animal arrival
Dosing
www.huntingdon.com
Uterotrophic Assay Endpoints
UTEROTROPHIC ASSAY ASSAY
ENDPOINTS Estr
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Ag
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Estr
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An
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Ag
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An
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An
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Th
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Ag
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Th
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An
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Ste
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ogenesis
Induction
Ste
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ogenesis
Inhib
itio
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Growth
Uterine Weights A
With Fluid
Without Fluid
A. Positive response Estrogen Agonist: Statistically significant increase of the mean uterus weight (wet and or
blotted)
www.huntingdon.com
Issues with Uterotrophic Assay 1. EPA has stated a preference for the ovariectomized female rat model while
OECD favors the immature rat model (based on animal welfare concerns).
2. Immature model appears more sensitive to dietary phytoestrogen content, to
body weight influences on uterine weight, and to systemic toxicity.
3. Ovariectomy increases animal manipulation/stress, introduces an artificial loss
of organ function to the assay, and requires additional acclimatization after
surgery as well as the requirement to confirm complete ovariectomy prior to the
assay and at necropsy; small ovarian remnants can alter assay outcome.
4. EPA favors subcutaneous route while OECD states most relevant route of
exposure should be used with consideration given to first pass metabolism.
5. For substances rapidly deactivated by first-pass hepatic metabolism, the
subcutaneous route may produce positive results that are irrelevant for the
route of administration used in Tier 2 testing and for actual environmental
exposures.
Borgert et al. 2011. Regulatory Toxicology and Pharmacology 59: 397–411
www.huntingdon.com
Tier 1 screening tests are intended to minimise false
negatives – focus is on sensitivity, not specificity
In vitro tests indicate mode of action, not endocrine
activity
In vivo tests include influence of kinetics / metabolism /
endocrine feedback control systems – but also non-
endocrine responses
Tests are complementary and ―redundant‖ – each
mode of action is assessed in more than one test
Look for consistent / inconsistent results
Consider the range, nature and magnitude of effects
Weight-of-Evidence Considerations
www.huntingdon.com
Flowchart for assessment of endocrine
disrupting properties for human health Multi-endpoint studies
(apical, in vivo) Targeted endpoint studies
(mechanistic, in vitro & in vivo)
No ED concern
per Weybridge?
Adverse health effect in apical
study supported by mechanistic
evidence of endocrine mediated
effect
No or insufficient
evidence of ED MoA
per Weybridge
Supporting studies
(non apical, in vivo)
No adverse health
effects giving
concern for
endocrine activity
Endocrine activity
giving concern for
endocrine toxicity
Adverse effects
giving concern for
endocrine toxicity
Endocrine activity
giving concern for
endocrine toxicity
Endocrine activity
giving concern for
endocrine toxicity
No evidence of
Endocrine activity
B A C D E
C. When adverse effects on endocrine relevant endpoints in apical or supporting non-apical in vivo studies are supported
by mechanistic data from in vitro and in vivo studies, (i.e. the sequence of the biochemical and cellular events that
underlies the adverse effect is described and understood, then conclusive proof of endocrine disruption can be considered
as established.
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Adverse health effect in apical study supported by
mechanistic evidence of endocrine mediated effect
Sufficient evidence of
ED as per Weybridge
Determine potency of ED
according to proposed
criteria
Relevance of ED mechanism
to humans?
(unless exposure is negligible)
Risk assessment based on
non-endocrine endpoint
Risk assessment based on
endocrine endpoint with
assessment factors according
to potency
Are adverse effects
specific?
Yes
Yes
Yes
No
No
Flowchart for Assessment of Endocrine
Disrupting Properties for Human Health
Modified from ECETOC Technical Report 106, June 2009
Bars R et al. 2011. Reg Toxicol Pharmaco 37–46.
www.huntingdon.com
Relevance and strength Weighting of
Tier 1 Endocrine Screening Endpoints
Borgert et al. (2011a) proposed a framework were the relevance of
each endpoint is assigned a weight according to its importance for
evaluating a specific hypothesis (described above).
―Weight‖ implies that all data do not contribute equally to answering
the question posed. Thus, ―weighting‖ involves a careful consideration
of the specific hypothesis to be evaluated and how each particular
measurement (data) informs that hypothesis. Ideally, weight would be
assigned quantitatively based on objective measurements of
predictive power, false positive and negative detection rates, and
potency or strength of the response. This value is deemed a relevance
weight, designated ―WREL.‖
The strength of response produced by the test chemical in a particular
assay or endpoint is also given weight, a value deemed the response
weight, ―WRES‖.
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ANY BURNING
QUESTIONS?
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References
Bars R, Broeckaert F, Fegert I, Gross M, et al. 2011. Science based guidance for the assessment of
endocrine disrupting properties of chemicals. Regulatory Toxicology and Pharmacology 59 (2011) 37–46.
Borgert CJ, Mihaich EM, Ortego LS, et al. 2011a. Hypothesis-driven weight of evidence framework for
evaluating data within the US EPA's Endocrine Disruptor Screening Program. Regul Toxicol Pharmacol.
61:185-191.
Borgert CJ, Mihaich EM, Quill TF, Marty MS et al. 2011b. Evaluation of EPA's Tier 1 Endocrine Screening
Battery and recommendations for improving the interpretation of screening results. Regul Toxicol
Pharmacol. 59:397-411.
Borgert CJ, Baker SP, Matthews JC. 2013. Potency matters: Thresholds govern endocrine activity. Regul.
Toxicol.Pharmacol., 67(1):83–88.
Detailed Review Paper on the State of the Science on Novel In Vitro and In Vivo Screening and Testing
Methods and Endpoints for Evaluating Endocrine Disruptors. Series on Testing & Assessment, No. 178;
ENV/JM/MONO(2012)23.
ECETOC Guidance on Identifying Endocrine Disrupting Effects .Technical Report 106 (June 2009)
http://www.ecetoc.org/technical-reports
Gray, LE, Wilson V, Noriega N et al. 2004. Use of the Laboratory Rat as a Model in Endocrine Disruptor
Screening and Testing. ILAR, Journal 45(4): 425-437.
Kortenkamp A, Martin O, Faust M, Evans R, McKinlay R, Orton F and Ro E. 2011. State of rhe Art
Assessment of Endocrine Disrupters: Final Report . European Commission, DG Environment , Section
7.2.2., p.127.
www.huntingdon.com
References Laws SC, Stoker TE, Ferrell JM, Hotchkiss MG and Cooper RL. 2007. Effects of altered food intake during
pubertal development in male and female wistar rats. Toxicol Sci 100:194-202.
Marty MS, Johnson KA and Carney EW. 2003. Effect of feed restriction on Hershberger and pubertal male
assay endpoints. Birth Defects Research Part B: Developmental and Reproductive Toxicology 68(4), 363-
374.
Marty MS, Carney EW and Rowlands JC. 2011 Endocrine Disruption: Historical Perspectives and Its
Impact on the Future of Toxicology Testing. Toxicol Sci 120(S1), S93–S108 .
OECD (Organization for Economic Cooperation and Development). 2012. Guidance Document on
Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption no 150. Organisation for
Economic Cooperation and Development, Paris, 24-Aug-2012.
Picut CA, Remick AK, Asakawa MG, Simons ML and Parker GA. 2013) Histologic Features of Prepubertal
and Pubertal Reproductive Development in Female Sprague-Dawley Rats. Toxicol Pathol
U.S. EPA (Enviromental Protection Agency) 2011a. Weight-of-Evidence: Evaluating Results of EDSP Tier
1 Screening to Identify the Need for Tier 2 Testing. EPA-HQ-OPPT-2010-0877-0021.
US EPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013.
Weybridge, 1996. European Workshop on the impact of endocrine disrupters on human health and wildlife.
2–4 December 1996, Weybridge, UK. In: Report of Proceedings EUR 17549 Copenhagen, Denmark:
European Commission DG XII, April 16, 1997). Available from: European Environment Agency, Kongens
Nytorv 6, DK-1050 Copenhagen K, Denmark.
Zoeller RT, Brown TR,L. Doan LL, Gore AC et al. 2012. Endocrine-Disrupting Chemicals and Public
Health Protection: A Statement of Principles from the Endocrine Society. Endocrinology 153: 4097–4110.
www.huntingdon.com
Other webinars this week
Thursday 23rd
Amphibian metamorphosis assay for the
EPA’s EDSP
Carole Jenkins
www.huntingdon.com
HLS EDSP expert team
Ephi Gur – Team lead and Regulatory
Bob Parker – Toxicology
Will Davies – Toxicology
John Carter – In vitro technologies
Carole Jenkins – Aquatic toxicology
Contact via me
+44 (0) 1480 892031