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

og

en

Ag

on

ist

Estr

og

en

An

tag

on

ist

An

dro

ge

n

Ag

on

ist

An

dro

ge

n

An

tag

on

ist

Estr

og

en

An

dro

ge

n

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

og

en

Ag

on

ist *

Estr

og

en

An

tag

on

ist *

An

dro

ge

n

Ag

on

ist

An

dro

ge

n

An

tag

on

ist

Th

yro

id

Ag

on

ist

Th

yro

id

An

tag

on

ist

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

og

en

Ag

on

ist

Estr

og

en

An

tag

on

ist

An

dro

ge

n

Ag

on

ist *

An

dro

ge

n

An

tag

on

ist *

Th

yro

id

Ag

on

ist

Th

yro

id

An

tag

on

ist

Ste

roid

ogenesis

Inhib

itio

n

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

og

en

Ag

on

ist

Estr

og

en

An

tag

on

ist

An

dro

ge

n

Ag

on

ist

An

dro

ge

n

An

tag

on

ist

Th

yro

id

Ag

on

ist

Th

yro

id

An

tag

on

ist

Ste

roid

ogenesis

Inhib

ition

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

og

en

Ag

on

ist

Estr

og

en

An

tag

on

ist

An

dro

ge

n

Ag

on

ist

An

dro

ge

n

An

tag

on

ist

Th

yro

id

Ag

on

ist

Th

yro

id

An

tag

on

ist

Ste

roid

ogenesis

Induction

Ste

roid

ogenesis

Inhib

itio

n

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.

www.huntingdon.com

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‖.

www.huntingdon.com

ANY BURNING

QUESTIONS?

www.huntingdon.com

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

reganj@ukorg.huntingdon.com

+44 (0) 1480 892031