Images of Endocrine-active Changes

Reimschuessel, Renate RREIMSCH at CVM.FDA.GOV
Tue Sep 9 12:54:06 EST 2003


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Request for Images for Use in Round-Robin Evaluation of Gonadal 
Tissues of Fish Exposed to Endocrine Disrupting Chemicals  
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As you may be aware, OECD has initiated validation activities for a fish 
assay for detection of endocrine active chemicals.  One of the goals of 
the preliminary (phase 1A) activities is to assess the value and 
feasibility of including gonad histopathology as an endpoint in the 
assay.  However, at present no standardized approach to evaluation of 
abnormalities and gonad stage has been agreed upon.  Some 
progress has been made toward this goal as the result of the OECD 
Workshop On Gonadal Histology Of Small Laboratory Fish As A Tool 
For Endocrine Disruption Tests (5-6 Sep 2002, RIVM, Bilthoven, the 
Netherlands) and the initial validation work, but detailed guidance is still 
lacking, and there is no clear consensus on the  approach to be taken 
in assessments.   

To gain some of the information that is needed to develop such 
guidance, a series of activities is being undertaken in cooperation with 
the OECD working group that is drafting the 21-day fish ED screening 
protocol. One of these activities is to carry out a round-robin 
histopathological evaluation of that involves:  

*   obtaining a series of digital images of abnormalities and lesions in 
the gonads of fathead minnows, medaka, and zebrafish exposed to 
endocrine disruptors  

*   providing copies of these images to a panel of expert fish 
histopathologists  

*   obtaining each expert's independent evaluation of the image  

*   compiling results and
identifying areas of agreement and disagreement 

*   holding a follow-up meeting to attempt to obtain concensus on 
evaluations, and to articulate the process leading to the concensus  

Laboratories in Japan and Germany that are involved in phase 1A of 
the validation of the OECD fish assay will provide images of all types of 
lesions and abnormalities that they observe in the gonads of fathead 
minnows, medaka, and zebra fish that they have exposed to endocrine 
active chemicals. However, these studies involve exposing fish to only 
two chemicals, estradiol and trenbolone.  For the round-robin we need 
to have more comprehensive coverage of the effects of different 
modes of action so that we can include examples of  the additional 
types of lesions and abnormalities that might be observed if the test is 
widely used. We need your assistance in obtaining  images of these 
features. Specifically, if you have examples of gonadal lesions or 
abnormalities that you think might be due to the action of an endocrine 
active material, would you be willing to share these examples with the 
participants of the round-robin? By providing examples of gonad 
lesions and abnormalities for use in the round robin, you will be 
assisting the regulatory community in the collection of information that 
is needed to proceed with development of a fish screening test for 
endocrine disruption.  Please note the following:  

*   The images you provide will be used in the round-robin and may be 
included in subsequent publications by OECD, but it cannot be 
guaranteed that every image will be used.  

*   Images of your examples will be attributed to you whenever possible 
in subsequent write-ups or publications.  

*   Participants in the round-robin evaluations are being selected by 
OECD, and providing images for use in the round robin does not affect 
whether you will be asked to participate in the evaluation of the 
examples.  

*   Individuals who contribute slides will be kept up-to-date on the 
findings of the round-robin and on progress toward the developmenent 
of guidance on the evaluation of gonad histopathology.  

Thanks for considering our request.  If you are able to contribute 
images, please let us know by contacting Jeff Wolf at 
Jwolfepl at aol.com. Additional information on the round-robin and on 
details of handling of slides/images is included in the following text.   

The overall plan for the round-robin evaluation is described Appendix I. 
The round-robin will be coordinated jointly by Wildlife International Ltd., 
USA, Experimental Pathology Laboratories (EPL), USA, the Chemicals 
Evaluation and Research Institute, Japan (CERI), and the OECD 
secretariat. If you have questions, please contact  

Jeff Wolf (Jwolfepl at aol.com),
Tim Springer (tspringer at wildlifeinternational.com),
or Masanori Seki ( seki-masanori at ceri.jp)


Selection of images: 

Each of the labs participating in the phase 1A validation efforts has 
been asked to provide at least one image of each type of lesion that 
has been observed in each combination of species, sex, and test 
substance in the phase 1A tests (including images of abnormalities 
observed in control fish). If a lab chooses to do so, it may send images 
that help demonstrate different severity grades of the same lesion.  

Our request to labs that are not involved in the phase 1A validation is a 
bit different.  From these labs we are seeking examples of lesions and 
abnormalities that might result from chemicals that have do not have 
the same modes of action as estrogen and trenbolone. Slides or 
images of gonads of fathead minnows, medaka, and zebra fish of 
either sex are all useful.  

Slides and Images:

If you have glass slides but cannot provide digital images we can make 
digital photos from the slides. However, sending glass slides back and 
forth involves some risk to the slides, and we would prefer to receive 
digital images of the lesions or abnormalities.  

We would prefer to receive images in JPEG or TIFF format. However, 
we can translate images between different most standard electronic 
formats. Avoid using GIF format files, because they have limited 
resolution.  

If possible, we would like to receive two versions of each image, one 
with arrows and labeling indicating the lesion that has been identified, 
and one without arrows and labelling. For each image please identify 
the test substance, its concentration, fish species, its state of maturity 
or age, and any other facts that might be relevant.  Information on the 
suspected mode of action of the test substance would be helpful for 
less well known chemicals.   

Schedule: 

During the last phone conference of the OECD fish drafting group, it 
was decided to try to get the pathologists participating in the round 
robin together at end of September for a follow-up meeting on the 
evaluations of the lesions. To allow time to organize, circulate, and 
evaluate images, we need to have all images sent to us by September 
12.  

Directions for sending images to Wildlife International Ltd.

Several methods can be used to transfer digital images to Wildlife 
International, Ltd.  

*   Images can be sent on CD to

Tim Springer
Wildlife International Ltd.
8598 Commerce Drive
Easton, MD 21601

*   Images can be sent as attachments to e-mail, if the total size of 
attached files is less than 5 megabytes per e-mail.  Email images can 
be sent to tspringer at wildlifeinternational.com  

*   If many image files or large image files are to be transferred via the 
Internet, it will be preferable to send them by FTP.  We have set up an 
FTP site that is dedicated to receiving images for the round robin.  

Please e-mail tspringer at wildlifeinternational.com if additional 
assistance is needed.  


Appendix I

Summary of Plans for a Round-Robin Evaluation of Histopothological 
Assessments of Gonadal Tissues in Fish Exposed to Endocrine 
Disruptors  

Objectives

(1) Identify main features being used for evaluation of the status of the 
gonadal tissues by expert fish pathologists of sections of gonadal 
tissues from fathead minnows, medaka, and zebrafish exposed 
according to the draft 21-day fish ED screen protocol. (2) Evaluate 
uniformity of assessments by expert fish pathologists of sections of 
gonadal tissues from fathead minnows, medaka, and zebrafish 
exposed according to the draft 21-day nonspawning fish screening 
assay protocol.  The objective is not to evaluate expertise or levels of 
competence, but to discover when and why assessments differ. (3) 
Develop guidance that will help standardize histopathological 
assessments of gonad sections.  


Introduction

The need to develop a standardized approach to scoring changes in 
the gonads of fish exposed in the 21-day screening study has been 
recognized, and some progress has been made in this direction. 
However, more detailed guidance is needed.  The histological 
assessment of gonads of fish from the 21-day ED non-spawning fish 
screening assay must be considered from two perspectives, (1) the 
assessment of abnormalities and (2) assessment of gonadal stage. 
Each perspective has its own difficulties, and will present different 
demands in terms of guidance.   

An important step toward developing such guidance is to develop a 
better understanding of the logic used by experienced fish pathologists 
in evaluating histological changes and whether such changes should 
be considered indicative of endocrine disrupting activity.  A series of 
activities designed to develop this understanding have been developed 
in cooperation with the OECD working group drafting the 21 day fish 
ED screening protocol.  These activities will be coordinated jointly by 
Wildlife International Ltd., USA, Experimental Pathology Laboratories 
(EPL), USA, the Chemicals Evaluation and Research Institute, Japan 
(CERI) , and OECD to obtain this understanding. The approach that is 
being followed is to provide each member of a panel of expert fish 
pathologists with the same set of images or slides of gonad tissue from 
fish exposed to endocrine disruptors, to acquire each pathologists 
independent assessment of each image, and then to collate the 
assessments. The collated finding from the group of fish pathologists 
will be used to determine the extent of agreement or disagreement in 
the evaluation of histopathological lesions in the gonads, and to help 
determine the reasons for disagreement. (This is sometimes referred 
to as a round-robin evaluation). A similar evaluation of assessment of 
agreement on gonad staging will be performed using histological 
sections of the gonads. Areas of agreement in the interpretation of 
findings will be recorded and form the basis for more concrete 
guidance. Follow-up discussions will be held to focus on the reasons 
for disagreement and attempt to develop consensus decisions.  


The Round Robin Evaluation

The round robin evaluation will consist of two parts, a lesion evaluation 
(LE) phase focused on interpreting digital images of gonad lesions and 
abnormalities resulting from exposure to endocrine disruptors, and a 
section evaluation (SE) phase focused on evaluating entire gonad 
sections, particularly with regard to gonadal staging.  

Steps in the lesion evaluation (LE) phase:

(1) Digital images of gonad lesions observed by the laboratories 
participating in phase 1-A of the validation of the 21 day fish test will be 
sent to Wildlife International Ltd. where they will be organized (in 
cooperation with EPL) and put into a common format. Details 
concerning selection of images and methods of transmitting images 
can be found in Annex 1 (attached).  Communications with 
participating Japanese laboratories will be coordination by CERI. 
Example lesions will be from male and female gonads of fathead 
minnows, medaka, and zebrafish.  Because phase 1-A lesions all 
result from exposure to estrogen or trenbolone, other laboratories that 
have tested endocrine disruptors that may act by other modes of action 
will be contacted to obtain additional images of other types of lesions 
that have been observed. (2)  The digital images of lesions from 
gonads of treated and untreated fish will be organized by EPL and 
Wildlife International Ltd. and sent to a panel of expert fish pathologists 
selected by OECD. Each image will be accompanied by a 
questionnaire which directs the pathologist to answer specific 
questions about the lesion being observed. Questions to be answered 
for each image will include those given in Box 1, below. (3) Completed 
questionnaires will be returned to Wildlife International Ltd., where 
responses will be compiled and summarized. (4) A follow-up meeting 
will be organized to allow the panel of expert pathologists to reconcile 
differences in interpretation, and to use areas of agreement to develop 
guidance on a standardized approach for use in routine screening. (5) 
A description of the pathologists' comparative findings will be written to 
support phase 1A validation efforts. The images evaluated and the 
consensus evaluations will be made available for future reference.  

Preliminary discussions have suggested that developing an all-
encompassing quantitative scoring scheme for gonad abnormalities is 
not feasible. However, a flow chart of the logic used in scoring can be 
developed (See Fig. 1 for a starting point), and some components of 
the assessment (e.g. severity) can be expressed as a qualitative 
scoring scheme.  In contrast, fairly straightforward criteria for staging 
fish gonads (perhaps using a scale of 1 to 5) can probably be 
proposed for each species.  An example of a staging scheme that has 
been proposed for fathead minnow gonads (developed by EPL based 
on criteria developed by USGS for carp gonads) is attached (Table 1). 
Proposed species-specific staging criteria could be agreed upon and 
made as consistent across species as possible.  


          Box 1. Questions for the LE phase of the round robin
          evaluation.


I.  Do you consider the observed histopathologic change to be an 
"abnormal" finding for the [testis/ovary] of laboratory reared adult 
[male/female] [species]? 

a) Always 
b) Only if present at a relatively high degree of severity (e.g., moderate 
to severe). 
c) Only if present at a significantly increased incidence in the treated 
fish compared to the control fish. 
d) Only under certain other conditions (please state) ______________ 
e) b and c 
f) b, c, and d 
g) Never 
h) Unsure  

II. On a scale of 1 to 5 (1=minimal, 2=mild, 3=moderate, 4=moderately 
severe, and 5=severe) how would you score the severity of the 
abnormal finding? _____________  

III. Do you believe that there is sufficient scientific evidence to justify 
the view that this particular histopathological change should be 
considered indicative of endocrine disrupting activity? (if possible, 
please support your answer with appropriate literary references). 

a) Yes. 
b) Not currently; however, this change should be identified when 
present because it is a potential indicator of endocrine disrupting 
activity. 
c) No.  

IV. Description of abnormality or abnormalities observed: 
_____________  


Even if the LE phase of the round-robin fails to lead directly to concrete 
guidance on identifying endocrine disruption in gonad tissues, a set of 
reference images with consensus interpretation by the expert panel 
would be available for training and standardization purposes.  

Steps in the section evaluation (SE) phase:

The SE phase of the round robin will have two parts, an assessment of 
abnormalities and assessment of gonad stage.  

Assessment of gonad abnormalities:

(1) The SE phase of the round robin will be combined with the follow 
up meeting being held to discuss the LE phase evaluations. (2) For the 
SE phase, example slides supplied by participating labs involved in the 
validation tests bearing whole gonad sections also will be examined by 
the panel of pathologists. These slides will be hand-carried to the 
meeting. (3) The set of slides will contain gonad sections from male 
and female fathead minnows, and longitudinal whole body sections (if 
available) that include gonads from male and female medaka and 
zebrafish.  These sections will be obtained from fish that were exposed 
for 21 days at the highest concentrations used in phase 1-A of 
validation to estradiol or an androgen such as trenbolone. Reference 
sections from untreated fish will also be included.  (4) Panel members 
will independently evaluate the gonad sections on each slide for 
abnormalities. Questions to be answered for each slide will include 
those given in Box 2, below.  (5) Disagreements in findings will be 
identified, and a follow-up meeting will be arranged to try to develop 
consensus evaluations of the lesions involved.  At this time, digital 
images of key lesions will be made for future reference, and the 
consensus evaluations will be made available for future reference. A 
description of the pathologists' comparative findings will be written to 
support phase 1A validation efforts. The panel will attempt to 
incorporate the logic used to develop their consensus interpretations of 
slides into a guidance document for use in routine screening.  

  Box 2 . Questions for the SE phase of the round-robin evaluation.

I.  Do you consider any observed histopathologic changes to be an 
"abnormal" finding for the [testis/ovary] of laboratory reared adult 
[male/female] [species]? 

a) Always 
b) Only if present at a relatively high degree of severity (e.g., moderate 
to severe). 
c) Only if present at a significantly increased incidence in the treated 
fish compared to the control fish. 
d) Only under certain other conditions (please state) ______________ 
e) b and c 
f) b, c, and d 
g) Never 
h) Unsure 

II.     Do you believe that there is sufficient scientific evidence to justify 
the view that any of the particular histopathologic changes should be 
considered indicative of endocrine disrupting activity? (if possible, 
please support your answer with appropriate literary references). 

a) Yes.  (Which changes? ____________________) 
b) Not currently; however, this change should be identified when 
present because it is a potential indicator of endocrine disrupting 
activity. (Which changes? ____________) 
c) No. 

III.    On a scale of 1 to 5 (1=minimal, 2=mild, 3=moderate, 
4=moderately severe, and 5=severe) how would you score the severity 
of the abnormal finding? _____________  

IV. Description of abnormality or abnormalities observed: ___________


Assessment of gonad stage:

(1) Pathologists will assign each gonad represented in the slides to one 
of 4 to 5 developmental stages described in Table 1.  

(2) Disagreements in stage assignments will be identified, and an 
attempt will be made to develop consensus on stage assignments. 
Consensus staging criteria will be developed for each species.  

Figure 1.  Conceptual flow chart for interpreting gonad tissue exposed 
to possible endocrine disruptors. [NOTE: This chart could not be 
included in the body of an e-mail message]  


Table 1.  Staging of Gonads.  Based on criteria in "Biomonitoring of 
environmental status and trends (BEST program): Selected Methods 
for Monitoring chemical contaminants and their effects in aquatic 
ecosystems" USGS/BDR/ITR - 2002-0005.  

Testes Stage   
Criteria
0 - Undeveloped Exclusively immature phases (spermatocytes to 
spermatids) with no spermatozoa 
1 - Early spermatogenic Immature phases predominate, but 
spermatazoa may also be observed 
2 - Mid spermatogenic   Spermatocytes, spermatids, and spermatozoa 
are present in roughly equal proportions 
3 - Late spermatogenic  All stages may be observed, however, mature 
sperm predominate 
4 - Spent   Loose connective tissue with some remnant sperm.  


Stage   Criteria

0-Undeveloped   Perinuclear pre-vitellogenic oocytes exclusively; no 
cortical alveoli 
1-Early development >90% pre-vitellogenic, predominantly perinuclear 
through cortical alveolar 
2-Mid development At least half of observed follicles are early and mid 
vitellogenic 
3-Late development  Majority of developing follicles are late vitellogenic 
4- Late development/hydrated    Majority are late vitellogenic; follicles 
are much larger 
5- Post ovulatory   Spent follicles, remnants of theca externa and 
granulosa Ovaries  

-- 
Jeffrey C. Wolf, DVM, Dipl. ACVP
Experimental Pathology Laboratories, Inc.
22866 Shaw Rd.
Sterling, VA, 20166 USA
Tel: 703-471-7060 Ext 242
Fax: 703-471-8447
jwolfepl at aol.com







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