Images of Endocrine-active Changes

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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
[Only registered users see links. ]. 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 ([Only registered users see links. ]),
Tim Springer ([Only registered users see links. ]),
or Masanori Seki ( [Only registered users see links. ])


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 [Only registered users see links. ]

* 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 [Only registered users see links. ] 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
[Only registered users see links. ]