MMT ISR Pathology Appendix DRAFT 1 March 29, 2013 Appendix F

. Pathology Guidance

Section 1 MMT Histotechnology

1.1 Euthanasia and necropsy

1.2 Histotechnology

1.2.1 Transverse

- Oblique Sectioning (Subadults)

1.2.2 Sagittal Sectioning (Adult)

Section 2 MMT Pathology

2

. General approach to reading studies

2 .2 Data Recording Insert an example histopathology spreadsheet

2.3 Statistical analysis

RSCABS

2 .3 Histopathology Report Format.

2 .4 Gonadal staging criteria

2 .4.1 Criteria for staging testes in Medaka.

2 .4.2 Criteria for staging ovaries in Me daka.

2 .5 Diagnoses of Pathologies Induced by Endocrine Disruptor s

2.5.1 Testis Pathology

Increased interstitial cells

Increased germ cells

Testicular generation

Etc.

2.5.2 Ovary Pathology

Oocyte atresia

Decreased yolk formation

Granulosa cell hypertrophy/hyperplasia

E tc.

2.5.3 Intersex

2.5.4 Kidney Pathology

2.5.5 Liver Pathology

Section 3 OATPR (Online Aquatic Toxicology Pathology Reference)

Section 4 ReferencesMMT ISR Pathology Appendix DRAFT 2 March 29, 2013 Section 1 MMT Histotechnology

M edaka were sampled at two lifestages for MMT histology analysis. While the fish’s age at

sampling varied slightly across the various MMTs, these differences did not have an impact on the

histotechnique used

. Initially, both adult and subadult fish were histologically processed using a

trans verse

- oblique (frontal) sectioning protocol for both life

- stages. The transverse

- oblique sectioning

plane provides excellent sections of the gonad in particular . However, to facilitate pathology

assessments of multiple organs including the gonads, later ve rsions of the MMT protocol employed

sagittal sectioning of adult samples. D etails of the histotechniques employed are outlined below.

Sect ion 1.1 Euthana sia and necropsy

Objectives

(1) Provide for the humane sacrifice of fish.

(2) Obtain necessary body weights and measurements.

(3) Excise liver tissue. ( Sub

- adult sampling )

(4) Provide for adequate fixation of the tissues within the carcass.

E uthana s i a s o l u t i on ( M S

- 222; t r i ca i n e m e t h anes u lf o nat e )

Tricaine methanesulfonate 100 mg

Sodium bicarbonate 200 mg

Tank or reservoir water 1 L

The buffered MS

- 222 should be buffered to the pH of the test dilution water to avoid distress.

Fixation of tissues for histopathology

T e chn i ques w e r e s e l e c t e d t hat w o u l d m ost o p ti m a ll y:

(1) Preserve the cellular structure of target tissues;

(2) Maximize the amount of target tissues available for analysis;

(3) Sample the target tissues available for analysis;

(4) Allow the pathologist to examine at least 3 step sections of target tiss ues.

Gonads should be pre

- fixed in situ and then be processed in the whole body. However, in order to

allow optimal penetration of the fixative, tail and /or head portions should be removed by means of

sharp razor blades or scissors.

Bouin ’ s fi x a t i ve or M o d i fi ed D a v i dso n  ́ s fi x a ti v e a r e t he r e co m m ended f i x a ti v e s

. C o m pa r e d t o

Bouin’s fixative

, the advantages of Modified D a v i dson ’ s f i xa t i v e a r e as f oll o w s :

(1) the morphologic appearance of gonad sections is generally considered to be comparable to

sections fixed in Bouin’s fixative and superior to sections fixed in formalin;

(2) compared to Bouin’s fixative, which contains picric acid, Davidson’s fixative is

generally considered to be less noxious, less hazardous, and more easily disposed of;MMT ISR Pathology Appendix DRAFT 3 March 29, 2013 (3) specimens fixed in Bouin’s fixative require multiple rinses in alcohol prior to transfer to

formalin.

F o r m u l a t i on o f M o d i fi e d D av i dso n ’ s F i x a tiv e ( 1 L ) :

For m a l deh y de ( 37

- 40 % ) 220 m l

G l ac i al ac e t i c a c i d 115 m l

95% E t h y l a l coh o l 330 m l

D i s t i l l ed w a t e r 335 m l

F o r m u l a t i on o f Bouin ’ s F i x a tiv e ( 1 L ) :

Saturated Picric acid solution 750 ml

Formaldehyde (37

- 40 %) 250 ml

Glacial acetic acid 50 ml

Pl e a s e be aw a r e t hat d i f f

e r ent r e ci p e s and p r odu c t s t h a t a r e d e s i g na t ed a s “ D a v i dson ’ s f i xa t i v e”

m ay ac t u a l l y be m od i f i c a ti o ns o f t he o ri g i n al f o r m u l a; i f a n alternative D av i dson ’ s f i x a ti v e formulation

i s u s e d, t h i s sho u l d be n o t ed by t he l abo r a t o r y

. I f nece s s a r y

, a dec a l c i f i c a ti on so l u t i on ( E D T A

, ac e t i c

a c i d) m ay be us e d. Fa c t o r s t h at m ay a ff ect t h e need f or d e c a l c i f i c a t i on i n c l ude t he s i z e of t he

i nd i v i d u a l f i sh, t he l e n g t h of ti m e t h a t t he c a r cass w a s i m m e r s e d i n f i x a ti v e, and t he ex t ent t o w h i c h t he

a b do m i nal ca v it y ca m e i n t o con t a ct w it h t h e f i x a ti v e.

Necropsy Proced u r e

( 1) Fi s h sho u l d be s a c r i f i ced w it h i n one t o t w o m i nu t es p r i or t o n e c r o p s y

. The r e f o r e, un l e s s

m

u l t i p l e p r os e c t o r s a r e a v a i l ab l e, m u l t i p l e f i sh sho u l d n o t be s a c r i f i ced s i m u l t a n eou s l y

.

( 2) U s i ng a s m a l l d i p n e t

, a s i n g l e f i sh i s r e m o v ed fr om t he exp e ri m en t a l c ha m ber and

t r a n sp o rt e d t o t h e n e c r o psy a r e a i n t he tr a n sp o r t con t a i n e r

. F or e a c h t e s t c ha m be r

, t he s e x of

ea ch f i sh i s de t e r m i ned by ex t e r n a l body cha r ac t e r i s ti c s ( sh a pe of d o r s al a nd a n al fi n s )

.

( 3) T he fish is placed into buffered MS

- 222 ( t r i c a i ne methanesulfonate ) solution

. T h e f i sh i s

r e m o v ed fr om t he so l u t i on w hen t h e r e i s ce s s a t i on o f r e s p i r a t i on and t he f i sh i s un r e s pon s i v e

t o ex t e r n a l s t i m u li

.

( 4) T he f i sh i s w e t w e i g hed and m ea s u r ed a cc o r d i ng t o p r o t oc o l

.

( 5) T he fi s h i s p l aced on a c ork boa r d on t he s t a g e of a d i s s e c t i ng m i c r os c ope.

a) For subadult animals, two transverse cuts are performed to remove the head and tail. The

head is removed by a transverse cut at the posterior margin of the operculum. The liver is

collected by applying slight pressure on the abdomen and separating the liver from the

body with forceps. Some liver tissue may also be contained in the head depending on the

location of the cut . The liver is then placed in a micro centri fuge tube in * RNA stabilizing

solution

. The tail is removed by a transverse cut posterior to vent and saved for preparing

the anal fins for counting papillae. The a nal fin is flared out on a dish and a few drop s of

Davidson’s fixative are applied to the fin. After approximately 30 seconds to a minute,MMT ISR Pathology Appendix DRAFT 4 March 29, 2013 the tail is placed in a small microcentrifuge tube and filled with Davidson’s fixative.

b) Adult animals are prepared by having tails removed as in the subadults abo ve, and then

the abdomen is opened by a small incision along the ventral midline starting at the vent

and ending midway along the abdomen. Using a syringe, approximately 0.5 mL of

fixative is then gently perfused into the viscera and buccal cavity

.

( 6) The c a r c a s s a nd a b do m i nal v i s c e r a a r e p l a ced i n an individual labeled histology cassette and

placed in a bottle o f fi x a t i v e. T he v o l u m e of fi xa t i v e i n t he con t a i n e r sho u l d be a t l ea st 10

t i m es t he app r o x i m a t ed v o l u m e of t h e t i s s u e s

. The fixative container is gently agitated for

five seconds to dislodge air bubbles from the cassette.

( 7 ) All tissues remain in Davidson’s fixative overnight, followed by transfer to 10 %

neutral buffered formalin the next day. Bouin’s fixed tissues remain in fixative for 72 hours,

before being washed twice overnight with a 70% ethanol solution saturated with lithium

carbonate and then transferred to 10% neutral buffered formalin. Fixative c on t a i ne r s ar e

g en tl y a git a t ed f or 5 s ec onds t o en sur e ad e qu at e pen etr a ti on o f solutions i n t o c a ss e tt es.

Section 1.2.1 Preparation of slides from subadult specimens

Typically, subadult fish shall be sectioned such that the gonads are sectioned on their long axis

along a plane roughly parallel to a frontal plane of the fish. The pathology evaluation shall be limited to

diagnosing effects on kidney, gonadal development , and grading the gonadal phenotype. The trunk

portion of each specimen shall be trimmed and embedded to produce sections that are roughly parallel

to the fish’s frontal plane. The specific trimming protocol is illustrated below. Cuts 1, 2, and 3 are

per formed at necropsy. Cut 4 is done post

- fixation prior to processing and embedment

.

a. Trimming: The fish shall be trimmed such that “cut 4”, which defines the sectioning plane, is

parallel to the long axis of the gonad. Note that the gonad is “tilted” with respect to the spine so

the trim

- plane must traverse the spine in an oblique fashion. Cuts 1, 2, and 3 are made during

necropsy. Cut 4 is done post

- fi xation, pre

- paraffin processing (Figure 1

- 1).

Figure 1

- 1 . Sequential cuts associated with trimming sub

- adult (9 wpf) medaka in preparation for

histopathology. Cut 4 should be performed by the Contractor prior to processing and embedding.MMT ISR Pathology Appendix DRAFT 5 March 29, 2013 b. Processing: The trimmed medaka shall be processed in an automatic tissue processor and

infiltrated with paraffin.

c. Embedding: Each trimmed and processed medaka shall be embedded such that the dorsal cut

surface of the fish is down and shall be sectioned first.

d

. Microtomy: Section thickness for this protocol shal l be set at 4

- 5 microns. The block shall be

faced from the dorsum toward the ventrum of the fish until the kidney tissue appears the section.

From this landmark, ~12 microns of tissue are microtomed from the block, and the first section

with kidney shall be obtained. Another ~12 microns shall be microtomed and from there, obtain a

second section with kidney.

From here the microtomy protocol varies based on the gross sexual phenotype of the

specimen (i.e., male or female).

Phenotypic males: The intent is to obtain, as best as possible, longitudinal sections of the testis.

To accomplish this, the microtomist shall be experienced and proficient in sectioning medaka of

this age. Sectioning continues until the testis is reached. Once the testis is reached, the

microtomist shall obtain 9 sections at 10 micron intervals to sample the middle half of the testis.

The distance to the middle of the gonad will vary depending on the size of the fish and its gonads.

Therefore the microtomist must estima te from experience and judgment where to begin taking

sections. The microtomist may choose to obtain more than 9 sections if necessary to ensure

sampling the central duct of the testis.

Phenotypic females: The intent is to obtain, as best as possible, longitudinal sections of the ovary.

To accomplish this, the microtomist shall be experienced and proficient in sectioning medaka of

this age. Sectioning continues until the ovary is reached whereupon the microtomist shall obtain 9

sections at 40 micron i ntervals to sample near the middle of the ovary. The distance to the middle

of the ovary will vary depending on the size of the fish and its gonads. Therefore the microtomist

must estimate from experience and judgment where to begin taking sections. The m icrotomist

may choose to obtain more than 9 sections if necessary to ensure adequate sampling of the ovary.

Following microtomy each paraffin block with residual tissue shall be sealed with paraffin.

e. Staining: The slides shall be stained with H&E on an automatic stainer. The tissue shall be

covered with an appropriate mounting medium and glass cover slips.MMT ISR Pathology Appendix DRAFT 6 March 29, 2013 Section 1.3 Preparation of slides from adult specimens (sagittal sectioning)

Typically adult fish shall be sagittally step

- sectioned and evaluated for pathology in target

tissues such as, the liver, gonad, kidney, inter

- renals, pituitary, and spleen

. Each specimen shall be

paraffin embedded so that 5 slides with sagittal and parasagittal sections are produced for pathology

evaluation. The detailed histotechnical protocol is illustrated below.

a. Processing: The trimmed medaka shall be processed in a n automatic tissue processor and

infiltrated with paraffin. Due to the large size of some of the specimens, it assumed that

decalcification and possibly longer processing times than usual will be required.

b. Embedding: Each sample shall be embedded wit h its left side down, oriented in a manner that the

spine will be approximately parallel to the sectioning plane.

Figure 1

- 2 . Embedded Adult Medaka in trimmed block.

c. Microtomy: Section thickness shall be 4

- 5 microns.

Slide 1. The block shall be faced starting from the left side of the fish. Trim until sections reveal

the left eye; continue to trim midway into the lens. At this point the lens will be a ring within the

eye. The ring can be seen in the sections and the block. The lens will be hard, the microtome

blade will produce a scratch that can be heard and felt as the blade cuts through. At this point the

sections should also reveal the visceral cavity. Collect a ribbon of 3

- 4 s erial sections and mount on

a glass slide (Figure 1

- 3).

Figure 1

- 3 . Images of the blocks, ribbons and section showing the location of the sectioning plane in

the lens and other tissues for slide 1.

Slide 2. Continue to trim through the left eye until it is no longer in the sections. The bla ck pigment

of the eye will diminish. Collect a ribbon of 3

- 4 serial section and mount on a glass slide (Figure

1

- 4).MMT ISR Pathology Appendix DRAFT 7 March 29, 2013 Figure 1

- 4. Image of the section indicating the location of the section plane for slide 2.

Slide 3. The target organ for this slide is the pituitary gland, which is located midway between the

eyes. From the point where the sections for slide 2 were taken, trim 200 microns and take a

section. Continue to take 2 or 3 more sections at 200 micron in tervals and mount them in order

(Figure 1

- 5).

Figure 1

- 5. An image of a mid

- sagittal section of the medaka that includes the pituitary gland,

which is the target organ for the sections on slide 3.

Slide 4. Trim to the medial portion of the right eye, the bl ack pigment can be seen in the sections.

Take a ribbon of 3

- 4 serial sections and mount on glass slide (Figure 1

- 6).

Figure 1

- 6. Image of a representative section showing the plane for collecting sections for slide 4.

Slide 5. Continue trimming into the right eye until midway through the lens. At this point, light

can be seen through the lens. Take a ribbon of 3

- 4 serial sections. Following microtomy, the

tissue blocks can be discarded (Figure 1

- 7).MMT ISR Pathology Appendix DRAFT 8 March 29, 2013 Figure 1

- 7. Image of a representative section showing the plane for collecting sections for slide 5.

d. Staining: The slides shall be stained with H&E on an automatic stainer. The tissue shall be

covered with an appropriate mounting medium and glass cover slips.

Section 2 : Pathology

Slides are evaluated for treatment

- induced lesions in gonad, liver, kidney, and thyroid. The

pathologist should also scan other tissues (e.g., pituitary, spleen, inter

- renal tissue) for treatment

induced lesions. Pathology is to be defined according to three general criteria:

1) The pathologist’s judgment,

2) Pathology described in the Draft OECD Guidance Document for the Diagnosis of Endocrine

Related Histopathology of Fish Gonads ( Johnson et al

. 2010 ), and

3) Histomorphological effects observed in treated specimens compare d to the controls.

The slides shall be evaluated by a pathologist for treatment

- induced effects. The pathologist shall

perform the evaluation according to the Best Practices Guideline: Toxicologic Histopathology

( Crissman et al. 2004 ). It is recommended that evaluation begin with examining representative slides

from specimens selected from control and the two highest treatments to determine likely treatment

related effects. Then, images of representative treatment

- related effects and associated severity scores

shall be prepared to be used as references for scoring the remainder of the specimens. Finally, after the

initial evaluation, the study pathologist shall perform a masked re

- evaluation of all the specimens, but

for only the previously observed treat ment

- related lesions.

Each section shall be evaluated for gonad pathology, whereas kidney, reproductive duct, and liver

pathologies shall be based on the assessment of one representative section. The prevalence and extent of

the effects shall be assessed in the various treatments as compared to controls. Effects data containing

diagnoses and associated severity grades shall be reported in an Excel spreadsheet similar to the

example provided below. Diagnoses of treatment effects shall be based on the colu mns in the example

pathology spreadsheet. However, for pathologies not listed in the spreadsheet, the pathologist shall

provide new diagnostic criteria to the spreadsheet. Pathology not related to treatment shall also be

noted as a comment in the spreads heet. Create digital images of representative diagnostic features in an

uncompressed file format. Images of representative control tissues shall be included for comparison.

Each image shall be at an appropriate magnification to illustrate the features usin g the best judgment ofMMT ISR Pathology Appendix DRAFT 9 March 29, 2013 the pathologist. An embedded calibrated scale bar shall be included in all pictures. The spreadsheet

shall indicate imaged sections.

A report shall be prepared which includes a short summary of the protocols used in slide preparatio n

and a description of treatment

- related effects based on concentration responses and comparisons with

control frogs. The narrative report shall include tables, figures, figure legends, and citations, as

necessary to describe treatment effects. Citations, tables, figures, figure legends, shall be placed at the

end of the narrative report. Other supporting histopathology data, images, and histology technical

reports shall be provided as spreadsheets, images without legends or annotations, and documents. Th e

titles and file names of reports and documentation shall include: laboratory; species name;

developmental stage; test chemical; and tissue examined. The file names of digital images shall identify

the test chemical, treatment, and specimen ID. A tabular cross

- reference of image files and report figure

numbers and titles shall be provided.

Section 2 .1 General approach to reading studies

 Adapted from OECD guidance document for the Diagnosis of Endocrine

- Related

Histopathology of Fish Gonads. (2010 )

Studies are to be read by individuals experienced in reading toxicologic pathology studies, and who are

familiar with normal small fish gonad histology, with gonadal physiology, and with general responses

of the gonads to toxicologic insult. Pathologists may be b oard certified (e.g. American College of

Veterinary Pathologists, The European Centre of Toxicologic Pathology, or other certifying

organizations); however, certification is not a requirement as long as the pathologist has obtained

sufficient experience wi th, and knowledge of, fish histology and toxicologic pathology. Technicians

should not be used to conduct readings due to the subtle nature of some changes and the need for

subjective judgments based on past experience.

It is recognized that there is a lim ited pool of pathologists with the necessary training and experience

that are available to read the gonadal histopathology for the Medaka Multigenerational Test (MMT). If

an individual has toxicological pathology experience and is familiar with gonadal his tology in small

fish species, he/she may be trained to read the fish assay. If pathologists with little experience are used

to conduct the histopathological analysis, informal peer review may be necessary.

It is preferable for pathologists to read the stud ies non

- blinded (i.e. with knowledge of the treatment

group status of individual fish). This is because endocrine effects on histomorphology tend to be

incremental, and subtle differences between exposed and unexposed animals may not be recognizable

unless tissue sections from high dose animals can be knowingly compared to those from controls. Thus,

the aim of the initial evaluation is to ensure that diagnoses are not missed (i.e., to avoid false

- negative

results). However, it is expected that any potential compound

- related findings will be re

- evaluated by

the pathologist in a blinded manner prior to reporting such findings, when appropriate. Certain

diagnostic criteria, such as relative increases or decreases in cell populations, cannot be read in aMMT ISR Pathology Appendix DRAFT 10 March 29, 2013 blinded manner due to the diagnostic dependence on control gonads. As a rule, treatment groups should

be evaluated in the following order: control, high

- dose, intermediate

- dose, and low

- dose.

It is suggested that the pathologists be provided with all available in formation related to the study prior

to conducting their readings. Information regarding gross morphologic abnormalities, mortality rates,

and general test population performance and health are useful for pathologists to provide

comprehensive reports and t o aid in the interpretation of findings. For a more comprehensive discussion

of standard reading approaches for toxicologic pathology studies, please refer to th e Society of

Toxicologic Pathology Best Practices for reading toxicologic histopathology studie s (Crissman al.,

2004).

Severity Grading

In toxicologic pathology, it is recognized that compounds may exert subtle effects on tissues that are not

adequately represented by simple binary (positive or negative) responses. Severity grading involves a

semi

- quantitative estimation of the degree to which a particular histomorphological change is present in

a tissue section (Shackelford et al., 2002). The purpose of severity grading is to provide an efficient,

semi

- objective mechanism for comparing changes (inc luding potential compound

- related effects)

among animals, treatment groups, and studies.

Severity grading will employ the following system:

Not remarkable

Grade 1 (minimal)

Grade 2 (mild)

Grade 3 (moderate)

Grade 4 (severe)

A grading system needs to be fl exible enough to encompass a variety of different tissue changes. In

theory, there are three broad categories of changes based on the intuitive manner in which people tend

to quantify observations in tissue sections:

(1) Discrete : these are changes that ca n be readily counted. Examples include atretic follicles,

oocytes in the testis, and clusters of apoptotic cells.

(2) Spatial : these are changes that can be quantified by area measurements. This includes lesions

that are typically classified as focal, multifocal, coalescing, or diffuse. Specific examples

include granulomatous inflammation and tissue necrosis.

(3) Global : these are generalized changes that would usually require more sophisticated

measurement techniques for quantification. Examples include increased hepatocyte basophilia,

Sertoli cell/interstitial cell hypertrophy, or quantitative alterations in cell populations.MMT ISR Pathology Appendix DRAFT 11 March 29, 2013 General severity grading scale

• Not Remarkable : This grade is used if there are no findings associated with a particular

diagnostic criterion.

• Grade 1: Minimal . Ranging from inconspicuous to barely noticeable but so minor, small, or

infrequent as to warrant no more than the least assignable grade. For discrete changes, grade 1 is

used when there are fewer than 2 occurrenc es per microscopic field, or 1

- 2 occurrences per

section. For multifocal or diffusely

- distributed alterations, this grade is used for processes where

≤ 20 % of the tissue in the section is involved.

• Grade 2: Mild . A noticeable feature of the tissue. Fo r discrete changes, grade 2 is used when

there are 3

- 5 occurrences per microscopic field or per tissue section. For multifocal or

diffusely

- distributed alterations, this grade is used for processes where 20

- 50 % of the tissue in

the section are involve d.

• Grade 3: Moderate . A dominant feature of the tissue. For discrete changes, grade 3 is used

when there are 6

- 8 occurrences per microscopic field or per tissue section. For multifocal or

diffusely

- distributed alterations, this grade is used for proces ses where 50

- 80 % of the tissue in

the section are involved.

• Grade 4: Severe. An overwhelming feature of the tissue. For discrete changes, grade 4 is

used when there are more than 9 occurrences per microscopic field or per tissue section. For

multifoca l or diffusely

- distributed alterations, this grade is used for processes where > 80 %

of the tissue in the section are involved.

At least some of the histomorphological changes that have been associated with EDCs in fish are

considered to be exacerbations of “normal”, physiologic findings (e.g., oocyte atresia [Nagahama, 1983;

Tyler and Sumpter, 1996]). At the discretion of the pathologist, the severity of a given change should

be scored according to one of the following two methods :

(1) score compound

- exposed animals relative to the severity of the same change in control

animals, or

(2) score all animals relative to “normal” as determined by the pathologist’s experience.

For each important (i.e., treatment

- associated) finding, the method that w as used should be stated in the

Materials and Methods section of the pathology narrative report. By convention, severity grading

should not be influenced by the estimated physiologic importance of the change, since this would add a

further layer of subject ivity to the findings that complicates inter

- laboratory results comparisons. For

example, the presence of two oocytes in the testis should not be graded as “severe”, even if the

pathologist considers this finding to be highly significant in terms of endocr ine modulation.MMT ISR Pathology Appendix DRAFT 12 March 29, 2013 Section 2 .2 Data Recording.

An Excel worksheet form has been created that includes worksheets for primary, secondary, and

additional diagnoses to facilitate histopathology data collection. In this worksheet, each data entry cell

represents an individual fish. Additional sheets are available for comments and additional findings. For

each fish, the pathologist records a severity score associated with the diagnosis. Diagnostic criteria with

non

- remarkable findings shall be denoted us ing (

- ). If there is no reasonably appropriate diagnostic term

for a particular finding, the pathologist can create a term that can be recorded in the “Additional

diagnoses” worksheet. If insufficient tissue is available for diagnosis, this should be recor ded as IT

(insufficient tissue). If a target tissue is missing, this should be recorded as MT (missing tissue).

( Attach sample spreadsheets: Medaka pathology .xls )

Se ction 2 .3 Histopathology Report Format.

Each histopathology narrative report should contain the following five sections:

Introduction

Materials and Methods

Results

Discussion

Summary/Conclusions

(References)

The Introduction section briefly outlines the experimental design. The Materials and Methods section

describes any items or procedur es that are essentially different from Section 1: Post

- mortem and

Histotechnical Procedures. As applicable, specific severity grading criteria (see Severity Grading)

should also be listed in this section. The Results section should report findings that are : (1) compound

related; (2) potentially compound

- related; (3) novel or unusual. Detailed histomorphological

descriptions need only be included for findings that differ substantially from diagnoses presented in

Section 4 (Glossary and diagnostic criteria). It is intended that the Results section should be as objective

as possible (i.e., opinions and theories should be reserved for the Discussion section). The Discussion

section, which contains subjective information, should address relevant findings that wer e reported in

the Results section. Opinions and theories can be included in this section, preferably backed by

references from peer

- reviewed sources, but unsupported speculation should be avoided. The

Summary/Conclusions section should encapsulate the most important information from the Results and

Discussion sections.MMT ISR Pathology Appendix DRAFT 13 March 29, 2013 Section 2 .4 Gonadal staging criteria

T he g oal of g onad a l s t a g i n g i s t o de t e r m i ne i f t he ad m i n i s t r a t i on of a p a r t i c u l a r e ndoc r i n e

- ac ti v e

s u b s t a n ce a f f ec t s t he r e p r odu c ti v e cy c l e s t a t u s o f ad u l t m a l e and f e m a l e f i s h

. T he p u r p ose o f t h i s

s e c t i on i s t o de s c ri b e a r a p i d, s e m i

- quan t i t a t i v e m e t hod f or a s s e s s i n g t he p r op o r t i ons o f v a ri o u s

g a m e t o g en i c c e l l t y pes ( g onadal s t a g i n g ) ba s e d on t he li g ht m i c r o s co p i c ex a m i na ti on of h e m a t ox y li n

a nd eo s i n

- ( H& E

- ) s t a i ned h i s t o l o g i c al s e c ti o ns.

Se m i

- quan ti t a t i v e g onadal s t a g i ng has been p r op os ed f o r

, or e mp l o y ed i n, s t ud i es i n v o l v i ng

f

a t h e ad m i nnow and o t her f i sh sp e c i es ( A n k l ey et a l

., 2002; J e ns e n et a l

., 2001; M i l e s

- R i ch a r dson e t

a l

., 1 9 99 a ; N i cho l s et a l

., 200 1 ; U S E n v ir on m en t al P r o t e c t i on A g enc y

, 2002 )

. A lt h ou g h su c h

studies generally i n c l uded exc e l l ent de s c r i p t i o n s of t he d i f f

e r e n t g a m e t o g en i c m a t u r a t i on s t a g es ( e. g

.,

s p e r m a t o g on i um t h r ou g h sp e r m a t o z oa f o r t he t e s t i s )

, t hey d i d n o t i nc o r po r a t e p r e

- de fi n e d c a t e g o r i c a l

g u i d e l i nes fo r e v a l ua t i ng and r ep o r t i ng t h e r ep r odu c ti v e c y c l e s t a t us of an i n d i v i du a l f i sh. T o m a i n t a i n

s c i en t i f i c i n t e g rit y ac r o ss t he bo a r d i n a p r o gr am t hat i n v o l v es m u l t i p l e s t u d i e s

, m u lt i p l e l abo r a t o r i e s

, and l a r g e nu m be r s o f an i m a l s, i t i s e s s en t i a l t h a t obs e r v a ti o n s a r e r e c o r d ed on a fi s h

- b y

- fi sh ba s i s. T h e

use of a c a t e g o r i za t i on s y s t em can i m p r o v e t he con s i s t ency and o b j e c t i v it y of r ep o rt e d

obs e r v a t i ons wi t h i n and a m ong expe r i m en t s ; co n s equ e n t l y , co mpa ri so n s of t h e r e su l t s a r e m o r e

m ean i n g f u l

.

C a t e g o ri z a t i on s y s t e m s a l s o ha v e s o m e d r awbac k s a n d l i m it a t i on s

, t he m ost s i gn i f i ca n t o f w h i ch a r e ( 1)

t he po t e n t i al l o s s of d i s c ri m i n a t o r y d a t a w hen s i m il a r

, b ut n o t i d e n t i c a l

, t ypes o f o bs er v a t i ons a r e

c o m b i ned ( b i n n ed) i n t o a s i n g l e c l a ss ; ( 2 ) t he q u e s ti o n ab l e b i o l og i cal r e l e v ance o f t he c l a s s i f i c a ti o n

c ri t e r i a i n so m e cas e s; and ( 3) t he i n ab i l i t y of any s i n g l e c l a s s i f i c a t i on s y s t em t o add r e s s e v e r y t ype of

obs e r v a t i on ( e i t her p r e d i c t ed o r u n f o r e s e e n )

.

T o add r e s s t h i s l a st li m i t a ti on, g onadal s t a g i ng i s a cc o m pan i ed by a co m p l e t e h i s t op a t h o l o g i c a l

e v a l u a t i on of t h e g onad s ; i n t h i s m an n e r

, t h e l o ss o r o v e r abu n da n ce of a sp e c i f i c g a m e t o g en i c ce l l t ype,

f or e x a mp l e, can be docu m en t ed.

T he s e m i

- quan ti t a t i v e g onadal s t a g i ng s c h e m e s el e c t e d f or an a l y s i s o f fi s h g onads i s a m od i f i c a ti o n of

a s y s t em adop t e d by t h e U n i t ed S t a t es D ep a rt m ent o f t he I n t e r i o r

, U

. S

. G e o l og i cal S u r v e y

, B i o l o g i ca l

R e s ou r ces D i v i s i on as p a r t of t he “U. S

. B i o m on it or i ng of En v ir on m en t a l S t a t us and Tren d s ( B E S T)

P r o g r a m ” ( M c D on a l d e t a l

. , 2000 )

. T h e a u t h o r s o f t h e B E ST s y s t em c r ed i t p r e v i ous w o r k by T r e a s u r er

and H o li day ( 1981 )

, N a g aha m a ( 19 8 3 )

, R o d r i quez e t a l

. ( 199 5 )

, a nd G ood b r ed et a l

. ( 199 7 )

. T h e

f

o r e m ost bene f i t s of t h i s s y s t em a r e sp e ed a nd e a se of u s e

, e s p ec i a l l y w hen co mpa r ed t o f u ll y

quan ti t a t i v e s t a g i n g

. T he ba s i s o f t he B E S T s y s t em i s a v i su a l a s s e s s m ent o f t h e de n s i t y of

g a m e t o g en i c p r e cu r so r s as c o m pa r e d t o m a t u r e g a m e t oc y t es i n o ne or m o r e g onad s e c t i ons.

A cco r d i n g l y

, t he s t a g e n u m be r s (t e s t i s: S t a g es 0 t o 4; o v a r y : S t a g es 0 t o 5) i n cr ease i n d i r ect

r e l a ti o ns h i p t o t he r e l a t i v e p r o po r ti o n of m a t u r e c e l l s.

A lt hou g h t h e B E S T s y s t e m w as i n i t i a l l y de v e l oped t o a s s e s s r e p r od u c t i v e f un c ti on i n s eas o nal

sp a w n er s su c h a s c a rp ( C yp ri n i d a e) and b l ack ba s s e s ( C e n t r a r c h i da e )

, t he s a m e s t a g e ca t eg o ri es c a n be

a p p l i ed t o fr a c t i on a l s pawn e r s su c h a s f a t h e ad m i nno w

. T h i s w as de m ons t r a t ed at t he O c t ober 2003

m ee t i ng of t h e h i s t op a t h o l o g y sub c o m m itt ee o f t he Fi sh D r a f t i ng G r oup a t t he P a r i s m ee t i n g

, at

w h i ch t he p a r t i c i pa n t s w e r e a s k ed t o b r i e f l y e v a l u a t e t he ap p l i ca b i l it y o f a m od if i ed B E S T s y s t em

us i ng a c t u a l h i s t o l o g i c a l sp e c i m ens of t he t hree s pe c i es f a t h e ad m i nno w

, m eda k a and z eb r a f i s h

. I nMMT ISR Pathology Appendix DRAFT 14 March 29, 2013 g ene r a l

, t h e p a r t i c i pa n t s a g r eed t h a t t hey cou l d r ea d il y r e c o g n i z e t he v a ri ous g onadal s t a g es as

de f i ned by m o d i f i ed B E S T c r i t e ri a

. T h e pa r t i c i pa n t s ac k no wl ed g ed t h a t t he t e r m i nal s t a g es of t he

s y s t em (t e s t i s: S t a g e 4, “Spen t ”; o v a r y : St a g e 5, “Pos t

- o v u l a t o r y ”) w ou l d be m i n i m a ll y p r e s e nt ( or

po ss i b l y n o t p r e s e nt at a l l ) a m ong r ep r o d uc t i v e l y

- ac ti v e adu l t f a t head m i nno w

, m eda k a and z eb r a fi s h,

b eca us e t h ey a r e n o t s e a son a l s p awner s

. Si m i l a r l y

, i t w as t hou g ht t h a t S t a g e 4, “L a t e

de v e l op m en t / h y d r a t e d ”, w ou l d b e r a r e l y obs e r v ed i n t he t e s t fi s he s

, due t o t h e

v e r y tr an s i e n t n a t u r e of t h i s s t a g e i n f r a c ti o nal sp a w n e r s.

A f ew m od if i ca t i o n s ha v e been m ade t o t h e B E S T s y s t em t o ad a pt i t f o r u s e. For exa m p l e, t h e r e

i s cu r r en t l y no p r o v i s i on i n t he s y s t em f or g onads t h a t a r e c o m p ri s e d en t i r e l y of s pe r m a t o g on i a o r

oo g on i a

. A lt hou g h i t i s i n t e nded t h a t r e p r od u c t i v e l y m a t u r e f i s h a r e u s ed, i t i s p o s s i b l e t hat an

o cca s i o n al a n i m al m a y not a t t a i n s e xu a l m a t u r i t y by t he t i m e t he e x p e ri m ent i s t e r m i na t ed, o r t h at

c e r t a i n t e s t co m pounds m i g ht ca u se r e v e r s i on of t he g onads t o a j u v en i l e p he n o t ype. The r e f o r e, a p r e

s t a g i ng ca t e g o r y ca l l e d “ j u v en i l e” has b e en a d ded f or b o t h m a l e and f e m a l e f i s h as one m od i f i c a t i on o f

t h e B E S T s y s t em. A no t h er m od ific a ti o n t o t he s y s t em i n v o l v es an app a r e n t d i s c r epancy b e t w e e n t he

B E S T s y s t em and G ood b r ed e t a l

. ( 199 7 ) c onc e r n i ng t he t h i c k ne s s of t he t e s t i c u l a r g e r m i nal

ep i t h e l i um as a s t a g i ng c ri t e r i on. A s i nd i ca t ed by G oodb r ed et a l

. ( 1 997 )

, t he g e r m i nal ep i t h e li um

b eco m es t h i nn e r a s t he t e s ti s s t a g e i n c r e a s e s, w h e r e as t he r e v e r se o cc u r s a cco r d i n g t o t he B E S T

s y s t em ( as p r e s en t ed i n M c D o n a l d e t a l

. , 20 0 0 )

. Al t hou g h i t i s d i f fi c u l t t o f i nd co r r ob o r a ti ng s t a t e m en t s

i n t he s c i en t i f i c l i t e r a t u r e, e m p ir i cal e v i de n ce i n d i c a t es t ha t G oodb r ed e t a l

. ( 1 9 97) i s c o rr e ct on t h i s

po i n t

. A t h i r d m od ifi c a t i on t o t h e s y s t em i s t h e o p t i on t o sub d i v i d e a s t a g e i n t o t w o sub o r d i na t e s t a g es

( e. g

., St a g es 3A and 3 B )

, i f t h e p a t ho l o g i st be l i e v es t hat t h i s t a c t i c w ou l d r e v e a l a su b t l e,

co m poun d

- r e l a t ed e ff e ct t h a t m i g ht o t h e r wi se be m i s s ed. O t her m od i f i c a t i ons t o t h e s y s t em a r e

r e l a ti v e l y m i nor and p r i m a ril y i n v o l v e r ew o r d i ng f o r c l a r i f i c a t i on.

T he c e l l d i s t r i b u ti o n pa t t e r n i s li k e l y t o v a r y t h r ou g hout a g i v en ti s s ue s e c t i on, t h e g onad shou l d be

s t a g ed acc o r d i ng t o t he p r edo m i nant p a t t e r n i n t h at s e c t i on. B o t h g onads sho u l d be s t a g ed as a s i n g l e

o r ga n acc o r d i ng t o t he p r e do m i nant pa t t e r n. G o n ads t h at c a nnot be r e a son a b l y s t a g ed f or v a r i o u s

r ea s ons ( e. g

., i ns u f f i c i ent t i s su e , or e x t e n s i v e n e c r o s i s, i n f l a m m a ti o n, or a rt if a c t ) s hou l d be re co r d e d a s

UT S ( una b l e t o s t a g e )

.MMT ISR Pathology Appendix DRAFT 15 March 29, 2013 Section 2 .4.1 Criteria for staging testes in Medaka.

T o d e ri v e e ach s t a g e s c o r e, t he e s ti m a t e d w i d t h of t h e g e r m i nal e p it h e l i um ( EW G ) c a n b e

co mpa r e d t o t he e s t i m a t ed wi d t h o f t h e t e s t i s ( E W T) i n t h e f o l l o w i ng m anne r :

• Stage 1 : E WG > 2⁄3 E W T

• Stage 2 : E WG 2⁄3 to > 1⁄2 E WT

• Stage 3 : E WG 1⁄2 to > 1⁄4 E WT

• Stage 4 : E WG < 1⁄4 E W T

Fi g ure 2

- 1. Exa m p l es of s t a g i ng s y s t em app li e d t o t e s t e s fr om J a pan e se m eda k a : B l ack a rr o w s ( ↔ )

r ep r e s e nt t he e s ti m a t ed w i d t h o f t he t e s t i s ( E W T )

, and r ed a r r o w s ( ↔ ) r ep r e s e n t t he e s t i m a t ed w i d t h of t he

g e r m i nal ep i t h e li um ( EW G ; m easu r e m en t s a r e i ll u s t r a t ed u n il a t e r a ll y f or s i m p li c it y )

. I n o r der t o ob t a i n

co m pa r a b l e s e c ti o ns, i t i s i m pe r a ti v e t h a t e ac h s e c t i on c o n t a i ns a p o r t i on of t he c e n t r al d uc t ( CD )

,

p r e f e r a b l y at i t s wid e st a n d l o n g e s t e x t e n t ( pa r a f f i n, H& E).MMT ISR Pathology Appendix DRAFT 16 March 29, 2013 Section 2 .4.2 Criteria for staging ovaries in Medaka.

T he f o ll o w i ng a r e m o r pho l o g i c c r i t e r i a f or s t a g i ng f e m a l e J a pa n e s e m eda k a:

• Juven i l e : g onad con s i s t s o f oo g on i a ex c l u s i v e l y ; i t m ay be d if f i c u l t o r i m pos si b l e

t o c on f ir m t he s ex o f t h e s e i n d i v i du a l s.

• Stage 0

– un d ev e l op e d : en t ir e l y i m m a t u r e p h a s e s ( oo g on i a t o p e r i nu c l e o l a r

ooc y t e s ) ; no co r t i c al a l v eo l i

• Stage 1

– ea r l y d e v e l o

p m e n t : v a s t m a j or it y ( e. g

., > 90 % ) a r e p r e

v it e l l o g en i c f o l l i c l e s, p r edo m i na n tl y pe r i n u c l e o l a r t h r ou g h co r t i c a l a l v eo l a r

.

• Stage 2

– mi d

- de v e l op m en t : at l e a s t h a l f o f o b s er v ed f o l l i c l es a r e ea r l y and m i d

v it e l l o g en i c.

• Stage 3

– l a t e dev e l o

p m e n t : m a j o r i t y of de v e l o p i ng f o l li c l e s a r e l a t e v it e ll o g en i c.

• Stage 4

– l a t e de v e l op m e n t/ h

y dr a t e d : m a j o r i t y of f o l l i c l es a r e l a t e

v it e ll o g en i c a n d m a t u re/ s p awning f o l l i c l e s ; f o l l i c l e s a r e l a r g er as co m pa r ed t o

S t a g e 3.

S t age 0 S t age 1

S t age 2 S t age 4

Fi g ure 2

- 2. Exa m p l es o f t h e s t a g i ng s y s t em app l i ed t o t he o v a ri e s of ad u l t J a pa n e s e m eda k a ( pa r a f f i n, H& E)MMT ISR Pathology Appendix DRAFT 17 March 29, 2013 Section 4: WebPath

The valid and consistent evaluation of pathology effects among pathologists and labs requires

that each pathologist be familiar with the diagnostic features and the appropriate severity scores

of EDC

- induced pathology. Accurate and consistent pathology is necessary to perform valid

statistical analyses of EDC and chemical induced treatment effects. To ensure that

histopathology is performed to the highest standards of quality and consistency, it is imperative

that persons performing the microscopic examinations are fully trained and that standardized

diagnostic criteria and terminology are available for these evaluations. Toward this end, EPA is

creating an interactive online website (figure 1) for viewing virtual images of scanned whole

histologic sections (figure 2) from different animal species. These sections have been selected to

demonstrate various types of findings that have been associated with endocrine disruption

activity, and annotations h ave been added to the images to describe the changes and provide

context. As time permits and the knowledge base expands, further images may be added to this

site. Although these images are intended primarily for use by professional scientists who are

acti vely involved in EDSP research, EPA is currently maintaining this as an open site.

Figure 1

. Beta version of the homepage for the Online Pathology Reference Atlas curr ently under

development by EPA.

Section 5: References cited in text

• Johnson R, Wolf, J, and Braunbeck T, 2009 . OECD Guidance Document for the Diagnosis of

Endocrine

- Related Histopathology of Fish Gonads. OECD Environmental Health and Safety

Monograph Series on Testing and Assessment.

• Crissman, J.W., Goodman, D.G., Hildebrandt, P.K., Maronpot, R. R., Prater, D. A., Riley, J. H.,

Seaman, W. J., and Thake, D. C. (2004). “Best Practices Guideline: Toxicologic Pathology.” Toxicol

Pathol 32:126

- 131.

• MMT pathology summary octylphenol

a. Gray MA, Metcalfe CD, 1997. Induction of testis

- ova in Japanese medaka ( Oryzias latipes )

exposed to p

- nonylphenol. Environ Toxicol Chem 16: 1082

- 1086.

b. Karels AA, Manning S, Brouwer TH, Brouwer M, 2003. Reproductive effects of estrogenic

and antiestrogenic chemicals on sheepshead minnows ( Cyprinodon variegate s ). Environ

Toxicol Chem 22: 855

- 865.

c. Knörr S and Braunbeck T, 2002. Decline in reproductive success, sex reversal, and

developmental alterations in Japanese medaka ( Oryzias latipes ) after continuous exposure to

octylphenol. Ecotoxicol and Environ Safet y 51: 187

- 195.

d. Vajda AM, Barber LB, Gray JL, Lopez EM, Woodling JD, Norris DO, 2008. Reproductive

disruption in fish downstream from an estrogenic wastewater effluent. Environ Sci Technol

42: 3407

- 3414.

e. Van den Belt K, Verheyen R, Witters H, 2001. Repro ductive effects of ethynylestradiol and

4t

- octylphenol on the zebrafish ( Danio rerio ). Arch Environ Contam Toxicol 41: 458

- 467.

f. Yokota H, Seki M, Maeda M, Oshima Y, Tadokoro H, Honjo T, Kobayashi K, 2001. Life

cycle toxicity of 4

- nonylphenol to medaka ( O ryzias latipes ). Environmental Toxicology and

Chemistry, 20: 2552

- 2560.

• MMT pathology summary trenboloneMMT ISR Pathology Appendix DRAFT 18 March 29, 2013 a. Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny

JS, Leino RL, Wilson VS, Cardon MC, Hartig PC, Gray LE, 2003. Effe cts of the androgenic

growth promoter 17β

- Trenbolone on fecundity and reproductive endocrinology of the fathead

minnow. Environmental Toxicology and Chemistry 22, 1350

- 1360.

b. Örn S, Yamani S, Norrgren L, 2006. Comparison of Vitellogenin Induction, Sex Rat io, and

Gonad Morphology Between Zebrafish and Japanese Medaka After Exposure to 17α

Ethinylestradiol and 17β

- Trenbolone. Arch Environ Contam Toxicol 51, 237

- 243.

c. Seki M, Fujishima S, Nozaka T, Maeda M, Kobayashi K, 2006. Comparison of response to

17β

- es tradiol and 17β

- trenbolone among three small fish species. Environmental Toxicology

and Chemistry 25, 2742

- 2752.

• MMT pathology summary procholoraz

a. Andersen HR, Vinggaard AM, Rasmussen TH, Gjermandsen IM, Bonefeld

- Jorgensen EC

(2002) Effects of currently used pesticides in assays for estrogenicity, androgenicity, and

aromatase activity in vitro. Toxicol Appl Pharmacol, 179(1):1

- 12.

b. EPA (2005) Phase 1

- B Pathology Analysis in Fish Exposed to Endocrine Disruptors. Study

No. WA 0 5

- 1 2, Test PROCHL

- 1 , Medaka / Prochloraz. USEPA Mid

- Continent Ecology

Division, Duluth, MN.

c. Mason JI, Carr BR, Murry BA (1987) Imidazole antimycotics: selective inhibitors of steroid

aromatization and progesterone hydroxylation. Steroids, 50(1

- 3):179

- 189.

d. Sanderson JT, Boerma J, Lans bergen GW, van den Berg M (2002) Induction and inhibition of

aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical

carcinoma cells. Toxicol Appl Pharmacol, 182(1):44

- 54.

e. Vinggaard AM, Hnida C, Breinholt V, Larsen JC (200 0) Screening of selected pesticides for

inhibition of CYP19 aromatase activity in vitro. Toxicol In Vitro, 14(3):227

- 234.

f. Vinggaard AM, Nellemann C, Dalgaard M, Jorgensen EB, Andersen HR (2002)

Antiandrogenic effects in vitro and in vivo of the fungicide p rochloraz. Toxicol Sci.,

69(2):344

- 53.