Male P ubertal Assay ( [ year of study ] ) / Page 1 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## Primary Reviewer: Signature :

[Insert Name of Organization] Date:

Secondary Reviewer: Signature :

[Insert Name of Organization] Date:

Template version 0 8 / 2011



## DATA EVALUATION RECORD

STUDY TYPE : Male P ubertal Assay ; O CSPP 890.1 5 0 0

PC CODE : (if applicable) DP BARCODE : (if applicable)

TXR# : (if applicable) CAS No. : [ # ]

TEST MATERIAL (PURITY) : ( use name of material tested as referred to in the study

(common agency chemical name in parenthesis) ) (% purity)

SYNONYMS : ( Other names and codes )

CITATION : Author ( up to 3, see SOP for exact format )

. ( [Study Year] ). Title. Laboratory

name and location. Laboratory report number, study completion date. MRID (if

applicable) ( no hyphen ) . Unpublished

. (OR if published, list Journal name,

vol.:pages)

SPONSOR : (Name of Study Sponso r )

TEST ORDER # : [ T est O rder R ecipient or the C onsortium No. ] ( e.g., EDSP

- PC Code

- ###)

EXECUTIVE SUMMARY : In a M ale P ubertal A ssay ( M RID [number] ( if applicable ) )

, [ # ]

[ strain ] rats /dose group were treated daily via [ oral gavage ] with [ch emical name (% purity,

batch/lot #)] in [ vehicle ] at doses of 0 ( [ vehicle ] )

, [ # ] or [#] mg/kg/day from post

- natal day

( PND) [ 23 ] to [ 53 ]

. Animals were examined for preputial separation (PPS) daily beginning on

PND 30 and weight at day of attainment was recorded. Following sacrifice on PND [ 53 ] or [ 54 ]

, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were

analyzed using a [ type ] assay. Urogenital organ weights were recorded and microscopic

examination of the [ testes, epididymides, prostate, thyroid, and kidneys ] were performed.

Provide a brief summary of the results and a concise discussion

. Discuss any major deficiencies,

failure to meet performance criteria, or any problems encountered in this study.

This assay [ s atisfies/does not satisfy ] the Test Order requirement for a Male P ubertal A ssay in

rats (OCSPP 890.1500)

. If it does not satisfy the requirement, concisely list only the major

deficiencies and refer to deficiency section.

COMPLIANCE : Signed and dated GLP and Quality Assurance statements [ were /were not ]

pro vided.Male P ubertal Assay ( [ year of study ] ) / Page 2 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Name of the Facility

Location: Location of the Facility

Study Director: Name

Other Personnel: Name and study responsibility

Study Period: Study start and end dates



## 2

. Test S ubstance : Common name as used by Agency

Description: e.g. technical, nature, color, odor, and molecular weight

Source: Company (and catalog number if available)

Lot/Batch #: include expiration date

Purity: %

Stability: Provide available data

CAS #: CAS # or Not available

Structure: [Structure] or Not available



## 3

. V ehicle : [ Corn oil, Water, or Carboxymethylcellulose ]

. Solvents such as Acetone

and DMSO should be avoided

. Include expiration date (if applicable)



## 4

. Test A nimals :

Species: Rat

Strain: [ Sprague

- Dawley (preferred),Wistar, or Long

- Evans ]

Age/ W eight at S tudy

I nitiation: PND [ # ] / [ #]

– [#] g males only

Source: Supplier (city, state and country , if outside U.S. )

Housing: ##/cage, type of cage, and bedding, etc. [e.g., 3/cage in stainless steel cages, suspended

above cage board,]

Recommended housing is 2

- 3 animals of the same dose group /cage ) with heat

- treated

laboratory

- grade wood shavings (not cedar) as bedding. Corn cob bedding is NOT

recommended due to potential to disrupt endocrine activity.

Diet: [ Diet name, source ]

, ad libitum

Water: Phytoestrogen content [#] μg of genistein equivalents/gram diet

Source, treatment [e.g., Reverse

- osmosis filtered tap water]

, ad libitum

Environmental

C onditions: Temperature:

Humidity:

Air changes:

Photoperiod: [#] °C

[#] %

[#] /hr

[ # ] hrs light/ [ # ] hrs dark



## B. STUDY DESIGN

1. In

- L ife D ates : Start: [Month/day/Year] End: [Month/day/year]

2. Mating : Describe procedure for obtaining juvenile animals (e.g., bred in

- house, including

determination of confirmation of pregnancy, OR received pregnant time

- mated dams from

suppl ier on GD 7, 8, 9, or 10) and standardizing litter size to 8

- 10 pups by culling on PND

3

- 5

. Example text is included below.Male P ubertal Assay ( [ year of study ] ) / Page 3 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## 3

. Animal A ssignment : Offspring were weaned on PND [ 21 ] and weighed. The rats from the

extreme ends of the weight range were eliminated to achieve a mean body weight and

variance across dose groups at study initiation. Animals were assigned ( note how assigned,

e

. g., randomized complete block design (time

- separated necropsy is the blocking factor) ) to

the test groups noted in Table 1. Littermates were not assigned to the same treatment group.

( The treatment groups include: (1) the vehicle

- treated and (2) xenobiotic

- treated with at

least two dose lev els. )

TABLE 1. Study D esign a



### Test group Dose (mg/kg/day) # of Males

Control 0 

### 15

Low [#] 

### 15

High [#] 

### 15

a Data were obtained from page [ # ] of the study report.



## 4

. Dose S election R ationale : Briefly describe any range

- finding study, including information

regarding the study identification (laboratory report or MRID number), study type (i.e.,

duration, route of administration, species), dose levels, effects, and conclusions. T he

guideline reco mmends that the highest dose level be at or just below the Maximum Tolerated

Dose (MTD) but need not exceed the Limit Dose (1000 mg/kg/day); however, typically, the

Agency also considers the toxicity profile of the chemical (i.e., cholinesterase inhibition

, target organ toxicity, etc

. ) in dose selection. The second dose level should typically be

spaced to produce a lesser degree of toxicity relative to the high dose unless justification is

provided for testing at a different level. Example text is provided below.

5

. D ose P reparation and A nalysis : D ose formulations were prepared daily by mixing

appropriate amounts of test substance with [ vehicle ]

. Homogeneity and stability were tested

at ( state when, what dose levels, duration, and temperature ) . During the study, samples of

dose formulations were analyzed ( state when and at what dose levels ) for achieved

concentration.

Results of D ose A nalysis

Homogeneity: concentration range as percent of nominal and/or coefficient of variation for

differen t strata (e.g., top, middle, and bottom)

Stability: range of values for each temperature and duration tested, expressed as percent of

initial (preferable) or percent of nominal concentration

Concentration: range of values expressed as percent of nominal

The analytical data indicated that the mixing procedure was [ adequate /inadequate] and that

the varia tion between nominal and actual dosage to the animals was

[ acceptable /unacceptable]

. Describe any deficiencies noted

.Male P ubertal Assay ( [ year of study ] ) / Page 4 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## 6

. Dosage A dministration : All doses were administered once daily by gavage from PND 23

through PND 53 , in a volume of [# ] ( recommended range from 2.5 to 5.0 ) mL/kg of body

weight. Dosing was performed between [ 0700 ] and [ 0900 ] hours daily.

7

. Statistics : The following describes the statistical analyses recommended by the test

guideline and may be used by the reviewer for verification. The DER should l ist parameters

that were analy z ed and the statistical methods used and include a statement as to whether or

no t the Reviewer considers the analyses to be appropriate. If inappropriate, provide

alternative/rationale

. The data should be analyzed for normal distribution and h om ogeneity

of variance in order to satisfy the assumptions of Analysis of Variance (ANOVA ) and

Analysis of Covariance (ANCOVA)). It is recommended that all data (i.e., initial body weight

[PND 23], body weight gain, age and body weight at preputial separation, terminal body

weight, organ weights, and serum hormones be analyzed by ANOVA. If th e study was

conducted in blocks, then the recommended analysis is to use to a two

- way ANOVA with

Block and Treatment as main effects. It is recommended a ge and body weight at preputial

separation and all organ weights also be analyzed by ANCOVA, using the body weight at

PND 2 3 as the covariate. When statistically significant effects among groups are observed

(p<0.05), appropriate pair

- wise comparison tests would be needed in order to identify which

dose group is significantly different from the control group. Where there is heterogeneity of

variance, data should be transformed appropriately to achieve homogeneous variances prior

to ANOVA/ANCOVA, or analyze d using an appropriate nonparametric test. However,

non

- parametric analysi s would generally be the method of last resort as it does not allow

analysis of covaria nce . In addition to ANOVA and ANCOVA, it is recommended that the

unadjusted and adjusted valu es be examined for linear trend with dose level.Male P ubertal Assay ( [ year of study ] ) / Page 5 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## C

. METHODS

1. Mortality and C linical E xaminations : All animals were examined [ frequency; e.g. twice

daily ] for mortality and moribundity. Clinical examinations were conducted [ frequency ]

.

2. Body W eight : Animals were weighed daily prior to dosing.

3. Preputial S eparation (PPS) : Beginning on PND [ 30 ] , all animals were examined daily for

onset of PPS . Age and weight at on the day of completion of PPS were recorded.

4. Sacrifice and P athology : On the day of termination, rats were removed one at a time from

their home cages to a separate room for euthanasia and terminal procedures so as not to

induce stress

- related responses in other animals which may affect hormone measurements.

All surviving a nimals were sacrificed [ ( describe method; preferred method is decapitation

without anesthesia ) ] on PND [ 53 ] approximately 2 hours post

- dosing

. ( The guideline

recommends s acrifices be completed by 1300 hours due to normal diurnal fluctuation in

thyroid hormone levels ) Blood from the trunk of the animals was collected immediately into

serum separation tubes

, processed [method]

, and stored at [temperature ] ; ( should be

- 20°C or

colder ) f or subsequent hormone and clinical chemistry evaluations

. ( Historical control data

should be provided. )

a. Hormone A nalysis : Total testosterone, total thyroxine (T 4 

## ), and thyroid stimulating

hormone (TSH) levels were analyzed using [ assay type; radioimmunoassay (RIA),

immunoradiometric assay (IRMA), enzyme

- linked immunosorbent assay (ELISA), or time

resolved immunofluorescent procedures. ]

b. Clinical C hemistry : The following CHECKED (X) parameters were examined.

X ELECTROLYTES X OTHER

Calcium Albumin

Chloride Creatinine*

Magnesium Urea nitrogen*

Phosphorus Total cholesterol

Potassium Globulins

Sodium Glucose

ENZYMES Total bilirubin

Alkaline phosphatase (ALK) Total protein

Cholinesterase (ChE) Triglycerides

Creatine phosphokinase Serum protein electrophoresis

Lactic acid dehydrogenase (LDH)

Alanine aminotransferase (ALT/also SGPT)

Aspartate aminotransferase (AST/also SGOT)

Sorbitol dehydrogenase

Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended based on guideline 890.1500.Male P ubertal Assay ( [ year of study ] ) / Page 6 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## c. Organ W eights and H istopathology : The following CHECKED (X) tissues were collected

and weighed . T he (XX) organs, in addition, were subjected to histological examination ( note

if not all collected tissues were examined

. )

X UROGENITAL X OTHER

Testes (left and right separately) * + Thyroid * +

Epididymides (left and right separately) * + Liver *

Seminal vesicle plus coagulating glands (with and without fluid) * Adrenals (paired) *

Ventral prostate * Pituitary *

Dorsolateral prostate *

Levator ani/bulbocavernosus (LABC) muscle complex *

Kidneys (paired) * +

* Weights required based on guideline 890.1500

+ Histopathological examination required based on guideline 890.1500



## This is the information that you would be found in the study report conducted following

guideline recommendations. Thyroid sections are subjectively evaluated for follicular cell

height and colloid area , preferably using a five point grading scale (1 = shortest/smallest; 5

= tallest/largest)

, and any abnormalities/lesions noted. At least two sections from each of

the two lobes of the thyroid should be examined in order to obtain representative sample of

the thyroid tissue from each lobe. Guidance related to the histological evaluation of the

testis and epididymis is given in EPA’s Health Effects Test Guideline OPPTS 870.3800:

Reproduction and Fertility Effects (US EPA, 1998). Report the histopathological findings i n

Section II. F

.

II. RESULTS D escribe findings

. I nclude tables if needed; tables are recommended to depict any

treatment

- related findings, thus limiting use of text to highlight specific points

.



## A. Mortality : Report any mortality and assess whether any animal deaths were due to

treatment with the test substance or other circumstances (e.g., gavage errors)

.

B

. Clinical S igns of T oxicity : Report any clinical signs of toxicity, including information on

the nature, incidence, severity, onset, and duration. As with the mortality data, the

Reviewer should assess whether the findings are considered adverse and related to

treatment and if the y had an impact on dose selection

.

TABLE 2

. Incidence of Clinical Observations a

Observation Dose L evel (mg/kg bw/day)

Vehicle Control Low [ # ] High [#]

# Observed # Examined # Observed # Examined # Observed # Examined

[observation type]

a Data were extracted from page [#] of the study report



## If clinical signs of toxicity are not observed include the following example text.Male P ubertal Assay ( [ year of study ] ) / Page 7 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## C

. General G rowth and P reputial S eparation : Body weights, body weight gains, age of

attainment of PPS and weight at day of PPS are presented in Table 3 below. Table 4

provides the proportion/incidence of unseparated

. ( T h e se table s are MANDATORY ]

T ABLE 3 . General G rowth and P reputial S eparation (PPS) a



## .

Parameter

Evaluated Vehicle C ontrol Low Dose ( [#] mg/kg/day) High Dose ( [#] mg/kg/day)

Number of

Animals

Examined Mean SD CV Number of

Animals

Examined Mean SD CV Number of

Animals

Examined Mean SD CV

Initial body

weight

(PND 2 3 ; g) U

A

Body weight

at PPS (g) U

A

Final body

weight(g) U

A

Final body

weight

(% of

control) U

A

Body weight

gain

(final

– initial; g) U

A

Age at PPS

(PND) U

A

a Data were obtained from page [ # ] of the study report, and are the average of [#] male rats per dose group.

U = Unadjusted for body weight on PND 2 3

A = Adjusted for body weight on PND 2 3

SD = Standard Deviation

CV = Coefficient of Variation

* Significantly different from controls at p<0.05.

NA Not applicable



## TABLE 4

. Preputial Separation

Vehicle Control Low Dose

( [#] 

### mg/kg/day) High Dose

( [#] 

### mg/kg/day)

N umber of Animals

Examined

IncidenceMale P ubertal Assay ( [ year of study ] ) / Page 8 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## D

. Organ W eights : Organ weights at necropsy are presented in Table 5 below.

( T his table is MANDATORY . Describe any treatment

- related effects. )

TABLE 5 . Organ Weights at Necropsy a

Organ Vehicle C ontrol Low Dose ( [#] m g/kg/day) High Dose ( [#] mg/kg/day)

Number of

Animals

Examined Mean SD CV Number of

Animals

Examined Mean SD CV Number of

Animals

Examined Mean SD CV

Liver

(g) U

A

R

Kidney s

(g) U

A

R

Pituit ary

(mg) U

A

R

Adrena ls

(mg) U

A

R

Semina l vesicle

+ coagulating

gland, with

fluid (mg) U

A

Seminal vesicle

+ coagulating

gland, without

fluid (mg) U

A

Ventral

prostate (mg) U

A

Dorsolateral

prostate (mg) U

A

LABC

(mg) U

A

Epididymis, left

(mg) U

A

Epididymis,

right (mg) U

A

Testis, left (mg) U

A

Testis, right

(mg) U

A

Thyroid, fixed

(mg) U

A

a Data were obtained from page [#] of the study report, and are the average of [#] male rats per dose group.

U = Unadjusted for body weight on PND 2 3

A = Adjusted for body weight on PND 2 3

R = Organ

- to

- body weight ratio (relative to body weight)

SD = Standard Deviation

CV = Coefficient of Variation

* Significantly different from controls at p<0.05.Male P ubertal Assay ( [ year of study ] ) / Page 9 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## E

. Clinical C hemistry and H ormone L evels :

( T his table is MANDATORY . Add rows as necessary to include any additional parameters

examined. Describe any treatment

- related effects )

TABLE 6 . Hormone Levels and C linica l C hemistry a



### Parameter

Evaluated Vehicle C ontrol Low Dose ( [#] 

### mg/kg/day) High Dose ( [#] 

### mg/kg/day)

Number of

Animals

Examined Mean SD CV Number of

Animals

Examined Mean SD CV Number of

Animals

Examined Mean SD C V

Hormones

Serum T

4 , Total (μg/dL)

Serum TSH

(ng/mL)

Serum

t estosterone

(ng/mL)

Clinical Chemistry

Creatinine

(μmol/L)

Blood urea

nitrogen

(mmol/L)

a Data were obtained from page [#] of the study report, and are the average of [#] male rats per dose group.

SD = Standard Deviation

CV = Coefficient of Variation

* Significantly different from controls at p<0.05.



## F

. Histopathology : The incidence of h istopathological findings of the thyroid gland are

presented below in Table 7

. ( T his table is MANDATORY ; limit text to integration of

findings, highlights ; relate with other findings, as appropriate )Male P ubertal Assay ( [ year of study ] ) / Page 10 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## T ABLE 7

. Incidence of Histopathological Lesions of the Thyroid G land a



### Treatment

Groups 

## Parameter Evaluated

Colloid Quality Follicular Cell

Height (Increase) Follicular Cell

Height (Decrease) Follicular Cell

Shape

Severity b 

### Incidence Severity 

### Incidence Severity 

### Incidence Severity 

### Incidence

O E O E O E O E



### Vehicle

Control 

## 0 0 0 0

1 1 1 1

2 2 2 2

3 3 3 3

Low Dose

( [#] mg/kg/day) 

## 0 0 0 0

1 1 1 1

2 2 2 2

3 3 3 3

High Dose

( [#] mg/kg/day) 

## 0 0 0 0

1 1 1 1

2 2 2 2

3 3 3 3

a Data were obtained from page [#] of the study report

. b Thyroid histopathology is graded 0

– 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe.

See OECD No. 82 for reference.

O = No. Observed

E = No. Examined



## The incidence of histopathological findings of the testes, epididymides and kidneys are presented

in Table 8.

T ABLE 8

. Incidence of Histopathological Lesions of the Testes, Epididymides and Kidney a

Findings Dose L evel ( # mg/kg bw/day)

Vehicle Control Low ( [ # ] ) High ( [ # ] )

# Observed # Examined # Observed # Examined # Observed # Examined

Testes

[observation type]

Epididymides

[observation type]

Kidney

[observation type]

a Data were obtained from page [#] of the study report

.Male P ubertal Assay ( [ year of study ] ) / Page 11 of 11

[ NAME OF TEST MATERIAL] / [ PC Code] (if applicable) O CSPP 8 9 0. 1 500 / OECD None



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Provide a brief paragraph of the investigator’s

conclusions.

B. AGENCY COMMENTS : B rief ly summarize the results and discuss the following:

Evaluate the adequacy of the dose levels tested.

Discuss the s ystemic toxicity observed (i.e., survival, body weight, body weight gain,

target organ toxicity etc)

Was age and weight at preputial separation within acceptable ranges? Report findings

relative to performance criteria ?

Were there any changes in organ weights compared to controls ? Report findings relative

to performance criteria ?

Were there any effects on hormone levels?

Wer e there any effects on clinical chemistry parameters ?

Were there any changes in histopatholog ical findings?

C. STUDY DEFICIENCIES : List each deviation from the protocol and classify the deviation

as major or minor. Also report any rationale provided by the investigator for the deviation.

Similarly list, classify, and discuss all other deficiencies with the conduct, results, and

repo rting of the study. Discuss the possibility of resolving the deficiencies and what would

be required. Major deficiencies may be presented and discussed in paragraph form, whereas

minor deficiencies can be presented in a bulleted list.