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

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

Primary Reviewer: Signature:

[Insert Name of Organization] Date: 

Secondary Reviewer: Signature:

[Insert Name of Organization] Date:

Template version 08/2011

DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500

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 Unpublished.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 Sponsor)

TEST ORDER #: [Test Order Recipient or the Consortium No.] (e.g., EDSP-PC Code-###)

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID [number] (if applicable)), [#]

[strain] rats/dose group were treated daily via [oral gavage] with [chemical 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 [satisfies/does not satisfy] the Test Order requirement for a Male Pubertal Assay 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]

provided.
Male Pubertal Assay ([year of study]) / Page 2 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / 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 Substance: 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. Vehicle: [Corn oil, Water, or Carboxymethylcellulose]. Solvents such as Acetone 

and DMSO should be avoided. Include expiration date (if applicable)

4. Test Animals:

Species: Rat

Strain: [Sprague-Dawley (preferred),Wistar, or Long-Evans]

Age/Weight at Study 

Initiation:

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 

Conditions:

Temperature:

Humidity:

Air changes:

Photoperiod:

[#]°C

[#]%

[#]/hr

[#] hrs light/ [#] hrs dark 

B. STUDY DESIGN

1. In-Life Dates: 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 

supplier 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 Pubertal Assay ([year of study]) / Page 3 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

3. Animal Assignment: 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 levels.)

TABLE 1. Study Design 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 Selection Rationale: 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. The 

guideline recommends 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. Dose Preparation and Analysis: Dose 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 Dose Analysis

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

different 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 variation between nominal and actual dosage to the animals was 

[acceptable/unacceptable]. Describe any deficiencies noted.
Male Pubertal Assay ([year of study]) / Page 4 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

6. Dosage Administration: 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 list parameters 

that were analyzed and the statistical methods used and include a statement as to whether or 

not the Reviewer considers the analyses to be appropriate. If inappropriate, provide 

alternative/rationale. The data should be analyzed for normal distribution and homogeneity 

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 the 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 age and body weight at preputial 

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

PND 23 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 analyzed using an appropriate nonparametric test. However, 

non-parametric analysis would generally be the method of last resort as it does not allow 

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

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

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

C. METHODS

1. Mortality and Clinical Examinations: All animals were examined [frequency; e.g. twice 

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

2. Body Weight: Animals were weighed daily prior to dosing.

3. Preputial Separation (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 Pathology: 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 animals were sacrificed [(describe method; preferred method is decapitation 

without anesthesia)] on PND [53] approximately 2 hours post-dosing. (The guideline 

recommends sacrifices 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) for subsequent hormone and clinical chemistry evaluations. (Historical control data 

should be provided.)

a. Hormone Analysis: Total testosterone, total thyroxine (T4), and thyroid stimulating 

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

immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay (ELISA), or timeresolved immunofluorescent procedures.]

b. Clinical Chemistry: 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 Pubertal Assay ([year of study]) / Page 6 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected 

and weighed. The (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 in 

Section II.F.

II. RESULTS Describe findings. Include 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 Signs of Toxicity: 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 they had an impact on dose selection.

TABLE 2. Incidence of Clinical Observations a

Observation

Dose Level (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 Pubertal Assay ([year of study]) / Page 7 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

C. General Growth and Preputial Separation: 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. (These tables are MANDATORY]

TABLE 3. General Growth and Preputial Separation (PPS)a.

Parameter 

Evaluated

Vehicle Control 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 23; 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 23

A = Adjusted for body weight on PND 23

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)

Number of Animals 

Examined

Incidence
Male Pubertal Assay ([year of study]) / Page 8 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

D. Organ Weights: Organ weights at necropsy are presented in Table 5 below.

(This table is MANDATORY. Describe any treatment-related effects.)

TABLE 5. Organ Weights at Necropsya

Organ

Vehicle Control 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

Liver 

(g)

U

A

R

Kidneys 

(g)

U

A

R

Pituitary 

(mg)

U

A

R

Adrenals 

(mg)

U

A

R

Seminal 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 23

A = Adjusted for body weight on PND 23

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 Pubertal Assay ([year of study]) / Page 9 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

E. Clinical Chemistry and Hormone Levels: 

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

examined. Describe any treatment-related effects)

TABLE 6. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated

Vehicle Control 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

Hormones

Serum T4, 

Total (μg/dL)

Serum TSH 

(ng/mL)

Serum 

testosterone 

(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 histopathological findings of the thyroid gland are 

presented below in Table 7. (This table is MANDATORY; limit text to integration of 

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

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

TABLE 7. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups

Parameter Evaluated

Colloid Quality Follicular Cell 

Height (Increase)

Follicular Cell 

Height (Decrease)

Follicular Cell 

Shape

Severityb 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.

TABLE 8. Incidence of Histopathological Lesions of the Testes, Epididymides and Kidneya

Findings

Dose Level (# 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 Pubertal Assay ([year of study]) / Page 11 of 11

[NAME OF TEST MATERIAL]/[PC Code] (if applicable) OCSPP 890.1500 / OECD None

III. DISCUSSION AND CONCLUSIONS

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

conclusions.

B. AGENCY COMMENTS: Briefly summarize the results and discuss the following:

Evaluate the adequacy of the dose levels tested. 

Discuss the systemic 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?

Were there any effects on clinical chemistry parameters?

Were there any changes in histopathological 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 

reporting 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.