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CFSAN/Office of Food Additive Safety
March 2004

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Introduction to the Template For Subchronic Toxicity Study in Rodents

Toxicology Template Introduction

Table Of Contents

  1. Identification of Study
  2. Good Laboratory Practice
  3. Executive Summary
  4. Materials and Methods
    1. Test Substance
    2. Test Substance As Administered
    3. Animal Diet
    4. Test Animals
    5. Experimental Design
    6. Body Weight And Feed Intake
    7. Cage-Side Observations
    8. Opthalmological Examination
    9. Hematology
    10. Clinical Chemistry
    11. Urinalysis
    12. Other Tests
    13. Necropsy (Interim Sacrifice)
    14. Necropsy (Terminal)
    15. Gross Pathology Observations
    16. Histopathology Observations
    17. Statistical Methods
  5. Results
    1. Dose Verification
    2. Feed Consumption Changes
    3. Intake Of Test Substance
    4. Feed Efficiency
    5. Body Weight Changes
    6. Cage-Side Observations
    7. Mortality
    8. Opthalmological Examination
    9. Hematology
    10. Clinical Chemistry
    11. Urinalysis
    12. Other Tests
    13. Organ Weights
    14. Gross Pathology Changes Observed
    15. Histopathology Changes Observed
    16. Neurotoxicity
  6. Evaluation And Comment On Study
  7. Summary And Conclusions
    1. Brief Summary Of Major Findings From The Study
    2. Relationship Between Dose And Incidence/Severity Of Lesions Or Abnormalities
    3. Was There A Target Organ?
    4. NOEL
  8. References
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Subchronic Toxicity Study in Rodents Template

Date of Submission:

Title of Petition or Notification:     

Name and Address of Petitioner or Notifier:         

I. Identification of Study1 

A. Study File Location:
B. Study Title/Report Number:
C. Name and Address of Testing Facility:
D. Date of Study Report:
E. Dates Study Conducted:
F. Study Objective:
G. Comments:

II. Good Laboratory Practice2 

A. Good Laboratory Practice (GLP) Compliance?
B. Quality Assurance (QA) Statement?
C. Availability and Location of Original Data/Specimens/Test Substance:

III. Executive Summary

IV. Materials and Methods

A.   Test Substance3 

  1. CAS name:
  2. Other name(s):
  3. CAS registry number:
  4. Molecular structure: http://www.chemfinder.com4 /
  5. Purity:            
  6. Impurities:    
  7. Stability:       
  8. Comments:     

B.   Test Substance As Administered5 

  1. Batch/lot number:
  2. Route:      
  3. Vehicle used:
  4. Tested adequately for concentration?
  5. Tested for homogeneity?       
  6. Tested for stability?  
  7. Problems with storage?         

C.   Animal Diet,

  1. Feed
    1. Type:               
    2. Name:             
    3. Availability:   
    4. Analysis for contaminants:
    5. Comments:     
  2. Water
    1. Source:           
    2. Availability:   
    3. Analysis for contaminants:  
    4. Comments:     

D.   Test Animals6 

  1. Species/strain/substrain:
  2. Sex:
  3. Age range at initiation of study:      
  4. Weight range at initiation of study:
  5. Quarantine/acclimation?    
  6. Physical examination times:
  7. Number per cage:       
  8. Environmental conditions:  
  9. Comments:

E.   Experimental Design7 

1. Targeted dose levels:

Table # [Heading]

 

test group

conc. in diet
(ppm or mg/kg)

dose to animals
(mg/kg body-weight/day)

number of males

number of females

Control

 

 

 

 

Low

 

 

 

 

Mid

 

 

 

 

High

 

 

 

 

2. Total number of animals:      
3. Duration of study (including recovery period, if any):       
4. Length of exposure to test substance:        
5. Were animals randomized?   
6. Recovery period:        
7. Comments:   

F.    Body Weight And Feed Intake

1. Parameter examined:

Table # [Heading]

 

examined

not examined

 

Feed Intake*
Feed Spillage*
Water Intake*
Body Weight*
Body Weight Changes*

*These parameters are recommended in REDBOOK for subchronic toxicity studies.

2. Comments:(e.g., list frequency)

G.   Cage-Side Observations8 

1. Parameter examined: 

Table # [Heading]

 

examined

not examined

 

Appearance*
Abnormal Stool*
Morbidity*
Mortality*
Neurotoxicity Screening (Specify parameters)*, ** 

*These parameters are recommended in REDBOOK for subchronic toxicity studies. Add additional parameters tested.

**The parameters for neurotoxicity screening may include, but are not limited to, the following:

2. Comments: (e.g., list frequency)

H. Opthalmological Examination

  1. Parameter examined:  
  2. Comments: (e.g., list frequency)

I. Hematology9 

  1. Fasting duration prior to blood collection:
  2. When in the study were the blood samples collected?        
  3. How were the blood samples drawn?            
  4. Dose groups and number of animals tested:
  5. Parameter examined:  

Table # [Heading]

measurement
related to

examined

not examined

red blood cells

 

  

Hematocrit (Hct)*
Hemoglobin Conc. (Hb)*
Mean Corp. Hb. (MCH)*
Mean Corp. Hb. Conc. (MCHC)* Mean Corp. Volume (MCV)*
Total Erythrocyte Count (RBC)*

white blood cells

 

  

Basophils, Eosinophils* Lymphocytes*
Macrophage/Monocytes*
Neutrophils*
Total Leukocytes (WBC)*

clotting potential

 

  

Activated Partial-Thromboplastin Time*
Clotting Time*
Platelet Count*
Prothrombin Time*

others

  

Bone marrow cytology*
Reticulocyte counts*

    *These parameters are recommended in REDBOOK for subchronic toxicity studies. Add additional additional parameters tested.

6. Comments:      

J.Clinical Chemistry10 

  1. Fasting duration prior to blood collection:  
  2. When in the study were the blood samples collected?
  3. How were the blood samples drawn?
  4. Dose groups and number of animals tested:
  5. Parameter examined:  

Table # [Heading]

measurement related to

examined

not examined

electrolyte balance

  

Calcium* Chloride*,** Phosphorus* Potassium*,** Sodium*,**

carbohydrate metabolism

  

Glucose*,**

liver function:
  A) hepatocellular
      (recommend at  
      least 3 out of 5 
      RED BOOK   
        PARAMETERS )
  B) hepatobiliary
      (recommend at  
      least 3 out of 5  
      RED BOOK
        PARAMETERS)

  

Alanine Aminotransferase
   (ALT or SGPT)*,**
Aspartate Aminotransferase
   (AST or SGOT)*
Glutamate Dehydrogenase* Sorbitol Dehydrogenase*
Total Bile Acids*
 

Alkaline Phosphatase (ALP)*,**
Gamma-Glutamyl Transferase (GGT)*,**
Total Bile Acids*
Total Bilirubin*
5' Nucleotidase*

kidney function

  

Creatinine*,**
Urea Nitrogen*,**

others

(acid/base balance, cholinesterases, hormones, lipids, methemoglobin, and proteins)

  

Albumin (A)*
Globulin (G, calculated) or A/G Ratio*
Total Cholesterol*
Cholinesterase*
Total protein*,**
Fasting Triglycerides*

*These parameters are recommended in REDBOOK for subchronic toxicity studies. Add additional additional parameters tested.

** These parameters should generally be given priority when adequate volumes of blood samples can not be obtained from test animals.  

6. Comments:  

K. Urinalysis11 

  1. When were urine samples collected?             
  2. Parameter examined:

 

Table # [Heading]

examined

not examined

 

Glucose*, Microscopic evaluation for sediment and presence of blood/blood cells*, pH*, Protein*, Specific Gravity*, Volume*

* These parameters are recommended in REDBOOK for subchronic toxicity studies.

3. Comments :

L. Other Tests12 

  1. Observations:
  2. Comments:

M. Necropsy (Interim Sacrifice)13 

  1. Was there an interim sacrifice?
  2. Dose groups and number of animals:
  3. Organs/Tissues weighed:

Table # [Heading]

examined

not examined

 

Adrenals*, Brain*, Epididymides*, Heart*,
Kidneys*, Liver*, Spleen*, Testes*, Thyroid/parathyroid*, Thymus*, Ovaries*,   Uterus*

* These parameters are recommended in REDBOOK for subchronic toxicity studies.

4. Comments:  

N. Necropsy (Terminal)

1. Organs/Tissues weighed:

Table # [Heading]

examined

not examined

 

Adrenals*, Brain*, Epididymides*, Heart*,
Kidneys*, Liver*, Spleen*, Testes*, Thyroid/parathyroid*, Thymus*, Ovaries*, Uterus*

*These parameters are recommended in REDBOOK for subchronic toxicity studies. Add additional additional parameters tested.

2. Comments:  

O. Gross Pathology Observations

  1. Organs/Tissues Examined:
  2. Comments:   

P. Histopathology Observations

  1. Organs/Tissues were collected from which dose groups?  
  2. Organs/Tissues were examined from which dose groups?
  3. How were the organs/tissues prepared for histopathology observation?  
  4. Organs/Tissues collected:

Table # [Heading]

system

examined

not examined

digestive

  

Salivary Gland*, Esophagus*, Stomach*, Duodenum*, Jejunum*, Ileum*, Cecum*, Colon*, Rectum*, Gall Bladder* (in case of mice), Liver* (middle, left and triangular lobes), Pancreas*

respiratory

  

Nasal Turbinates*, Trachea*,
Lung* (with main-stem bronchi)

cardiovascular

  

Aorta*, Heart*

reticulo- endothelial/
hematopoietic

  

Bone Marrow* (sternum),
Lymph Nodes* (1 related to route of administration, and 1 from a distant location),
Spleen*, Thymus*

urogenital

  

Kidneys*, Ovaries* and fallopian tubes*, Corpus Uteri*, Cervix Uteri*, Prostate*, Seminal Vesicle* (if present), Testes*, Urinary Bladder*, Vagina*

neurologic

  

Brain* (at least 3 different levels), Spinal-Cervical*,
Spinal-Lumbar*,
Spinal-Midthoracic*,
Sciatic Nerve*,
Harderian Gland* (if present)

glandular

  

Adrenals*, Mammary Glands*, Pituitary Glands*, Thyroid/Parathyroid Glands*, Thymus*,
Zymbal's Gland* (if present)

other

  

Bone (Femur)*, Eyes*, Skeletal Muscle*, Skin*, Epididymis*

*These parameters are recommended in REDBOOK for subchronic toxicity studies. Add additional additional parameters tested.

5. Comments:

Q. Statistical Methods

1. Methods of statistical analysis:

Table # [Heading]

methods of statistical analysis

parameters tested
   
   
   
   

2. Comments:

V. Results

A.   Dose Verification14 

1. Were doses verified?

Table # [Heading]

dose group

targeted concentration
 (ppm or mg/kg)

concentrations found in feed
(ppm or mg/kg)

standardDeviation

N*

low

 

 

 

 

mid

 

 

 

 

high

 

 

 

 

* Number of measurements (N)

  1. Verified by:      
  2. Comments:     

B.   Feed Consumption Changes15 

  1. Observations:
  2. Comments:

C.   Intake Of Test Substance16

1. Observations:

Table # [Heading]

dose group

daily dose
(mg/kg body-weight/day)

control

0

low

 

mid

 

high

 

2. Comments:

D.   Feed Efficiency17 

  1. Was feed efficiency calculated?
  2. Comments:  

E.   Body Weight Changes18 

  1. Observations:
  2. Comments:

F.    Cage-Side Observations19 

  1. Observations:            
  2. Comments:     

G.   Mortality20 

  1. Observations:            
  2. Comments:     

H. Opthalmological Examination

  1. Observations:
  2. Comments:

I.      Hematology21 

1. Observations:

Table # [Heading]

 SEX  males  females

DAILY DOSE
 (mg/kg body-weight/day)

0
CONTROL

 

 

 

0
CONTROL

 

 

 

NUMBER OF ANIMALS

 

 

 

 

 

 

 

 

red blood cells

 

 

 

 

 

 

 

 

Hematocrit (Hct)

%

 

 

 

 

 

 

 

 

Hemoglobin Conc. (Hb)

g/L

 

 

 

 

 

 

 

 

Mean Corp. Hb. (MCH)

 

 

 

 

 

 

 

 

 

Mean Corp. Hb. Conc. (MCHC)

 

 

 

 

 

 

 

 

 

Mean Corp. Volume (MCV)

L/L

 

 

 

 

 

 

 

 

Total Erythrocyte Count (RBC)

1012/L

 

 

 

 

 

 

 

 

white blood cells

 

 

 

 

 

 

 

 

Basophils

 

 

 

 

 

 

 

 

 

Eosinophils

 

 

 

 

 

 

 

 

 

Lymphocytes

109/L

 

 

 

 

 

 

 

 

Macrophage/
Monocytes

 

 

 

 

 

 

 

 

 

Neutrophils

109/L

 

 

 

 

 

 

 

 

Total Leukocytes (WBC)

109/L

 

 

 

 

 

 

 

 

clotting potential

 

 

 

 

 

 

 

 

Activated Partial-Thromboplastin Time

 

 

 

 

 

 

 

 

 

Clotting Time

 

 

 

 

 

 

 

 

 

Platelet Count

109/L

 

 

 

 

 

 

 

 

Prothrombin Time

 

 

 

 

 

 

 

 

 

others

 

 

 

 

 

 

 

 

Bone marrow cytology

 

 

 

 

 

 

 

 

 

Reticulocyte counts

1012/L

 

 

 

 

 

 

 

 

 (Specify a method of statistical analysis): * p<0.05, ** p<0.01

2. Comments:

J.    Clinical Chemistry22 

1. Observations:

Table # [Heading]

SEX

males

females

DAILY DOSE
 (mg/kg body-weight/day)

0
CONTROL

 

 

 

0
CONTROL

 

 

 

NUMBER OF ANIMALS

 

 

 

 

 

 

 

 

electrolyte balance

 

 

 

 

 

 

 

 

Calcium

mmol/L

 

 

 

 

 

 

 

 

Chloride

mmol/L

 

 

 

 

 

 

 

 

Phosphorus

mmol/L

 

 

 

 

 

 

 

 

Potassium

mmol/L

 

 

 

 

 

 

 

 

Sodium

mmol/L

 

 

 

 

 

 

 

 

carbohydrate metabolism

 

 

 

 

 

 

 

 

Glucose

mmol/L

 

 

 

 

 

 

 

 

liver function:

   A) hepatocellular

 

 

 

 

 

 

 

 

Alanine Aminotransferase

   (ALT or SGPT)

U/L

 

 

 

 

 

 

 

 

Aspartate Aminotransferase

   (AST or SGOT)

U/L

 

 

 

 

 

 

 

 

Glutamate Dehydrogenase

U/L

 

 

 

 

 

 

 

 

Sorbitol Dehydrogenase

U/L

 

 

 

 

 

 

 

 

liver function:
    B) hepatobiliary

 

 

 

 

 

 

 

 

Alkaline Phosphatase (ALP)

U/L

 

 

 

 

 

 

 

 

Gamma-Glutamyl Transferase (GGT)

U/L

 

 

 

 

 

 

 

 

Total Bile Acids

mmol/L

 

 

 

 

 

 

 

 

Total Bilirubin

mmol/L

 

 

 

 

 

 

 

 

5' Nucleotidase

U/L

 

 

 

 

 

 

 

 

kidney function

 

 

 

 

 

 

 

 

Creatinine

mmol/L

 

 

 

 

 

 

 

 

Urea Nitrogen

mg/dL

 

 

 

 

 

 

 

 

others

 

 

 

 

 

 

 

 

Albumin (A)

g/L

 

 

 

 

 

 

 

 

Globulin (G, calculated)

g/L

 

 

 

 

 

 

 

 

A/G Ratio

 

 

 

 

 

 

 

 

 

Total protein

g/L

 

 

 

 

 

 

 

 

Total Cholesterol

mmol/L

 

 

 

 

 

 

 

 

Fasting Triglycerides

mmol/L

 

 

 

 

 

 

 

 

Cholinesterase

U/L

 

 

 

 

 

 

 

 

            (Specify statistical method of analysis): * p<0.05, ** p<0.01

           

2. Comments:

K.   Urinalysis23 

1. Observations:

Table # [Heading]

SEX

males

females

DAILY DOSE
 (mg/kg body-weight/day)

0
CONTROL

 

 

 

0
CONTROL

 

 

 

NUMBER OF ANIMALS

 

 

 

 

 

 

 

 

Glucose

mmol/L

 

 

 

 

 

 

 

 

Microscopic evaluation for sediment and presence of blood/blood cells

 

 

 

 

 

 

 

 

 

pH

 

 

 

 

 

 

 

 

 

Protein

g/L

 

 

 

 

 

 

 

 

Specific Gravity

 

 

 

 

 

 

 

 

 

Volume

L/time

 

 

 

 

 

 

 

 

2. Comments:

L.    Other Tests24 

  1. Observations:
  2. Comments:

M.  Organ Weights25 

1. Observations:

 

Table # [Heading]

SEX

males

females

DAILY DOSE
 (mg/kg body-weight/day)

0
CONTROL

 

 

 

0
CONTROL

 

 

 

NUMBER OF ANIMALS

 

 

 

 

 

 

 

 

BODY WEIGHT (gram)a

 

 

 

 

 

 

 

 

BRAIN

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

ADRENALS

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

EPIDIDYMIDES

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

Per Body Weighta

%

 

 

 

 

Per Brain Weighta

%

 

 

 

 

HEART

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

SPLEEN

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

TESTES

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

Per Body Weighta

%

 

 

 

 

Per Brain Weighta

%

 

 

 

 

THYROID and PARATHYROID

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

THYMUS

 

 

 

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

 

 

 

 

Per Body Weighta

%

 

 

 

 

 

 

 

 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

OVARIES

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

Per Body Weighta

%

 

 

 

 

Per Brain Weighta

%

 

 

 

 

UTERUS

 

 

 

 

 

Absolute Weighta

gram

 

 

 

 

Per Body Weighta

%

 

 

 

 

Per Brain Weighta

%

 

 

 

 

a: Group means at the end of terminal necropsy are shown.
(Specify methods of statistical analysis): * p<0.05, ** p<0.01

2. Comments:

N.   Gross Pathology Changes Observed26 

  1. Observations:
  2. Comments:

O.   Histopathology Changes Observed27 

1. Observations:

Table # [Heading)

 

 

NUMBER OF ANIMALS
WITH, GROSS, NON-NEOPLASTIC, OR NEOPLASTIC LESIONS

SEX

males

females

DAILY DOSE
 (MG/KG BODY-WEIGHT/DAY)

0
CONTROL

 

 

 

0
CONTROL

 

 

 

NUMBER OF ANIMALS EXAMINED

 

 

 

 

 

 

 

 

DIGESTIVE SYSTEM

 

 

 

 

 

 

 

 

ORGAN/TISSUE #

 

 

 

 

 

 

 

 

GROSS LESION

 

 

 

 

 

 

 

 

NON-NEOPLASTIC LESION

 

 

 

 

 

 

 

 

NON-NEOPLASTIC LESION

 

 

 

 

 

 

 

 

NON-NEOPLASTIC LESION

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NEOPLASTIC LESIONS