Introduction to the Template for Subchronic Toxicity Study in Dogs

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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 (Terminal)
   14.       Gross Pathology Observations
   15.       Histopathology Observations
   16.       Statistical Methods
5. Results
   1.       Dose Verification
   2.       Feed Consumption
   3.       Body Weight
   4.       Intake Of Test Substance
   5.       Feed Efficiency
   6.       Cage-Side Observations
   7.       Mortality
   8.       Ophthalmological Examination
   9.        Hematology
   10.       Clinical Chemistry
   11.       Urinalysis
   12.       Neurotoxicity (If Indicated)
   13.      Other Tests
   14.       Organ Weights
   15.       Gross Pathology Changes Observed
   16.       Histopathology Changes Observed
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

Subchronic Toxicity Study in Dogs ¹

Date of Submission:

Title of Petition or Notification:                 

Name and Address of Petitioner or Notifier:         

I. Identification of Study² 

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 Practice³ 

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 Substance⁴ 

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

B.   Test Substance As Administered⁶ 

1. Batch/Lot Number(s):
2.   Route:
3.   Vehicle used:
4.   Tested adequately for concentration? (See Part V-A for calculation)
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 Animals⁷ 

1.   Species/strain:
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 Design⁸ 

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 Intake⁹ 

  Parameter examined:

Table # [Heading]

 

Examined	Not Examined
	Feed Intake* 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 Observations¹⁰ 

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.

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

• Changes in skin, fur, eyes, mucous membranes, gait, posture, and  response to handling,
• Occurrence of secretions/excretions or other evidence of autonomic activity such as lacrimation, piloerection, pupil size change, unusual respiratory pattern,
• Presence of clonic or tonic seizure,
• Stereotype behaviors such as excessive grooming and repetitive circling,
• Bizarre behavior such as self-mutilating and walking backwards,
• Gross tumor development.

2. Comments:

H.  Opthalmological Examination

1.   Parameter examined:
2.   Comments:

 

I.      Hematology¹¹ 

1. Fasting duration prior to blood collection:

2. When 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.

6. Comments:

J.    Clinical Chemistry¹² 

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)   B) Hepatobiliary       (Recommend At         Least 3 Out Of 5)		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.

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

6. Comments:

K.   Urinalysis¹³ 

1. When and how were urine samples collected?
2. Dose groups and number of animals tested:
3. 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.

4. Comments:

L.   Other Tests

1.   Observations:
2.   Comments:

 

M.  Necropsy (Terminal)

1. Organs 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.                                                               

2. Comments:

N.   Gross Pathology Observations

1.   Organs/Tissues examined:
2.   Comments:

 

O. Histopathology Observations

1. Organs were collected from which dose groups?
2. Organs 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*, 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*, Urinary Bladder*;  As applicable: fallopian tubes*, Corpus Uteri*, Cervix Uteri*, Vagina*, Prostate*, Seminal Vesicle*, Testes*
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*
Other		Bone (Femur)*, Eyes*, Skeletal Muscle*, Skin*, Epididymis*

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

5. Comments:

P. 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?    

We suggest using an Excel file to calculate the Means and Standard Deviations (SD).  (Optional): A sample Excel file (calculation.xls) is provided.

Table # [Heading]

 

Dose Groups	Targeted Daily Dose (Mg/Kg Body-Weight/Day)	Targeted Concentration In Feed (Ppm Or Mg/Kg)	Concentrations Found In Feed (Ppm Or Mg/Kg)	Standarddeviation	N*
Low
Mid
High

* Number of measurements (N)

2. Verified by:

3. Comments:

B.   Feed Consumption¹⁵ 

1. Observations:

Table # [Heading]

 

	Daily Feed Consumption (gram of feed consumed/day)
Sex	Males								Females
Targeted Daily Dose (mg/kg body-weight/day)	0 Control								0 Control
Number of Animals
	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD
0 week
1st week
2nd week
3rd week
4th week
5th week
6th week
7th week
8th week
9th week
10th week
11th week
12th week
13th week

X: Mean, SD: Standard Deviation

2. Comments:

C.   Body Weight¹⁶ 

1. Observations:

Table # [Heading]

	Body Weights (kg body weight/day)
Sex	Males								Females
Targeted Daily Dose (mg/kg body-weight/day)	0 CONTROL								0 CONTROL
Number Of Animals
	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD
0 week
1st week
2nd week
3rd week
4th week
5th week
6th week
7th week
8th week
9th week
10th week
11th week
12th week
13th week

X: Mean, SD: Standard Deviation

           

2. Comments:

D.   Intake Of Test Substance¹⁷ 

1. Observations:

            Table # [Heading]

	Daily Intake Of Test Material (mg/kg body-weight/day))
Sex	Males								Females
Targeted Daily Dose (mg/kg body-weight/day)	0 CONTROL								0 CONTROL
Number Of Animals
	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD	X	SD
0 week
1st week
2nd week
3rd week
4th week
5th week
6th week
7th week
8th week
9th week
10th week
11th week
12th week
13th week

X: Mean, SD: Standard Deviation

2. Comments:

E.   Feed Efficiency¹⁸ 

1.   Was feed efficiency calculated?
2.   Comments:

F.    Cage-Side Observations¹⁹ 

1.   Appearance
2.   Abnormal Stool
3.   Morbidity
4.   Signs of Neurotoxicity 
5.   Comments:

G.   Mortality²⁰ 

1.   Observations:
2.   Comments:

H.  Ophthalmological 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 Amino-transferase  (ALT or SGPT)	U/L
Aspartate Amino-transferase (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