Template for Chronic Toxicity Study with an in utero Phase

April 2005

Return to Toxicology Template Introduction

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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. MATING PROCEDURES FOR IN UTERO PHASE
   7. CULLING PROCEDURES FOR IN UTERO PHASE
   8. PARENTAL BODY WEIGHT AND FEED INTAKE FOR IN UTERO PHASE
   9. PARENTAL CAGE-SIDE OBSERVATIONS FOR IN UTERO PHASE
   10. PARENTAL REPRODUCTIVE PARAMETERS FOR IN UTERO PHASE
   11. OFFSPRING OBSERVATIONS AND BODY WEIGHT FOR IN UTERO PHASE
   12. BODY WEIGHT AND FEED INTAKE FOR CHRONIC PHASE
   13. CAGE-SIDE OBSERVATIONS FOR CHRONIC PHASE
   14. OPTHALMOLOGICAL EXAMINATION
   15. HEMATOLOGY
   16. CLINICAL CHEMISTRY
   17. URINALYSIS
   18. OTHER TESTS
   19. NECROPSY (INTERIM SACRIFICE)
   20. NECROPSY (TERMINAL)
   21. GROSS PATHOLOGY OBSERVATIONS
   22. HISTOPATHOLOGY OBSERVATIONS
   23. STATISTICAL METHODS
5. Results
   1. DOSE VERIFICATION
   2. INTAKE OF TEST SUBSTANCE
   3. PARENTAL FEED CONSUMPTION CHANGES FOR THE IN UTERO PHASE
   4. FEED EFFICIENCY FOR THE UTERO PHASE
   5. PARENTAL BODY WEIGHT CHANGES FOR THE IN UTERO PHASE
   6. PARENTAL ABNORMAL CAGE-SIDE OBSERVATIONS FOR THE IN UTERO PHASE
   7. MORTALITY IN ADULTS
   8. MATING, FERTILITY, AND REPRODUCTION
   9. OFFSPRING BODY WEIGHT CHANGES FOR IN UTERO PHASE
   10. OFFSPRING OBSERVATIONS FOR IN UTERO PHASE
   11. FEED CONSUMPTION CHANGES FOR THE CHRONIC PHASE
   12. FEED EFFICIENCY FOR THE CHRONIC PHASE
   13. BODY WEIGHT CHANGES FOR THE CHRONIC PHASE
   14. CAGE-SIDE OBSERVATIONS FOR THE CHRONIC PHASE
   15. MORTALITY IN ADULTS
   16. OPTHALMOLOGICAL EXAMINATION
   17. HEMATOLOGY
   18. CLINICAL CHEMISTRY
   19. URINALYSIS
   20. OTHER TESTS
   21. ORGAN WEIGHTS
   22. GROSS PATHOLOGY CHANGES OBSERVED
   23. HISTOPATHOLOGY CHANGES OBSERVED
   24. NEUROTOXICITY
6. Evaluation and Comments 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. IS THERE A TARGET ORGAN?
   4. NOEL
8. REFERENCES
9. APPENDIX
   1. HEMATOLOGY RESULTS
   2. CLINICAL CHEMISTRY RESULTS
   3. URINALYSIS RESULTS

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CHRONIC TOXICITY STUDY WITH AN IN UTERO PHASE² 

DATE OF SUBMISSION:
TITLE OF PETITION OR NOTIFICATION:
NAME AND ADDRESS OF PETITIONER OR NOTIFIER:

I. IDENTIFICATION OF STUDY³ 

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 PRACTICE⁴ 

A.    WAS THERE COMPLIANCE WITH GOOD LABORATORY PRACTICE REGULATIONS?
B.    WAS A QUALITY ASSURANCE (QA) STATEMENT INCLUDEDd?
C.    AVAILABILITY AND LOCATION OF ORIGINAL DATA/SPECIMENS/TEST SUBSTANCE:

III. EXECUTIVE SUMMARY⁵   

IV. MATERIALS AND METHODS

A. TEST SUBSTANCE⁶ 

1.     CAS NAME:
2.     OTHER NAME(S):
3.     CAS REGISTRY NUMBER:
4.     MOLECULAR STRUCTURE: ⁷ 
5.     PURITY:
6.     IMPURITIES:
7.     STABILITY:
8.     COMMENTS:

B. TEST SUBSTANCE AS ADMINISTERED⁸ 

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 ANIMALS⁹ 

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:

• TEMPERATURE:
• HUMIDITY:
• AIR CHANGES:
• PHOTOPERIOD:

9.     COMMENTS:

E. EXPERIMENTAL DESIGN¹⁰ 

1. TARGETED DOSE LEVELS; IN UTERO PHASE FOR PARENTAL ANIMALS.

TABLE # [HEADING]

TEST GROUP	CONC. IN DIET (ppm or mg/kg)	NUMBER OF MALES	DOSE TO MALES (mg/kg body-weight/day	NUMBER OF FEMALES	DOSE TO FEMALES (mg/kg body-weight/day)
CONTROL
LOW
MID
HIGH

2. TARGETED DOSE LEVELS FOR THE ANIMALS IN THE CHRONIC PHASE:

TABLE # [HEADING]

TEST GROUP	CONC. IN DIET (ppm or mg/kg)	NUMBER OF MALES	DOSE TO MALES (mg/kg body-weight/day)	NUMBER OF FEMALES	DOSE TO FEMALES (MG/KG BODY-WEIGHT/DAY)
CONTROL
LOW
MID
HIGH

3. TOTAL NUMBER OF ANIMALS:
4. DURATION OF STUDY (INCLUDING RECOVERY PERIOD, IF ANY):
5. LENGTH OF EXPOSURE TO TEST SUBSTANCE:
6. WERE ANIMALS RANDOMIZED
7. RECOVERY PERIOD:
8. COMMENTS:

IVA. MATERIALS AND METHODS - IN UTERO PHASE

F. MATING PROCEDURES FOR IN UTERO PHASE¹¹ 

1. DESCRIPTION:
2. COMMENT:

G. CULLING PROCEDURES FOR IN UTERO PHASE¹² 

1. DESCRIPTION:
2. COMMENT:

H. PARENTAL BODY WEIGHT AND FEED INTAKE FOR IN UTERO PHASE ¹³

1. PARAMETER EXAMINED:
2. FREQUENCY OF MEASUREMENT:

TABLE # [HEADING]

EXAMINED	NOT EXAMINED
	Feed Intake, Feed Spillage, Water Intake, Body Weight, Body Weight Changes

3. COMMENT:

I. PARENTAL CAGE-SIDE OBSERVATIONS FOR IN UTERO PHASE¹³

1. PARAMETER EXAMINED:
2. FREQUENCY OF MEASUREMENT:

TABLE # [HEADING]

EXAMINED	NOT EXAMINED
	Appearance, Abnormal Stool, Deficiencies in care** Morbidity, Mortality, Neurotoxicity Screening (Specify parameters)***

** Deficiencies in care (inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or inadequate lactation or feeding]
*** The parameters for neurotoxicity screening may include, but are not limited to, the following:

• Changes in skin, fur, eyes, mucous membranes, gut, 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.

3. COMMENT:

J. PARENTAL REPRODUCTIVE PARAMETERS FOR IN UTERO PHASE¹³ 

1. REPRODUCTIVE PARAMETERS EXAMINED:

TABLE # [HEADING]

EXAMINED	NOT EXAMINED
	Female fertility Index, Gestation Index, Gestation Length, Live-born Index, Number (Total and Per Litter) of Stillbirths and Live Births at Day 0

2. INDICES FORMULAS:
3. COMMENTS:

K. OFFSPRING OBSERVATIONS AND BODY WEIGHT FOR IN UTERO PHASE¹³

1. PARAMETER EXAMINED:
2. FREQUENCY OF MEASUREMENT:

TABLE # [HEADING]

OBSERVATION	EXAMINED	NOT EXAMINED
CAGE- SIDE		Appearance, Gross Anomalies, Morbidity, Mortality
OTHER		Litter size (Total: Day 0, 4, 7, 14, 21), Litter Weight (Day 0, 4, 7, 14, 21), Pup Body Weight and Sex (Day 0, 4, 7, 14, 21), Sex Ratio, Viability Index (Day 0-4, 4-7, 7-14, 14-21), Weaning Index

3. COMMENTS:

IVB. MATERIALS AND METHODS - CHRONIC PHASE

L. BODY WEIGHT AND FEED INTAKE FOR CHRONIC PHASE¹³

1. PARAMETER EXAMINED:
2. FREQUENCY OF MEASUREMENT:

TABLE # [HEADING]

EXAMINED*	NOT EXAMINED*
	Feed Intake, Feed Spillage, Water Intake, Body Weight, Body Weight Changes

*These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic toxicity studies.

3. COMMENTS:

M. CAGE-SIDE OBSERVATIONS FOR CHRONIC PHASE¹³ 

1. PARAMETER EXAMINED:
2. FREQUENCY OF MEASUREMENT:

TABLE # [HEADING]

EXAMINED*	NOT EXAMINED*
	Appearance, Abnormal Stool, Morbidity, Mortality, Neurotoxicity Screening (Specify parameters)**

* These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic 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.

3. COMMENTS:

N. OPTHALMOLOGICAL EXAMINATION

1. PARAMETER EXAMINED:
2. FREQUENCY OF MEASUREMENT:
3. COMMENTS:

O. HEMATOLOGY¹³ 

1. FASTING DURATION PRIOR TO BLOOD COLLECTION:
2. SCHEDULE INTERVAL FOR BLOOD SAMPLE COLLECTION:
3. METHOD OF BLOOD COLLECTION:
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 Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic toxicity studies.

6. Comment:

P. CLINICAL CHEMISTRY¹³ 

1.     FASTING DURATION PRIOR TO BLOOD COLLECTION:
2.     SCHEDULE INTERVAL FOR BLOOD SAMPLE COLLECTION:
3.     METHOD OF BLOOD COLLECTION:
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*,**
BLOOD SUGAR		Glucose*,**
liver function: A) HEPATO-CELLULAR (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 Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic toxicity studies.
** These parameters should generally be given priority when adequate volumes of blood samples can not be obtained from test animals.

6.     COMMENTS:

Q. URINALYSIS¹⁴ 

1.     Describe the frequency of urine sample collection:
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 Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic toxicity studies.

3.     COMMENTS:

R. OTHER TESTS¹⁵ 

1.     Observations:
2.     COMMENTS:

S. NECROPSY (INTERIM SACRIFICE)¹⁶ 

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 Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic toxicity studies.

4.     COMMENTS:

T. 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 Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic toxicity studies.

2.     COMMENTS:

 

U. GROSS PATHOLOGY OBSERVATIONS

1.     Organs/Tissues Examined:
2.     COMMENTS:

V. 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)
cardio-vascular		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 Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for chronic toxicity studies.

5.     COMMENTS:

W. STATISTICAL METHODS

1.     Methods of statistical analysis:

TABLE # [HEADING]

methods of statistical analysis	parameters tested

2.     COMMENTS:

V. Results

A. DOSE VERIFICATION¹⁷ 

1.     Were doses verified?

TABLE # [HEADING]

dose group	targeted concentration (ppm or mg/kg)	concentrations found in feed (ppm or mg/kg)	standard Deviation	N*
low
mid
high

* Number of measurements (N)

2.     Verified by:
3.     COMMENTS:

B. INTAKE OF TEST SUBSTANCE¹⁸

1.     Intake during in utero phase:

TABLE # [HEADING]

dose group	daily dose (mg/kg body-weight/day)
control	0
low
mid
high

2.     Intake during the chronic phase:

Table # [Heading]

dose group	daily dose (mg/kg body-weight/day)
control	0
low
mid
high

3.     COMMENTS:

Va. Results - In Utero Phase

C. PARENTAL FEED CONSUMPTION CHANGES FOR THE IN UTERO PHASE¹⁹ 

1.     Observations during Pre-mating and Post-mating:
2.     Observations during Gestation:
3.     Observations during Lactation:
4.     COMMENTS:

D. FEED EFFICIENCY FOR THE UTERO PHASE²⁰

1. Was feed efficiency calculated?
2. COMMENTS:

E. PARENTAL BODY WEIGHT CHANGES FOR THE IN UTERO PHASE²¹ 

1. Observations during Premating and Postmating:
2. Observations during Gestation:
3. Observations during Lactation:
4. COMMENTS:

F. PARENTAL ABNORMAL CAGE-SIDE OBSERVATIONS FOR THE IN UTERO PHASE²² 

1. Observations during Premating and Postmating:
2. Observations during Gestation:
3. Observations during Lactation:
4. COMMENTS:

G. MORTALITY IN ADULTS ²³ 

1.     Observations:
2.     COMMENTS:

H. MATING, FERTILITY, AND REPRODUCTION

1. Observations:

TABLE # [HEADING]

PREGNANCY

	Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)
Number of Females Paired
Number of Females Achieving Pregnancy
Female Fertility Index (%)

MATERNAL WASTAGE

	Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)
Number Died
Number Died Pregnant
Number Died Nonpregnant
Number Aborted
Number Premature Delivery

GESTATION

	Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)
Gestation Length	Mean (S.D.)
Gestation Index

CORPORA LUTEA

	Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)
Total Number Corpora Lutea
Corpora Lutea/Dam	Mean (S.D.)

IMPLANTATIONS

	Females
0 (Control)
Total Number Implantations
Implantations/Dam	Mean (S.D.)

BIRTHS - DAY 0

	Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)
Number Stillborn - Total Per Litter	Mean (SD) N
Number Live-Born - Total Per Litter	Mean (SD) N
Live-Born Index (%)

(Specify a method of statistical analysis): * p<0.05, **=""><>

2. COMMENTS:

I. OFFSPRING BODY WEIGHT CHANGES FOR IN UTERO PHASE²⁴

1. Observations:

 

TABLE # [HEADING]

Pup Weight (gm)

Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0	Mean (SD) N
Day 4	Mean (SD) N
Day 7	Mean (SD) N
Day 14	Mean (SD) N
Day 21	Mean (SD) N

(Specify a method of statistical analysis): * p<0.05, **=""><>

2. COMMENTS:

J. OFFSPRING OBSERVATIONS FOR IN UTERO PHASE ²⁵ 

1. Observations:

TABLE # [HEADING]

DAILY DOSE (mg/kg body-weight/day)	0 (Control)
LITTER SIZE
Number Born - Total Per Litter	Mean (SD) N
Day 0 - Total Per Litter	Mean (SD) N
Day 4 -Total Per Litter	Mean (SD) N
Day 7 -Total Per Litter	Mean (SD) N
Day 14 - Total Per Litter	Mean (SD) N
Day 21 - Total Per Litter	Mean (SD) N
LITTER WEIGHT (G)
Day 0	Mean (SD) N
Day 4	Mean (SD) N
Day 7	Mean (SD) N
Day 14	Mean (SD) N
Day 21	Mean (SD) N
VIABILITY INDICES
Day 0-4	Mean (SD) N
Day 4-7	Mean (SD) N
Day 7-14	Mean (SD) N
Day 14-21	Mean (SD) N
WEANING INDEX
SEX RATIO
Day 0
Day 21

(Specify a method of statistical analysis): * p<0.05, **=""><>

3.     COMMENTS:

Vb. Results - Chronic Phase

K. FEED CONSUMPTION CHANGES FOR THE CHRONIC PHASE²⁶

1. Observations:
2. feed consumption table (and/or graph)²⁷ :

TABLE # [HEADING]

FEED CONSUMPTION (gm feed/ kg body-weight/day)
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)			0 (Control)
Day 0	Mean (SD) N
Day A	Mean (SD) N
Day B	Mean (SD) N
Day C	Mean (SD) N
Day D	Mean (SD) N

(Specify a method of statistical analysis): * p<0.05, **=""><>

3. COMMENTS:

L. FEED EFFICIENCY FOR THE CHRONIC PHASE²⁸ 

1. Was feed efficiency calculated?
2. COMMENTS:

M. BODY WEIGHT CHANGES FOR THE CHRONIC PHASE²⁹ 

1. Observations:
2. body weight table (and/or graph)³⁰ :

TABLE # [HEADING]

body weight (gm/day)
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0	Mean (SD) N
Day A	Mean (SD) N
Day B	Mean (SD) N
Day C	Mean (SD) N
Day D	Mean (SD) N

(Specify a method of statistical analysis): * p<0.05, **=""><>

3. COMMENTS:

N. CAGE-SIDE OBSERVATIONS FOR THE CHRONIC PHASE

1. observation³¹ :

TABLE # [HEADING]

cage-side observation (specify parameters32 )
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0	N
Day A	N
Day B	N
Day C	N
Day D	N

N: Number of Animals with abnormal observation

(Specify a method of statistical analysis): * p<0.05, **=""><>

2. COMMENTS:

O. MORTALITY IN ADULTS³³

1. Observations:
2. mortality table (and/or graph)³⁴ :

TABLE # [HEADING]

mortality (%)
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0
Day A
Day B
Day C
Day D

(Specify a method of statistical analysis): * p<0.05, **=""><>

3. COMMENTS:

P. OPTHALMOLOGICAL EXAMINATION

1. Observations:
2. COMMENTS:

Q. HEMATOLOGY

1. Observations³⁵ :

TABLE # [HEADING]³⁶ 

(specify parameter)
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0	Mean (SD) N
Day A	Mean (SD) N
Day B	Mean (SD) N
Day C	Mean (SD) N
Day D	Mean (SD) N

(Specify a method of statistical analysis): * p<0.05, **=""><>

3. COMMENTS:

R. CLINICAL CHEMISTRY³⁷ 

1. Observations:

TABLE # [HEADING]³⁸ 

hematology results (specify parameter)
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0	Mean (SD) N
Day A	Mean (SD) N
Day B	Mean (SD) N
Day C	Mean (SD) N
Day D	Mean (SD) N

(Specify a method of statistical analysis): * p<0.05, **=""><>

2. COMMENTS:

S. URINALYSIS³⁹ 

1. Observations:

 

TABLE # [HEADING]⁴⁰ 

urinalysis results (specify parameter)
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0	Mean (SD) N
Day A	Mean (SD) N
Day B	Mean (SD) N
Day C	Mean (SD) N
Day D	Mean (SD) N

(Specify a method of statistical analysis): * p<0.05, **=""><>

2. COMMENTS:

T. OTHER TESTS⁴¹ 

1. Observations:

TABLE # [HEADING]⁴² 

specify parameter
Sex	Males				Females
DAILY DOSE (mg/kg body-weight/day)	0 (Control)				0 (Control)
Day 0	Mean (SD) N
Day A	Mean (SD) N
Day B	Mean (SD) N
Day C	Mean (SD) N
Day D	Mean (SD) N

(Specify a method of statistical analysis): * p<0.05, **=""><>

2. COMMENTS:

U. ORGAN WEIGHTS⁴³ 

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, **=""><>

2.     COMMENTS:

V. GROSS PATHOLOGY CHANGES OBSERVED⁴⁴ 

1.     Observations:
2.     COMMENTS:

W. HISTOPATHOLOGY CHANGES OBSERVED⁴⁵ 

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
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
RESPIRATORY SYSTEM
ORGAN/TISSUE #
GROSS LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
CARDIOVASCULAR SYSTEM
ORGAN/TISSUE #
GROSS LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
RETICULO-ENDOTHELIAL /HEMATOPOIETIC SYSTEM
ORGAN/TISSUE #
GROSS LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
UROGENITAL SYSTEM
ORGAN/TISSUE #
GROSS LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
GLANDULAR SYSTEM
ORGAN/TISSUE #
GROSS LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS

(Specify methods of statistical analysis): * p<0.05, **=""><>

# Organs/tissues listed under section IV.V.

In general, data at end of dosing period can be shown; however, if there were additional noteworthy findings at earlier timepoints, these should be included. Note severity of lesions as needed.

2.     COMMENTS:

X. NEUROTOXICITY⁴⁶ 

1.     Observations:

TABLE # [HEADING]

	NUMBER OF ANIMALS
SEX	males				females
DAILY DOSE (mg/kg body-weight/day)	0 (CONTROL)				0 (CONTROL)
NUMBER OF ANIMALS EXAMINED
OBSERVATIONS OF NERVOUS SYSTEM TOXICITY +
OBSERVATION
OBSERVATION
OBSERVATION
GROSS- AND HISTO-PATHOLOGY CHANGES IN THE NEUROLOGIC SYSTEM #
ORGAN/TISSUE
GROSS LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NON-NEOPLASTIC LESION
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS
NEOPLASTIC LESIONS

+ See under section IV.M for the types of observation for nervous system toxicity: List noteworthy findings. If additional parameters (other than those in the Template) showed noteworthy changes, these should be added to the tables. In general, include data at the end of the dosing period. However, if one observes additional noteworthy findings at earlier timepoints, these should be included. Footnotes should be used as needed to provide additional information about the tests or the results. Note the severity of the abnormal observations using the following scales; + Mild, ++ Moderate, and +++ Marked or other scales, as appropriate.

# Organs/tissues listed under section IV.V

2.     COMMENTS:

VI. Evaluation and Comment on Study⁴⁷ 

VII. Summary and Conclusions⁴⁸

A. BRIEF SUMMARY OF MAJOR FINDINGS FROM THE STUDY

B. RELATIONSHIP BETWEEN DOSE AND INCIDENCE/ SEVERITY OF LESIONS OR ABNORMALITIES

C. IS THERE A TARGET ORGAN?

D. NOEL

1.     Was there a no observed effect level?
2.     COMMENTS:

VIII. References

IX. Appendix

Vb.Q. HEMATOLOGY RESULTS

TABLE # [HEADING]

day of Sampling⁴⁹

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, **=""><>

Vb.R. CLINICAL CHEMISTRY RESULTS

TABLE # [HEADING]

day of Sampling⁴⁹

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, **=""><>

Vb.S. URINALYSIS RESULTS

TABLE # [HEADING]

day of Sampling⁴⁹

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

(Specify statistical method of analysis): * p<0.05, **=""><>

---

End Notes

This section includes some comments that are only relevant when using the Word template.

 ¹ Please refer to the Introduction to the Template for general instructions before you begin using this form

 ² Please refer to the Introduction to the Template for general instructions before you begin using this form

 ³Make note of: study file location (Volume, pages), study title/report #, testing facility name, publication dates of study, study objective, and comments, if needed.

 ⁴Indicate Yes or No for Questions A and B. However, you may want to elaborate on the type of GLP compliance (USFDA, OECD, etc.) or make other notations.

 ⁵The executive summary should be a brief, concise, stand alone summary describing the test system, the test protocol and the results. It contains three paragraphs: the first describing the test system, the second reporting and discussing the results and the third stating whether the study is acceptable or not. If not acceptable, state why not and what information could be supplied to make it acceptable. A typical executive summary should be less than one page long.

 ⁶Description of the test substance should be given, including purity, any possible contaminants or impurities, and any properties of the test substance that could affect its integrity. Were there factors that have affected the actual administered dose, as opposed to the intended dose?

 ⁷After you put a blinking cursor on the http address, (CTRL +) left-click once to follow the link and wait. Enter a chemical name, CAS Number, or molecular formula inside the search window and click 'search' button. Right-click inside the molecular structure and select 'copy' submenu. Return to your document and put the cursor underneath item #4 (molecular structure). Right click to open up the menu options and select 'paste' submenu. You can drag the structure to any position you want and resize.

If preferred, the reviewer is free to use other methods of depicting the molecular structure.

 ⁸Indicate how the test substance was given and whether any vehicle was used to dissolve/suspend the test substance (e.g., dissolved in corn oil and mixed into the feed). Also note if there was adequate testing for concentration and homogeneity (appropriate tests with replicates) and whether there were stability tests done. Was the strength of the test material checked over the period and under the conditions that the feed mix, test article in vehicle, etc., would be stored? Note anything that was not normal or routine.

 ⁹Note anything that might have affected the study. Use the comments to indicate extremes or other factors that might have impacted the study.

 ¹⁰Provide adequate details. Use the comments to indicate additional information about the experimental design. To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ¹¹Describe how animals were mated. Indicate when and how females were examined for pregnancy. Note if sibling mating was avoided.

 ¹²Standardization of the number of pups per litter is optional. If performed, describe how the litters were standardized (on what day, standardized to how many, if standardized in a random manner, etc.).

 ¹³Indicate the measurements or observations that were made by highlighting the parameter and dragging the term into the "Examined" side. Those parameters not examined will remain in the "Not Examined" side. Use the comments to indicate any other notable fact.

 ¹⁴Note when urine samples were collected for testing, the groups that were affected, and drag and drop the parameters that were tested. Make comments on anything significant or treatment-related.

 ¹⁵When other tests are conducted, make note of the tests and any significant treatment-related effects.

 ¹⁶If there was an interim sacrifice (e.g., at 3 month intervals), make notes on which groups were affected, and the number and sex of the animals that were tested.

 ¹⁷Note here whether there was a check or verification of the doses being administered (by what analytical methodology and in which laboratory). If the test substance was mixed in the feed, was there a verification for substance concentration in the feed? Was this verification done more than once? Were there adequate data to permit the calculation of the actual dose that was administered?

 ¹⁸Knowing what the intake of feed was and the level of test substance in the feed, what was the dose actually being delivered to the animals?

 ¹⁹Note any statistically or biologically significant feed consumption changes. Note feed/water consumption changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend.

 ²⁰Note in this section if feed efficiency was determined; when in the study determinations were made; if any changes were observed; if feed efficiency was calculated for all groups or only selected groups; and if calculations were made per group, per cage, or per animal.

 ²¹Note any statistically or biologically significant body weight changes. Note body weight changes that are not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. You may wish to insert graphs or tables in this section.

 ²²List significant, dose-related abnormal cage-side observations reported. This may include neurotoxicity endpoints.

 ²³For each animal with an unscheduled death, note the group, date of death, and any observations, including lesions observed at necropsy.

 ²⁴Note any statistically or biologically significant body weight changes. Also note body weight changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. The comment section is available to explain data findings, if needed.

²⁵List significant, dose-related abnormal observations reported.

²⁶Note any statistically or biologically significant feed consumption changes. Also note feed consumption changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend.

²⁷To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

²⁸Note in this section if feed efficiency was determined; when in the study determinations were made; if any changes were observed; if feed efficiency was calculated for all groups or only selected groups; and if calculations were made per group, per cage, or per animal.

 ²⁹Note any statistically or biologically significant body weight changes. Also note body weight changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. You may wish to insert graphs or tables in this section.

 ³⁰To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ³¹To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ³²List significant, dose-related abnormal cage-side observations reported. For neurotoxicological observations, also use a Table in Section V. X. (see Section IV. M. for parameters).

 ³³For each animal with an unscheduled death, note the group, date of death, and any observations, including lesions observed at necropsy.

 ³⁴To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ³⁵Note findings that were statistically or biologically treatment-related. Also note changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. The comment section is available to explain data findings, if needed. Note detailed results in Appendix.

 ³⁶To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ³⁷Note findings that were statistically or biologically treatment-related. Also note changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. The comment section is available to explain data findings, if needed. Note detailed results in Appendix.

 ³⁸To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ³⁹Note treatment-related findings that were statistically or biologically significant. Also note changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. The comment section is available to explain data findings, if needed. Note detailed results in Appendix.

 ⁴⁰To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ⁴¹When other tests are conducted, make note of the tests and any significant treatment-related effects.

 ⁴²To change the table, place cursor in the row or column that you wish to modify, then from the 'Table' menu, use 'Select Row' or 'Select Column' to highlight the row or column that you wish to change, and use the 'delete' or 'insert' functions to make your changes.

 ⁴³Note treatment-related findings that were statistically or biologically significant. Also note changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. The comment section is available to explain data findings, if needed.

 ⁴⁴Note treatment-related findings that were statistically or biologically significant. Also note changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. The type of lesion should be noted. The comment section is available to explain data findings, if needed. Also note the findings in Histopathology Section V.W. below.

 ⁴⁵Note treatment-related findings that were statistically or biologically significant. Also note changes that were not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. The type of lesion should be noted. The comment section is available to explain data findings, if needed.

 ⁴⁶ Summary tables of all positive effects should be prescribed. In addition, provide an explicit statement as to whether or not the test substance represents a potential neurotoxic hazard.

 ⁴⁷Give your evaluation of the study: what was done well; what the problems were; did the study accomplish what it was supposed to do?

 ⁴⁸Summarize the key findings from the study. Was there a dose-effect relationship? Were target organs identified? Was a NOEL achieved for each significant effect/observation? What was the NOEL? The whole study should be summarized in this section.

 ⁴⁹Specify day of sampling (e.g., day 0, day 90). And, create separate tables for each interval.

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