Food

Introduction to the 2-Generation Reproduction Toxicity Study Template

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. MATING PROCEDURES
    7. CULLING PROCEDURES
    8. PARENTAL BODY WEIGHT AND FEED/WATER INTAKE
    9. PARENTAL CAGE-SIDE OBSERVATIONS
    10. PARENTAL REPRODUCTIVE PARAMETERS
    11. PARENTAL ORGAN WEIGHTS
    12. PARENTAL GROSS PATHOLOGY OBSERVATIONS
    13. PARENTAL HISTOPATHOLOGY OBSERVATIONS
    14. OTHER PARENTAL TESTS
    15. OFFSPRING OBSERVATIONS/BODY WEIGHT
    16. OFFSPRING DEVELOPMENTAL ENDPOINTS AND NEUROTOXICITY SCREENING - OPTIONAL
    17. OFFSPRING IMMUNOTOXICITY - OPTIONAL
    18. OFFSPRING ORGAN WEIGHTS
    19. OFFSPRING GROSS PATHOLOGY OBSERVATIONS
    20. OFFSPRING HISTOPATHOLOGY OBSERVATIONS
    21. OTHER OFFSPRING TESTS
    22. STATISTICAL METHODS
  5. Results
    1. DOSE VERIFICATION
    2. PARENTAL FEED/WATER CONSUMPTION CHANGES
    3. PARENTAL INTAKE OF TEST MATERIAL
    4. PARENTAL FEED EFFICIENCY
    5. PARENTAL BODY WEIGHT CHANGES
    6. PARENTAL ABNORMAL CAGE-SIDE OBSERVATIONS
    7. PARENTAL MORTALITY
    8. MATING, FERTILITY, AND REPRODUCTION
    9. PARENTAL ORGAN WEIGHTS
    10. PARENTAL GROSS PATHOLOGY CHANGES OBSERVED
    11. PARENTAL HISTOPATHOLOGY CHANGES OBSERVED
    12. OTHER PARENTAL TESTS
    13. OFFSPRING BODY WEIGHT CHANGES
    14. OFFSPRING OBSERVATIONS
    15. OFFSRPING MORTALITY
    16. SEXUAL MATURATION
    17. ANOGENITAL DISTANCE
    18. OFFSPRING DEVELOPMENTAL ENDPOINTS AND NEUROTOXICITY - OPTIONAL
    19. OFFSPRING IMMUNOTOXICITY - OPTIONAL
    20. OFFSPRING ORGAN WEIGHTS
    21. OFFSPRING GROSS PATHOLOGY CHANGES OBSERVED
    22. OFFSPRING HISTOPATHOLOGY CHANGES OBSERVED
    23. OTHER OFFSPRING TESTS
  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. NOEL
  8. References

Two-Generation Reproduction Toxicity Study [1]

Date of Submission:

Title of Petition or Notification:

Name and Address of Petitioner or Notifier:

I. Identification of Study [2]

A Study File Location :
B. Study Title/Report Number:
C. Study Author:
D. Name and Address of Testing Facility:
E. Study Initiation Date [3]:
F. Study Completion Date [4]:
G. Study Objective:
H. Comments:

II. Good Laboratory Practice [5]

A. Good Laboratory Practice (GLP) Compliance Statement? [6]
B. Quality Assurance (QA) Statement? [7]
C. Availability and Location of Original Data/Specimens/Test Substance:
D. Describe any changes to the protocol that would affect the quality or integrity of the study:
E. Comments:

III. Executive Summary

IV. Materials and Methods

A. TEST SUBSTANCE [8]

1. CAS name:
2. Other name(s):
3. CAS number:
4. Molecular structure: http://www.chemfinder.com/[9]

5. Purity:
6. Impurities:
7. Stability:
8. Comments:

B. TEST SUBSTANCE AS ADMINISTERED [10]

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 [11]

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 ANIMALS [12]

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

  • Temperature:
  • Humidity:
  • Air changes:
  • Photoperiod:
  • Bedding:

11. Comments:

E. EXPERIMENTAL DESIGN [13]

1. Targeted dose levels:
2. Dose Selection Rationale:
3. Dosage Preparation: 

Table # [Heading]

test group- conc. in diet
(ppm or mg/kg)
dose to animals*
(mg/kg body-weight/day)
number assigned per group
f0 males f0 females f0 males f0 females
Control          
Low          
Mid          
High          
*Premating dose

Table # [Heading]

test group conc. in diet
(ppm or mg/kg)
dose to animals*
(mg/kg body-weight/day)
number assigned per group
f1 males f1 females f1 males f1 females
Control          
Low          
Mid          
High          
*Premating dose

4. Total number of parental animals:
5. Duration of study (including recovery period, if any):
6. Length of exposure to test substance:
7. Were animals assigned to test and control groups in a stratified random manner to minimize intergroup weight differences?
8. Comments: 

F. MATING PROCEDURES [14]

1. Description:
2. Comments: 

G. CULLING PROCEDURES [15]

1. Description:
2. Comments: 

H. PARENTAL BODY WEIGHT AND FEED/WATER INTAKE [16]

1. Parameters examined (in F0 and F1 animals): 

Table # [Heading]

examined parameters*
f0 parents f1 parents
    Body Weight
    Body Weight Changes
    Feed Intake
    Feed Spillage
    Water Intake
*These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for 2-generation reproduction toxicity studies.

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

I. PARENTAL CAGE-SIDE OBSERVATIONS [17]

1. Parameters examined (in F0 and F1 animals):

Table # [Heading]

examined parameters*
f0 parents f1 parents
    Appearance
    Abnormal Stool
    Deficiencies in Care**
    Morbidity
    Mortality
    Neurotoxicity Screening (specify parameters)***

*These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for 2-generation reproduction toxicity studies.

**Deficiencies in care include the following: 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, 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:

J. PARENTAL REPRODUCTIVE PARAMETERS [18]

1. Reproductive parameters examined (in F0 and F1 animals): 

Table # [Heading]

examined parameters*
f0 parents f1 parents
    Estrous Cycling
    Female Fertility Index
    Gestation Index
    Gestation Length
    Live-born Index
    Number of Primordial Follicles
    Number of Growing Follicles
    Number of Large Corpora Lutea of Lactation from the Post-Lactation Ovary
    Number (Total and Per Liter) of Stillbirths at Day 0
    Number (Total and Per Liter) of Live Births at Day 0
    Number of Implantation Sites
    Number of Resorptions (Total, Early, and Late)
    Sperm Count
    Sperm Motility
    Sperm Morphology

* These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for 2-generation reproduction toxicity studies.

2. Indices Formulas:
3. Methods used for Sperm Analysis:
4. Comments:

K. PARENTAL ORGAN WEIGHTS [19]

1. Organs/Tissues weighed (in F0 and F1 animals):

Table # [Heading]

examined parameters*
f0 parents f1 parents
    Adrenals
    Brain
    Epididymides (single and total weight)
    Kidneys
    Known Target Organs
    Liver
    Ovaries
    Pituitary Gland
    Prostate (ventral and/or dorsal and/or dorsolateral)
    Seminal Vesicles (with coagulating glands)
    Spleen
    Testes (both)
    Thymus
    Uterus

* These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for 2-generation reproduction toxicity studies.

2. Comments:

L. PARENTAL GROSS PATHOLOGY OBSERVATIONS

f0 animals

1. Organs/Tissues Examined:
2. Comments:

f1 animals

1. Organs/Tissues Examined:
2. Comments: 

M. PARENTAL HISTOPATHOLOGY OBSERVATIONS [20]

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 (in F0 and F1 animals): 

Table # [Heading]

SYSTEM examined in
f0 parents*
not examined in
f0 parents*
examined in
f1 parents*
not examined in
f1 parents*
digestive
  Liver, Stomach   Liver, Stomach
reticulo- endothelial/hematopoietic 
  Bone Marrow, Lymph Nodes, Peyer's Patch, Spleen, Thymus   Bone Marrow, Lymph Nodes, Peyer's Patch, Spleen, Thymus
urogenital  
  Epididymis, Kidneys, Ovaries, Primordial Follicles, Growing Follicles, and Large Corpora Lutea of Lactation from the Post-Lactation Ovary, Prostate, Seminal Vesicle with Coagulating Gland (if present), Testes, Uterus (with oviducts), Vagina   Epididymis, Kidneys, Ovaries, Primordial Follicles, Growing Follicles, and Large Corpora Lutea of Lactation from the Post-Lactation Ovary, Prostate, Seminal Vesicle with Coagulating Gland (if present), Testes, Uterus (with oviducts), Vagina
neurologic
  Brain (at least 3 different levels), Peripheral Nerve Tissue, Spinal Cord (at least 2 different locations)   Brain (at least 3 different levels), Peripheral Nerve Tissue, Spinal Cord (at least 2 different locations)
glandular
  Adrenals, Pituitary Gland, Thymus   Adrenals, Pituitary Gland, Thymus
other
  Target Organs   Target Organs

* These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for 2-generation reproduction toxicity studies.

5.    Comments:

N. OTHER PARENTAL TESTS [21]

1. Observations:
2. Comments: 

O. OFFSPRING OBSERVATIONS/BODY WEIGHT [22]

1. Parameters examined (in F1 and F2 offspring):[23] 

Table # [Heading]

observations examined in
f1 offspring*
not examined
in
f1 offspring*
examined in
f2 offspring*
not examined
in
f2 offspring*
cage-side
  Appearance
Gross Anomalies
Morbidity
Mortality
  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 (Day 0, 4, 7, 14, 21)
Viability Index (Day 0-4, 4-7, 7-14, 14-21)
Weaning Index
  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 (Day 0, 4, 7, 14, 21)
Viability Index (Day 0-4, 4-7, 7-14, 14-21)
Weaning Index

*These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for 2-generation reproduction toxicity studies.

2. Offspring Indices:
3. Sexual Maturation (in F1 offspring only):
4. Anogenital Distance (in F2 offspring only)[24] :
5. Comments: (e.g., list frequency)

P. OFFSPRING DEVELOPMENTAL ENDPOINTS AND NEUROTOXICITY SCREENING - OPTIONAL [25]

1. Parameters examined (in F1 and F2 offspring): 

Table # [Heading]

observations examined in
f1 offspring
not examined in
f1 offspring
examined in
f2 offspring
not examined in
f2 offspring
developmental endpoints
  Detachment of Pinna
Eye OpeningTooth Eruption
  Detachment of Pinna
Eye OpeningTooth Eruption
gross neurotoxicity screening
  Pupil Reflex
Startle Reflex
Surface and/or Mid-air ReflexVibrissae Placing
  Pupil Reflex
Startle Reflex
Surface and/or Mid-air ReflexVibrissae Placing

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

Q. OFFSPRING IMMUNOTOXICITY - OPTIONAL [26]

1. Parameters examined: 

Table # [Heading]

measurement related to examined in
f1 offspring
not examined in
f1 offspring
examined inf2 offspring not examined inf2 offspring
white blood cells 
  Basophils
Eosinophils
Lymphocytes
Macrophage/Monocytes
Neutrophils
Total Leukocytes (WBC)
  Basophils
Eosinophils
Lymphocytes
Macrophage/Monocytes
Neutrophils
Total Leukocytes (WBC)
organs/tissues(weight changes)
  Lymph Nodes
Peyer's Patch
Spleen
Thymus
  Lymph Nodes
Peyer's Patch
Spleen
Thymus
others 
  Acute Phase Protein
Albumin
Albumin-to-Globulin Ratio
Bone marrow cytology
Electrophoretic Analysis of Serum Proteins
Globulin
Immunoglobulin G
Immunostaining of Spleen and Lymph Nodes for B and T Cells
Total Serum Protein
  Acute Phase Protein
Albumin
Albumin-to-Globulin Ratio
Bone marrow cytology
Electrophoretic Analysis of Serum Proteins
Globulin
Immunoglobulin G
Immunostaining of Spleen and Lymph Nodes for B and T Cells
Total Serum Protein

2. Fasting duration prior to blood collection:
3. When in the study were the blood samples collected?
4. How were the blood samples drawn?
5. Comments: 

R. OFFSPRING ORGAN WEIGHTS [27]

1. Organs/Tissues weighed: 

Table # [Heading]

examined parameters*
f1 offspring f2 offspring
    Brain
    Spleen
    Thymus

*These parameters are recommended in Toxicological Principles for the Safety of Food Ingredients: Redbook 2000 for 2-generation reproduction toxicity studies.

2. Comments: 

S. OFFSPRING GROSS PATHOLOGY OBSERVATIONS

f1 offspring

1. Organs/Tissues Examined:
2. Comments: 

f2 offspring

1. Organs/Tissues Examined:
2. Comments: 

T. OFFSPRING HISTOPATHOLOGY OBSERVATIONS [28]

f1 offspring

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:
5. Comments: 

f2 offspring

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:
5. Comments: 

U. OTHER OFFSPRING TESTS [29]

1. Observations:
2. Comments: 

V. STATISTICAL METHODS

1. Methods of statistical analysis: 

Table # [Heading]

methods of statistical analysis parameters tested
   
   
   
   

2. Comments: 

V. Results

A. DOSE VERIFICATION [30]

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. PARENTAL FEED/WATER CONSUMPTION CHANGES [31]

f0 parents

1. Observations during Premating and Postmating (males):
2. Observations during Premating (female):
3. Observations during Gestation (female):
4. Observations during Lactation (female):
5. Comments: 

f1 parents

1.    Observations during Premating and Postmating (males):
2.    Observations during Premating (female):
3.    Observations during Gestation (female):
4.    Observations during Lactation (female):
5.    Comments: 

C. PARENTAL INTAKE OF TEST MATERIAL [32] 

1. Observations: 

Table # [Heading]

dose group f0 males
DAILY DOSE
(mg/kg body-weight/day)
f0 females
daily dose
(mg/kg body-weight/day)
f1 males
daily dose
(mg/kg body-weight/day)
f1 females
daily dose
(mg/kg body-weight/day)
control 0 0 0 0
low        
mid        
high        

2. Comments: 

D. PARENTAL FEED EFFICIENCY [33]  

f0 parents

1. Was feed efficiency calculated?
2. Comments: 

f1 parents

1.    Was feed efficiency calculated?
2.    Comments: 

E. PARENTAL BODY WEIGHT CHANGES [34] 

f0 parents

1. Observations during Premating and Postmating (males):
2. Observations during Premating (female):
3. Observations during Gestation (female):
4. Observations during Lactation (female):
5. Comments: 

f1 parents

1.    Observations during Premating and Postmating (males):
2.    Observations during Premating (female):
3.    Observations during Gestation (female):
4.    Observations during Lactation (female):
5.    Comments: 

F. PARENTAL ABNORMAL CAGE-SIDE OBSERVATIONS [35] 

f0 parents

1. Observations:
2. Comments: 

f1 parents

1. Observations:
2. Comments:

G. PARENTAL MORTALITY[36] 

f0 parents

1.    Observations:
2.    Comments: 

f1 parents

1. Observations:
2. Comments:

H. MATING, FERTILITY, AND REPRODUCTION

1.    Observations: 

Table # [Heading]

pregnancy
 GENERATION  F0  F1
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
Number of Females Paired                
Number of Females Achieving Pregnancy                
Female Fertility Index (%)                
 Maternal Wastage
 GENERATION  F0  F1
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
# Died                
# Died Pregnant                
# Died Nonpregnant                
# Aborted                
# Premature Delivery                
 corpora lutea
 GENERATION  F0  F1
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
Total # Corpora Lutea (N)                
Corpora Lutea/Dam (MEAN S.D.)                
 
implantations
 GENERATION  F0  F1
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
Total # Implantations (N)                
Implantations/Dam (MEAN
S.D.
)
               
 
births - day 0
 GENERATION  F0  F1
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
Number Stillborns - Total Per Liter (MEAN
S.D.
)
               
Number Live-born - Total Per Liter (MEAN
S.D.
)
               
Live-born Index (%)                

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

2.    Sperm Evaluation:

Table # [Heading]

 GENERATION  F0  F1
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
% Motile Sperm (N MEAN S.D.)
               
% Progressively Motile Sperm (N MEAN S.D.)
               
% Abnormal Sperm (N MEAN S.D.)
               
# Homogenization Resistant Spermatids per Testis (N MEAN S.D.)
               
# Homogenization Resistant Spermatids per mg Testis (N MEAN S.D.)
               
# Sperm per Cauda (N MEAN S.D.)
               
# Sperm per mg Cauda (N MEAN S.D.)
               

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

2.    Estrous Cycling Evaluation:
3.    Comments: 

I. PARENTAL ORGAN WEIGHTS [37] 

1.    Observations: 

Table # [Heading]

 SEX  f0 males  f0 females
 DAILY DOSE
(mg/kg body-weight/day)
 0CONTROL           0 CONTROL         
NUMBER OF ANIMALS                
BODY WEIGHT (gram)[a]                
BRAIN                
Absolute Weight[a] gram                
Per Body Weight[a] %                
ADRENALS                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
EPIDIDYMIDES[b]          
Absolute Weight[a] gram        
Per Body Weight[a] %        
Per Brain Weight[a] %        
KIDNEYS                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
LIVER                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
PITUITARY GLAND                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
PROSTATE[b]          
Absolute Weight[a] gram        
Per Body Weight[a] %        
Per Brain Weight[a] %        
SEMINAL VESICLES WITH COAGULATING GLAND[b]          
Absolute Weight[a] gram        
Per Body Weight[a] %        
Per Brain Weight[a] %        
SPLEEN                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
TESTES[b]          
Absolute Weight[a] gram        
Per Body Weight[a] %        
Per Brain Weight[a] %        
THYMUS                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
OVARIES[b]          
Absolute Weight[a] gram        
Per Body Weight[a] %        
Per Brain Weight[a] %        
UTERUS[b]          
Absolute Weight[a] gram        
Per Body Weight[a] %        
Per Brain Weight[a] %        

a: Group means at the end of terminal necropsy are shown.
b: Sexual organ weights should be recorded regardless of significant findings.
(Specify methods of statistical analysis): * p<0.05, ** p<0.01 

Table # [Heading] 

 SEX  f1 males  f1 females
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0 CONTROL         
NUMBER OF ANIMALS                
BODY WEIGHT (gram)[a]                
 BRAIN                
Absolute Weight[a]
gram                
Per Body Weight[a]
%                
ADRENALS
               
Absolute Weight[a]
gram                
Per Body Weight[a]
%                
Per Brain Weight[a]
%                
EPIDIDYMIDES[b]
         
Absolute Weight[a]
gram        
Per Body Weight[a]
%        
Per Brain Weight[a]
%        
KIDNEYS
               
Absolute Weight[a]
gram                
Per Body Weight[a]
%                
Per Brain Weight[a]
%                
LIVER
               
Absolute Weight[a]
gram                
Per Body Weight[a]
%                
Per Brain Weight[a]
%                
PITUITARY GLAND
               
Absolute Weight[a]
gram                
Per Body Weight[a]
%                
Per Brain Weight[a]
%                
PROSTATE[b]
         
Absolute Weight[a]
gram        
Per Body Weight[a]
%        
Per Brain Weight[a]
%        
SEMINAL VESICLES WITH COAGULATING GLAND[b]
         
Absolute Weight[a]
gram        
Per Body Weight[a]
%        
Per Brain Weight[a]
%        
SPLEEN
               
Absolute Weight[a]
gram                
Per Body Weight[a]
%                
Per Brain Weight[a]
%                
TESTES[b]
         
Absolute Weight[a]
gram        
Per Body Weight[a]
%        
Per Brain Weight[a]
%        
THYMUS
               
Absolute Weight[a]
gram                
Per Body Weight[a]
%                
Per Brain Weight[a]
%                
OVARIES[b]
         
Absolute Weight[a]
gram        
Per Body Weight[a]
%        
Per Brain Weight[a]
%        
UTERUS[b]
         
Absolute Weight[a]
gram        
Per Body Weight[a]
%        
Per Brain Weight[a]
%        

a: Group means at the end of terminal necropsy are shown.
b: Sexual organ weights should be recorded regardless of significant findings.
(Specify methods of statistical analysis): * p<0.05, ** p<0.01

1. Comments:

J. PARENTAL GROSS PATHOLOGY CHANGES OBSERVED [38] 

f0 parents

1. Observations:
2. Comments: 

f1 parents

1. Observations:
2. Comments:

K. PARENTAL HISTOPATHOLOGY CHANGES OBSERVED [39] 

1.    Observations: 

Table # [Heading]

 SEX  F0 MALES  F0 FEMALES
 DAILY DOSE
(mg/kg body-weight/day)
  0
CONTROL
           0
CONTROL
        
number of animals                
digestive system
               
name of organ/tissue[#]                
gross pathology
               
histopathology-number of animals w/non-neoplasticlesions
               
lesion
               
lesion
               
                 
number of animals w/
neoplastic lesions
               
lesion
               
lesion
               
                 
reticuloendothelial/
hematopoietic system
               
name of organ/tissue[#]
               
gross pathology
               
histopathology-number of animals w/non-neoplasticlesions
               
lesion
               
lesion
               
                 
number of animals w/neoplastic lesions
               
lesion
               
lesion
               
                 
urogenital system
               
name of organ/tissue[#]
               
gross pathology
               
histopathology-number of animals w/non-neoplasticlesions
               
lesion
               
lesion
               
                 
number of animals w/neoplastic lesions
               
lesion
               
lesion
               
                 
neurologic system
               
name of organ/tissue#
               
gross pathology
               
histopathology-number of animals w/non-neoplasticlesions
               
lesion
               
lesion
               
                 
number of animals w/neoplastic lesions
               
lesion
               
lesion
               
                 
glandular system
               
name of organ/tissue#
               
gross pathology
               
histopathology-number of animals w/non-neoplasticlesions
               
lesion
               
lesion
               
                 
number of animals w/neoplastic lesions
               
lesion
               
lesion
               
                 
other
               
name of organ/tissue#
               
gross pathology
               
histopathology-number of animals w/non-neoplasticlesions
               
lesion
               
lesion
               
                 
number of animals w/neoplastic lesions
               
lesion
               
lesion
               

(Specify methods of statistical analysis): * p<0.05, ** p<0.01
# Organs/tissues listed under section IV.M.
In general, data at end of dosing period can be shown; however, if there were additional noteworthy findings at earlier time points, these should be included. Note severity of lesions as needed.

Table # [Heading]

 SEX  F1 MALES  F1 FEMALES
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
number of animals                
digestive system                
name of organ/tissue[#]                
gross pathology                
histopathology-number of animals w/non-neoplasticlesions                
lesion                
lesion                
                 
number of animals w/neoplastic lesions                
lesion                
lesion                
                 
reticuloendothelial/hematopoietic system                
name of organ/tissue[#]                
gross pathology                
histopathology-number of animals w/non-neoplasticlesions                
lesion                
lesion                
                 
number of animals w/neoplastic lesions                
lesion                
lesion                
                 
urogenital system                
name of organ/tissue[#]                
gross pathology                
histopathology-number of animals w/non-neoplasticlesions                
lesion                
lesion                
                 
number of animals w/neoplastic lesions                
lesion                
lesion                
                 
neurologic system                
name of organ/tissue#                
gross pathology                
histopathology-number of animals w/non-neoplasticlesions                
lesion                
lesion                
                 
number of animals w/neoplastic lesions                
lesion                
lesion                
                 
glandular system                
name of organ/tissue#                
gross pathology                
histopathology-number of animals w/non-neoplasticlesions                
lesion                
lesion                
                 
number of animals w/neoplastic lesions                
lesion                
lesion                
                 
other                
name of organ/tissue#                
gross pathology                
histopathology-number of animals w/non-neoplasticlesions                
lesion                
lesion                
                 
number of animals w/neoplastic lesions                
lesion                
lesion                

(Specify methods of statistical analysis): * p<0.05, ** p<0.01
# Organs/tissues listed under section IV.M.
In general, data at end of dosing period can be shown; however, if there were additional noteworthy findings at earlier time points, these should be included. Note severity of lesions as needed.

2. Comments:

L. OTHER PARENTAL TESTS [40] 

1. Observations:
2. Comments: 

M. OFFSPRING BODY WEIGHT CHANGES [41] 

1.    Observations: 

Table # [Heading]

 GENERATION    F1 MALES  F1 FEMALES
 DAILY DOSE
(mg/kg body-weight/day)
   0
CONTROL
       0
CONTROL
     
pup weight (g)                  
Day 0
mean
S.D.
N
               
Day 4
mean
S.D.
N
               
Day 7
mean
S.D.
N
               
Day 14
mean
S.D.
N
               
Day 21
mean
S.D.
N
               

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

Table # [Heading]

 GENERATION    F2 MALES  F2 FEMALES
 DAILY DOSE
(mg/kg body-weight/day)
   0
CONTROL
       0
CONTROL
     
pup weight (g)                  
Day 0
mean
S.D.
N
               
Day 4
mean
S.D.
N
               
Day 7
mean
S.D.
N
               
Day 14
mean
S.D.
N
               
Day 21
mean
S.D.
N
               

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

2. Comments:

N. OFFSPRING OBSERVATIONS [42] 

1. Observations:

Table # [Heading]

 GENERATION    F1 LITTER  F2 LITTER
 DAILY DOSE
(mg/kg body-weight/day)
   0
CONTROL
       0
CONTROL
     
litter size                  
Number Born - TotalPer Litter N
MEAN
S.D.
               
Day 0 - TotalPer Litter N
MEAN
S.D.
               
Day 4 - TotalPer Litter N
MEAN
S.D.
               
Day 7 - TotalPer Litter N
MEAN
S.D.
               
Day 14 - TotalPer Litter N
MEAN
S.D.
               
Day 21 - TotalPer Litter N
MEAN
S.D.
               
litter weight (g)                  
Day 0 N
MEAN
S.D.
               
Day 4 N
MEAN
S.D.
               
Day 7 N
MEAN
S.D.
               
Day 14 N
MEAN
S.D.
               
Day 21 N
MEAN
S.D.
               
viability Indices                  
Day 0-4 N
MEAN
S.D.
               
Day 4-7 N
MEAN
S.D.
               
Day 7-14 N
MEAN
S.D.
               
Day 14-21 N
MEAN
S.D.
               
weaning Index N
MEAN
S.D.
               
Sex Ratio                  
Day 1                  
Day 21                  

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

2. Comments:

O. OFFSPRING MORTALITY [43] 

f1 offspring

1. Observations:
2. Comments: 

f2 offspring

1. Observations:
2. Comments:

P. SEXUAL MATURATION

1. Observations (in F1 offspring only): 

Table # [Heading]

 GENERATION    F1 MALES  F1 FEMALES
 DAILY DOSE
(mg/kg body-weight/day)
   0
CONTROL
       0
CONTROL
     
Age of Preputial separation mean
S.D.
N
         
Age of vaginal opening mean
S.D.
N
         

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

2. Comments: 

Q. ANOGENITAL DISTANCE [44] 

3. Observations (in F2 offspring only):
4. Comments: 

R. OFFSPRING DEVELOPMENTAL ENDPOINTS AND NEUROTOXICITY - OPTIONAL [45] 

f1 offspring

1. Developmental Endpoints: 

  • Detachment of Pinna:
  • Eye Opening:
  • Tooth Eruption:

2. Gross Neurotoxicity Observations: 

  • Pupil Reflex:
  • Startle Reflex:
  • Surface and/or Mid-air Reflex:
  • Vibrissae Placing:

3. Comments: 

f2 offspring

1. Developmental Endpoints: 

  • Detachment of Pinna:
  • Eye Opening:
  • Tooth Eruption:

2. Gross Neurotoxicity Observations: 

  • Pupil Reflex:
  • Startle Reflex:
  • Surface and/or Mid-air Reflex:
  • Vibrissae Placing:

3. Comments: 

S. OFFSPRING IMMUNOTOXICITY - OPTIONAL [46] 

1.    Observations: 

Table # [Heading] 

 SEX  f1 males  f1 females
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
NUMBER OF ANIMALS                
white blood cells                
Basophils                  
Eosinophils                  
Lymphocytes 10[9]/L                
Macrophage/Monocytes                  
Neutrophils 10[9]/L                
Total Leukocytes (WBC) 10[9]/L                
others                
Acute phase proteins                  
Total Serum Protein                  
Albumin                  
Albumin-to-Globulin Ratio                  
Bone marrow cytology                  
Globulin                  
Immunoglobulin G                  

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

Table # [Heading]

 SEX  f2 males  f2 females
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
NUMBER OF ANIMALS                
white blood cells                
Basophils                  
Eosinophils                  
Lymphocytes 10[9]/L                
Macrophage/Monocytes                  
Neutrophils 10[9]/L                
Total Leukocytes (WBC) 10[9]/L                
others                
Acute phase proteins                  
Total Serum Protein                  
Albumin                  
Albumin-to-Globulin Ratio                  
Bone marrow cytology                  
Globulin                  
Immunoglobulin G                  

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

2.    Noted Organ/Tissue Weight Changes: 

  • Lymph Nodes:
  • Peyer's Patches:
  • Spleen:
  • Thymus:

3.    Electrophoretic Analysis of Serum Proteins:
4.    Immunostaining of spleen and lymph nodes for B and T cells: 

T.  OFFSPRING ORGAN WEIGHTS [47] 

1.    Observations: 

Table # [Heading]

 SEX  f1 males  f1 females
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
NUMBER OF ANIMALS                
 BODY WEIGHT (gram)[a]                
 BRAIN                
Absolute Weight[a] gram                
Per Body Weight[a] %                
SPLEEN                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
THYMUS                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                

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

Table # [Heading]

 SEX  f2 males  f2 females
 DAILY DOSE
(mg/kg body-weight/day)
 0
CONTROL
          0
CONTROL
        
NUMBER OF ANIMALS                
 BODY WEIGHT (gram)[a]                
 BRAIN                
Absolute Weight[a] gram                
Per Body Weight[a] %                
SPLEEN                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                
THYMUS                
Absolute Weight[a] gram                
Per Body Weight[a] %                
Per Brain Weight[a] %                

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

2. Comments: 

U. OFFSPRING GROSS PATHOLOGY CHANGES OBSERVED [48] 

f1 offspring

1.    Observations:
2.    Comments: 

f2 offspring

1. Observations:
2. Comments:

V. OFFSPRING HISTOPATHOLOGY CHANGES OBSERVED [49] 

f1 offspring

1.    Observations:
2.    Comments: 

f2 offspring

1. Observations:
2. Comments:

W. OTHER OFFSPRING TESTS [50] 

1.    Observations:
2.    Comments: 

VI. Evaluation and Comment on Study [51] 

VII. Summary and Conclusions [52] 

A. BRIEF SUMMARY OF MAJOR FINDINGS FROM THE STUDY

1. Parental Toxicity:
2. Offspring Toxicity: 

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

1. Was there a parental no observed effect level?
2. Was there an offspring no observed effect level?
3. Comments: 

VIII. References

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]Make note of: study file location (Volume, pages), study title/report #, testing facility name, publication dates of study, study objective, and comments, if needed. Indicate Yes or No for the rest of this section. However, you may want to elaborate on the type of GLP compliance (USFDA, OECD, etc.) or make other notations. 

[3]Date protocol was signed by study director. 

[4]Date the final report was signed by the study director. 

[5]FDA Good Laboratory Practice (GLP) regulations became effective June 20, 1979 and include the requirement for a Quality Assurance Statement. FDA also recognizes EPA and OECD GLP standards as being equivalent to those of FDA. 

[6]Is a signed and dated Good Laboratory Practice Compliance Statement included in the study report? Answer Yes or No. If yes, provide the location of the statement in the study report and identify the GLP standard (FDA, EPA or OECD) 

[7]Is a signed Quality Assurance Statement, including dates of inspections, provided in the study report? Answer Yes or No. If yes, provide the location of the statement in the study report.  

[8]Description of the test substance should be given, including purity, any possible contaminants or impurities, and any properties of the test substance that might have affected its integrity. Are there factors that might have affected the actual administered dose, as opposed to the intended dose? 

[9]After 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, you are free to use other methods of depicting the molecular structure.  

[10]Indicate 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? 

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

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

[13]Provide adequate details so the information can be used to help prepare a toxicology evaluation report. 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. 

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

[15]Standardization 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.). 

[16]Indicate the measurements or observations that were made by placing an 'X' in the 'Examined' side. Those parameters not examined will not contain an 'X' in the 'Examined' side. Use the comments to indicate when observations were made and any other notable fact. 

[17]Indicate the measurements or observations that were made by placing an 'X' in the 'Examined' side. Those parameters not examined will not contain an 'X' in the 'Examined' side. Use the comments to indicate when observations were made and any other notable fact. 

[18]Indicate the measurements or observations that were made by placing an 'X' in the 'Examined' side. Those parameters not examined will not contain an 'X' in the 'Examined' side. Use the comments to indicate when observations were made and any other notable fact. 

[19]Indicate the measurements or observations that were made by placing an 'X' in the 'Examined' side. Those parameters not examined will not contain an 'X' in the 'Examined' side. Use the comments to indicate when observations were made and any other notable fact. 

[20]To split a table (between 2 pages): Place the cursor in the table. From the 'Table' menu, choose 'Select Table' to highlight. From the 'Format' menu, select 'Paragraph' then the 'Line and Page Breaks' tab. Uncheck the box 'Keep with next' to split the table. 

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

[22]Indicate 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 when observations were made and any other notable fact. 

[23]For litter weight, pup body weight and sex, sex ratio, and viability index, please list the days examined in parentheses, especially if different from those listed in the table below. 

[24]Anogenital distance should be measured at day zero for all F2 pups that show treatment-related effects in F1 sex ratio or sexual maturation. 

[25]This section is optional. Indicate 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 when observations were made and any other notable fact. 

[26]This section is optional. Indicate 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 when observations were made and any other notable fact. 

[27]Indicate the measurements or observations that were made by placing an 'X' in the 'Examined' side. Those parameters not examined will not contain an 'X' in the 'Examined' side. Use the comments to indicate when observations were made and any other notable fact. 

[28]For F1 and F2 weanlings, histopathology examination of treatment-related abnormalities noted at macroscopic examination should be considered, if deemed appropriate and would contribute to the overall quality of the study data. 

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

[30]Note 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 the level of the test substance in the feed analyzed? Was this done more than once? Are there adequate data to permit the calculation of the actual dose that was administered? 

[31]Note any statistically or biologically significant feed/water consumption changes. You may also want to note feed/water consumption changes that are not statistically significant (e.g., at low or mid dose) but could indicate a treatment-related trend. 

[32]Knowing what the intake of feed/water was and the level of test substance in the feed/water, what was the dose actually being delivered to the animals? This section would not be relevant for a gavage study. 

[33]Note 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. 

[34]Note any statistically or biologically significant body weight changes. You may also want to 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. 

[35]List significant, dose-related abnormal clinical observations reported. This may include neurotoxicity endpoints.  

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

[37]Note findings that are statistically or biologically treatment-related. Also note the changes that are 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. 

[38]Note findings that are statistically or biologically treatment-related. The type of lesion should be noted. The comment section is available to explain data findings, if needed. 

[39]Note findings that are statistically or biologically treatment-related. The type of lesion should be noted. The comment section is available to explain data findings, if needed. 

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

[41]Note findings that are statistically or biologically treatment-related. Also note the changes that are 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. If the study measures pup weight on days that are different than those listed, please correct the information so it corresponds with the study. 

[42]List significant, dose-related abnormal observations reported. If the study measures litter size, litter weight, viability, or sex ratio on days that are different than those listed, please correct the information so it corresponds with the study. 

[43]For each pup with an unscheduled death, note the group, date of death, and any observations, including lesions observed at necropsy. 

[44]Anogenital distance should be measured at day zero for all F2 pups that show treatment-related effects in F1 sex ratio or sexual maturation. 

[45]This section is optional.  List significant, dose-related developmental or neurotoxicity changes observed. 

[46]This section is optional. List treatment-related findings in the immune system that were noted under result sections V.T: Offspring Organ Weights, V.U: Offspring Gross Pathological Changes, and V.V: Offspring Histopathological Changes. Provide a statement about whether or not the test substance presents a potential hazard to immunology. 

[47]Note findings that are statistically or biologically treatment-related. Also note the changes that are 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. 

[48]Note findings that are statistically or biologically treatment-related. The type of lesion should be noted. The comment section is available to explain data findings, if needed. 

[49]Note findings only if treatment-related abnormalities were noted at macroscopic examination and contributed to the overall quality of the study data. The type of lesion should be noted. The comment section is available to explain data findings, if needed. 

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

[51]Give your impressions of the study: what was done well; what the problems were; did the study accomplish what it was supposed to do? 

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

(Return to Toxicology Template Introduction)

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