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Introduction to the 2-Generation Reproduction Toxicity Study Template


 

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 Study2 

A.     Study File Location :
B.     Study Title/Report Number:
C.     Study Author:
D.     Name and Address of Testing Facility:
E.     Study Initiation Date3 :
F.     Study Completion Date4 :
G.     Study Objective:
H.     Comments:
 

II. Good Laboratory Practice5 

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 SUBSTANCE8 

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 ADMINISTERED10 

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 DIET11 

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 ANIMALS12 

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 DESIGN13 

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 malesf0 femalesf0 malesf0 females
Control     
Low     
Mid     
High     

*Premating dose

Table # [Heading]

 

test groupconc. in diet
(ppm or mg/kg)
dose to animals*
(mg/kg body-weight/day)
number assigned per group
f1 malesf1 femalesf1 malesf1 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 PROCEDURES14 

1.     Description:
2.     Comments:
 

G.   CULLING PROCEDURES15 

1.     Description:
2.     Comments:
 

H.  PARENTAL BODY WEIGHT AND FEED/WATER INTAKE16 

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

 

Table # [Heading]

 

examinedparameters*
f0 parentsf1 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 OBSERVATIONS17 

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

 

Table # [Heading]

 

examinedparameters*
f0 parentsf1 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 PARAMETERS18 

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

 

Table # [Heading]

 

examinedparameters*
f0 parentsf1 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 WEIGHTS19 

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

 

Table # [Heading]

 

examinedparameters*
f0 parentsf1 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 OBSERVATIONS20 

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]

 

SYSTEMexamined 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 TESTS21 

1.     Observations:
2.     Comments:
 

O.   OFFSPRING OBSERVATIONS/BODY WEIGHT22 

 

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

 

Table # [Heading]

 

observationsexamined 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 - OPTIONAL25 

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

 

Table # [Heading]

 

observationsexamined 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 - OPTIONAL26 

1.     Parameters examined:
 

 

Table # [Heading]

 

measurement related toexamined in
f1 offspring
not examined in
f1 offspring
examined inf2 offspringnot 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 WEIGHTS27 

1.     Organs/Tissues weighed:
 

 

Table # [Heading]

 

examinedparameters*
f1 offspringf2 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 OBSERVATIONS28 

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 TESTS29 

1.     Observations:
2.     Comments:
 

V.   STATISTICAL METHODS

1.     Methods of statistical analysis:
 

 

Table # [Heading]

 

methods of statistical analysisparameters tested
  
  
  
  

 

2.     Comments:
 

V. Results

A.   DOSE VERIFICATION30 

1.     Were doses verified?
 

 

Table # [Heading]

 

dose grouptargeted 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 CHANGES31 

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 MATERIAL32 

1.     Observations:
 

 

Table # [Heading]

 

dose groupf0 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)
control0000
low    
mid    
high    

 

2.     Comments:
 

 

D.   PARENTAL FEED EFFICIENCY33 
 

f0 parents

1.     Was feed efficiency calculated?
2.     Comments:
 

 

f1 parents

1.       Was feed efficiency calculated?
2.       Comments:
 

E.   PARENTAL BODY WEIGHT CHANGES34 

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 OBSERVATIONS35 

f0 parents

1.     Observations:
2.     Comments:
 

 

f1 parents

1.     Observations:
2.     Comments:

G.   PARENTAL MORTALITY36 

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 WEIGHTS37 

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 Weightagram        
Per Body Weighta%        
ADRENALS        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
EPIDIDYMIDESb     
Absolute Weightagram    
Per Body Weighta%    
Per Brain Weighta%    
KIDNEYS        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
LIVER        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
PITUITARY GLAND        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
PROSTATEb     
Absolute Weightagram    
Per Body Weighta%    
Per Brain Weighta%    
SEMINAL VESICLES WITH COAGULATING GLANDb     
Absolute Weightagram    
Per Body Weighta%    
Per Brain Weighta%    
SPLEEN        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
TESTESb     
Absolute Weightagram    
Per Body Weighta%    
Per Brain Weighta%    
THYMUS        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
OVARIESb     
Absolute Weightagram    
Per Body Weighta%    
Per Brain Weighta%    
UTERUSb     
Absolute Weightagram    
Per Body Weighta%    
Per Brain Weighta%    

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 Weighta
gram        
Per Body Weighta
%        
ADRENALS
        
Absolute Weighta
gram        
Per Body Weighta
%        
Per Brain Weighta
%        
EPIDIDYMIDESb
     
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
%        
PITUITARY GLAND
        
Absolute Weighta
gram        
Per Body Weighta
%        
Per Brain Weighta
%        
PROSTATEb
     
Absolute Weighta
gram    
Per Body Weighta
%    
Per Brain Weighta
%    
SEMINAL VESICLES WITH COAGULATING GLANDb
     
Absolute Weighta
gram    
Per Body Weighta
%    
Per Brain Weighta
%    
SPLEEN
        
Absolute Weighta
gram        
Per Body Weighta
%        
Per Brain Weighta
%        
TESTESb
     
Absolute Weighta
gram    
Per Body Weighta
%    
Per Brain Weighta
%    
THYMUS
        
Absolute Weighta
gram        
Per Body Weighta
%        
Per Brain Weighta
%        
OVARIESb
     
Absolute Weighta
gram    
Per Body Weighta
%    
Per Brain Weighta
%    
UTERUSb
     
Absolute Weighta
gram    
Per Body Weighta
%    
Per Brain Weighta
%    

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 OBSERVED38 

f0 parents

1.     Observations:
2.     Comments:
 

 

f1 parents

1.     Observations:
2.     Comments:

K.   PARENTAL HISTOPATHOLOGY CHANGES OBSERVED39 

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 TESTS40 

1.     Observations:
2.     Comments:
 

M. OFFSPRING BODY WEIGHT CHANGES41 

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 OBSERVATIONS42 

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 LitterN
MEAN
S.D.
        
Day 0 - TotalPer LitterN
MEAN
S.D.
        
Day 4 - TotalPer LitterN
MEAN
S.D.
        
Day 7 - TotalPer LitterN
MEAN
S.D.
        
Day 14 - TotalPer LitterN
MEAN
S.D.
        
Day 21 - TotalPer LitterN
MEAN
S.D.
        
litter weight (g)         
Day 0N
MEAN
S.D.
        
Day 4N
MEAN
S.D.
        
Day 7N
MEAN
S.D.
        
Day 14N
MEAN
S.D.
        
Day 21N
MEAN
S.D.
        
viability Indices         
Day 0-4N
MEAN
S.D.
        
Day 4-7N
MEAN
S.D.
        
Day 7-14N
MEAN
S.D.
        
Day 14-21N
MEAN
S.D.
        
weaning IndexN
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 MORTALITY43 

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 separationmean
S.D.
N
     
Age of vaginal openingmean
S.D.
N
     

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

 

2.     Comments:
 

Q.   ANOGENITAL DISTANCE44 

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

R.   OFFSPRING DEVELOPMENTAL ENDPOINTS AND NEUROTOXICITY - OPTIONAL45 

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 - OPTIONAL46 

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         
Lymphocytes109/L        
Macrophage/Monocytes         
Neutrophils109/L        
Total Leukocytes (WBC)109/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         
Lymphocytes109/L        
Macrophage/Monocytes         
Neutrophils109/L        
Total Leukocytes (WBC)109/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 WEIGHTS47 

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 Weightagram        
Per Body Weighta%        
SPLEEN        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
THYMUS        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        

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

 

 

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 Weightagram        
Per Body Weighta%        
SPLEEN        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        
THYMUS        
Absolute Weightagram        
Per Body Weighta%        
Per Brain Weighta%        

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

 

2.     Comments:
 

U.   OFFSPRING GROSS PATHOLOGY CHANGES OBSERVED48 

f1 offspring

1.       Observations:
2.       Comments:
 

 

f2 offspring

1.     Observations:
2.     Comments:

V.   OFFSPRING HISTOPATHOLOGY CHANGES OBSERVED49 

f1 offspring

1.       Observations:
2.       Comments:
 

 

f2 offspring

1.     Observations:
2.     Comments:

W. OTHER OFFSPRING TESTS50 

1.       Observations:
2.       Comments:
 

VI. Evaluation and Comment on Study51 

 

 

 

 

 

VII. Summary and Conclusions52 

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.

 

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

 

 2Make 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.

 

 3Date protocol was signed by study director.

 

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

 

 5FDA 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.

 

 6Is 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)

 

 7Is 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.

 

 8Description 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?

 

 9After 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.

 

 10Indicate 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?

 

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

 

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

 

 13Provide 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.

 

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

 

 15Standardization 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.).

 

  16Indicate 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.

 

 17Indicate 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.

 

 18Indicate 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.

 

  19Indicate 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.

 

 20To 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.

 

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

 

 22Indicate 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.

 

 23For 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.

 

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

 

 25This 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.

 

 26This 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.

 

 27Indicate 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.

 

 28For 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.

 

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

 

 30Note 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?

 

 31Note 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.

 

 32Knowing 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.

 

 33Note 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.

 

 34Note 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.

 

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

 

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

 

 37Note 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.

 

 38Note 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.

 

 39Note 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.

 

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

 

 41Note 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.

 

 42List 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.

 

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

 

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

 

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

 

 46This 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.

 

 47Note 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.

 

 48Note 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.

 

 49Note 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.

 

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

 

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

 

 52Summarize 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.

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