Introduction to the 2-Generation Reproduction Toxicity Study Template

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Table of Contents

1. Identification of Study
2. Good Laboratory Practice
3. Executive Summary
4. Materials and Methods
   1. TEST SUBSTANCE
   2. TEST SUBSTANCE AS ADMINISTERED
   3. ANIMAL DIET
   4. TEST ANIMALS
   5. EXPERIMENTAL DESIGN
   6. MATING PROCEDURES
   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 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 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]

 

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 OBSERVATIONS¹⁷ 

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 PARAMETERS¹⁸ 

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 WEIGHTS¹⁹ 

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 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]

 

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 TESTS²¹ 

1.     Observations:
2.     Comments:

O.   OFFSPRING OBSERVATIONS/BODY WEIGHT²² 

 

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 - OPTIONAL²⁵ 

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 - OPTIONAL²⁶ 

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 WEIGHTS²⁷ 

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 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 analysis	parameters tested

 

2.     Comments:

V. Results

A.   DOSE VERIFICATION³⁰ 

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 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 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 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 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

 

 

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 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 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 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 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
Lymphocytes	109/L
Macrophage/Monocytes
Neutrophils	109/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
Lymphocytes	109/L
Macrophage/Monocytes
Neutrophils	109/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 Weighta	gram
Per Body Weighta	%
SPLEEN
Absolute Weighta	gram
Per Body Weighta	%
Per Brain Weighta	%
THYMUS
Absolute Weighta	gram
Per Body Weighta	%
Per Brain Weighta	%

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

 

 

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

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

 

2.     Comments:

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

 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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