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  6. Template for in vitro Mouse Lymphoma Thymidine Kinase Gene Mutation and Related Assays
  1. Ingredients, Additives, GRAS & Packaging Guidance Documents & Regulatory Information

Template for in vitro Mouse Lymphoma Thymidine Kinase Gene Mutation and Related Assays

March 2004

Introduction to the Template for in vitro Mouse Lymphoma Thymidine Kinase Gene Mutation and Related Assays

Return to Toxicology Template Introduction

Table Of Contents

  1. Identification Of Assay
  2. Good Laboratory Practice
  3. Materials And Methods
    1. Test Substance
      1. Chemical Name:
      2. Cas Registry Name:
      3. Cas Registry Number:
      4. Structure:
      5. Purity:
      6. Impurities:
      7. Physical Description:
      8. Stability:
      9. Comments:
    2. Test Substance As Administered
      1. Batch/Lot Number:
      2. Vehicle Used:
      3. Tested Adequately For Concentration?
      4. Tested For Stability?
      5. Problems With Storage?
    3. Experimental Methodology
      1. Test Mammalian Cells In Culture
      2. In Vitro Metabolic Activation System (S9 Mix)
      3. Control Materials
      4. Locus Examined
      5. Test Compound And Concentrations Used
    4. Test Performance For Mutation Assay
      1. Cell Treatment (Table 1a And 1b):
      2. Statistical Methods:
      3. Evaluation Criteria:
  4. Results
    1. Preliminary Cytotoxicity Assay
      1. Table 2. Summary Of Preliminary Cytotoxicity Assay
      2. Comments:
    2. Mutation Assay
      1. Table 3a. Summary Of Mutation Assay Without Metabolic Activation Or S9 Mix
      2. Comments:
      3. Table 3b. Summary Of Mutation Assay With Metabolic Activation Or S9 Mix
      4. Comments:
  5. Overall Assessment And Summary
    1. Assessment Of Study
    2. Summary
  6. References

Genetic Toxicity Studies: In Vitro Mouse Lymphoma Thymidine Knase Gene Mutation Assay And Related Assays [1]  

Date Of Submission:

Title Of Petition Or Notification:

Name And Address Of Petitioner Or Notifier:

I. Identification Of Assay

A. Assay File Location (Volume And Page Number(s)):
B. Assay Title/Report Number:
C. Assay Author Or Director:
D. Name And Address Of Testing Laboratory:
E. Date Of Assay Report:
F. Dates Assay Conducted:
G. Study Objective:
H. Comments:

II. Good Laboratory Practice [2]  

A. Is A Good Laboratory Practice (GLP) Compliance Statement Included?
B. Is A Quality Assurance (QA) Statement Included?
C. Availability And Location Of Original Data/Specimens/Test Substance:

III. Materials And Methods

A. Test Substance

  1. Chemical Name:   
  2. Cas Registry Name:  
  3. Cas Registry Number:  
  4. Structure: [3]
  5. Purity:
  6. Impurities:
  7. Physical Description:
  8. Stability:  
  9. Comments:

B. Test Substance As Administered [4]

  1. Batch/Lot Number:
  2. Vehicle Used:
  3. Tested For Concentration?
  4. Tested For Stability?
  5. Problems With Storage?  

C. Experimental Methodology [5]

1. Test Mammalian Cells In Culture

a. Type Of Cells:
Mouse Lymphoma L5178y Tk +/. -3.7.2c Cells
Other Cells (Specify):

b. Cell Culture Media:

c. Maintenance Of Cell Cultures:

Was The Following Information Provided In A Study Report? (Check 'Yes' Or 'No') 

Information Yes No
Properly Maintained?    
Periodically Checked For Mycoplasma Contamination?    
Periodically Checked For Karyotype Stability?    
Periodically "Cleansed" Against High Spontaneous Background?    

d. Comments:  

2. In Vitro Metabolic Activation System (S9 Mix)

a. Composition: [6]

Component

Concentration Or Amount (Unit)

Comment
     
     
     
S9 Fraction
(details on induction, see below)		    

b. s9 Fraction:   

Induced
Or Not

Inducer Used

Animal
Induced*

Animal Tissue Used For S9 Fraction
Induced Aroclor 1254 Rat Liver
  Phenobarbital Mouse Lung
 

Other (specify):

 Hamster

Other (specify):
   

Other (specify):

  
Non-induced None None None

* Specify strain and sex of the animal induced.

Strain: Sex:

c. Comments:  

3. Control Materials

a. Negative Or Solvent Control:  

Solvent [7]

Concentration
   
   

b. Positive Control(s) Without Metabolic Activation:

Chemical 

Concentration
Methylmethanesulfonate (MMS)  
Other    

c. Positive Control(s) With Metabolic Activation:

Chemical

Concentration
Cyclophosphamide (CP)  
3-Methylcholanthrene (3-MCA)  
Benzo(a)pyrene  
Other  

4. Locus Examined

Locus

Selection Agent

Concentration
Thymidine kinase (TK) Trifluorothymidine (TFT)  
Others Others  

5. Test Compound And Concentrations Used

a. Preliminary Cytotoxicity Assay: [8]

Condition

Test Compound

Concentration/Solvent
Non-Activated    
Activated    

b. Mutation Assay:

Condition

Test Compound

Concentration/Solvent
Non-Activated    
Activated    

D. Test Performance For Mutation Assay

1. Cell Treatment (Table 1a And 1b):

a. Method [9]  : Soft-Agar ______________ Or Micro-Well ________________

b. Table 1a. Summary Of Cell Treatment Without Metabolic Activation Or S9 Mix

Treatment

Without S9 Mix

headers="with">Number Of Cultures

Treatment Period (Hrs)

Expression Period (Days)

Number Of Cells Seeded Per
Plate Or Well

Culture Period (Days)

For
Cloning Efficiency

For
Mutant Detection

headers="trea">. (Solvent Control)

            

headers="trea">Test Dose 1

            

Test Dose 2

            

Test Dose 3

            
             

Positive Control 1

            
             

c. Table 1b. Summary Of Cell Treatment With Metabolic Activation Or S9 Mix

Treatment

id="with">With S9 Mix

Number Of Cultures

Treatment Period (Hrs)

Expression Period (Days)

Number Of Cells Seeded Per

Plate Or Well

Culture Period (Days)

For

Cloning Efficiency

For

Mutant Detection

headers="trea">. (Solvent Control)

            

Test Dose 1

            

Test Dose 2

            

Test Dose 3

            
             

Positive Control 1

            
             

d. Comments:  

2. Statistical Methods: [10]

3. Evaluation Criteria: [11]

IV. Results

A. Preliminary Cytotoxicity Assay [12]

1. Table 2. Summary Of Preliminary Cytotoxicity Assay

Treatment

(mG/Ml)

Without S9 Mix

Treatment

(mG/Ml)

With S9 Mix

  

RS

RTG

Other Factors

RS

RTG

Other Factors

0 (Solvent)

100

1.00

  

0 (Solvent)

100

1.00

  
               
               
               
                 

RS = Relative Survival (%) [13]  
RTG = Relative Total Growth [14]  
Other factors (if present, such as precipitation, nonphysiological pH, osmolality, etc.)

If other criteria are used for measurement of cytotoxicity, modify the table and enter those experimental data.

Detailed explanations can be found in the Redbook, Section IV.C.1.c., Mouse Lymphoma

Thymidine Kinase Gene Mutation Assay.

2. Comments:

B. Mutation Assay [15]

1. Table 3a. Summary Of Mutation Assay Without Metabolic Activation Or S9 Mix

Treatment (µG/Ml)

Without S9 Mix

RS

RTG

MF

(Note Statistical Significance

Number Or % Colonies

Large

Small

headers="treat">0 (Solvent Control)

headers="mix-rs">100

headers="mix-rtg">1.00

      
           
           
           
           

Linear Trend (If Conducted):

Positive Control

          

2. Comments:

3. Table 3b. Summary Of Mutation Assay With Metabolic Activation Or S9 Mix

Treatment (µG/Ml)

Without S9 Mix

RS

RTG

MF

(Note Statistical Significance

Number Or % Colonies

Large

Small

headers="treat">0 (Solvent Control)

headers="mix-rs">100

headers="mix-rtg">1.00

      
           
           
           
           
Linear Trend (If Conducted):

Positive Control

          
               

Refer to the Redbook guidelines at http://www.cfsan.fda.gov/~redbook/redivc1c.html for the definitions and considerations of the terminology below:
RS = Relative Survival (%) [16]  
RTG = Relative Total Growth [17]  
MF (Mutant Frequency) = Mutants/10 [6] viable cells
Notation of Statistical Significance
NS (Not Significant) or
S (Significant) at * (P-value < 0.05)="" or="" **="" (p-value=""><>

4. Comments:

V. Overall Assessment And Summary [18]  

A. Assessment Of Study
B. Summary

VI. References

End Notes

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

[1] FDA recommendations for conducting a gene mutation assay in mouse lymphoma cells in vitro are contained in http://www.cfsan.fda.gov/~redbook/redivc1c.html. FDA recommends use of the mouse lymphoma assay using L5178Y TK [+/-] -3.7.2 C cells because the assay can detect point mutations and chromosomal events.

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

[3] If the molecular structure is not provided with the study, it may be available from a source such as Chemfinder (http://www.chemfinder.com) or ChemIDplus (http://chem.sis.nlm.nih.gov/chemidplus/). The CAS number is the preferred search term if you have it, otherwise try the common name or any synonyms given. If the correct structure is found on Chemfinder or ChemIDplus, the image can be downloaded and inserted into your report. If the structure is not available, enter 'Not available'.

[4] Indicate how the test substance was administered and whether any vehicle was used to dissolve/suspend the test substance (e.g., dissolved in dimethylsulfoxide). Describe how adequate testings for concentration and stability were done. How was the test substance in vehicle, etc., stored before being used in the assay? Note anything that was not normal or routine.

[5] Provide adequate details so the information can be used to help prepare your 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.

[6] List the components and concentrations of cofactor solutions as well as the amount of S9 fraction in the S9 mix. If S9 mix was purchased, provide details.

[7] If all table rows are used, add additional rows by clicking the left mouse button in the last cell (bottom right) and pressing the TAB key.

[8] If no preliminary cytotoxicity assay was performed, enter 'not performed'.

[9] Either a soft-agar or micro-well method can be used for the detection of mutants and measurement of cloning efficiency. Copy and paste additional tables to this section, if needed.

[10] State which data were analyzed and what statistical methods were used.

[11] Summarize the testing laboratory's criteria for an acceptable assay and for a positive and negative response.

[12] Enter the data requested in the table. Additional rows can be added to the table using standard Word commands.

[13] Relative survival (RS):
The relative cloning efficiency of the test culture plated immediately after cell treatment and compared to the cloning efficiency of the negative control. http://www.cfsan.fda.gov/~redbook/redivc1c.html for more information.

[14] Relative total growth (RTG):
RTG is used as the measure of treatment-related cytotoxicity in the assay. It is a measure of growth of test cultures (relative to the vehicle control) both during the two-day expression and mutant selection cloning phases of the assay. The RSG of each test culture is multiplied by the relative cloning efficiency of the test culture at the time of mutant selection and expressed relative to the cloning efficiency of the vehicle control.
Relative suspension growth (RSG): Relative total two day suspension growth of the test culture compared to the total two-day suspension growth of the vehicle control. http://www.cfsan.fda.gov/~redbook/redivc1c.html for more information.

15 Enter the data requested in summary tables. If a linear trend test was conducted, enter the results. In addition, briefly describe any pertinent information, such as appropriateness of cloning efficiencies, appropriateness of control values (include the testing laboratory's historical control ranges if provided) and the testing laboratory's criteria for differentiating large versus small colonies. If more than one mutation assay is reported, copy and paste Tables 3A and 3B and make the appropriate changes (i.e., the headings of the new tables).

16  Relative survival (RS):
The relative cloning efficiency of the test culture plated immediately after the cell treatment and compared to the cloning efficiency of the negative control.

17 Relative total growth (RTG):
RTG is used as the measure of treatment-related cytotoxicity in the assay. It is a measure of growth of test cultures (relative to the vehicle control) both during the two-day expression and mutant selection cloning phases of the assay. The RSG of each test culture is multiplied by the relative cloning efficiency of the test culture at the time of mutant selection and expressed relative to the cloning efficiency of the vehicle control.
Relative suspension growth (RSG): Relative total two day suspension growth of the test culture compared to the total two-day suspension growth of the vehicle control.

18 Briefly summarize the study and discuss deficiencies. 

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