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March 2004

Introduction to the Template for in vitro Bacterial Reverse Mutation (Ames) Test

Toxicology Template Introduction

Identification Of Test
Good Laboratory Practice
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. Bacterial Strains Used:
  2. In Vitro Metabolic Activation System (S9 Mix):
  3. Control Agents:
  4. Test Compound Concentrations Used:
  5. Test Procedure Used:
  6. Protocol:
  7. Statistical Analysis:
  8. Evaluation Criteria:
Results
1. Preliminary Cytotoxicity (Range-Finding) Test
  1. Table 1. Summary Of Preliminary Cytotoxicity (Range-Finding) Test
  2. Comments On Cytotoxicity (Range-Finding) Test:
2. Mutation Tests
  1. Table 2. Summary Of Initial Mutation Test
  2. Comments On Initial Mutation Test:
  3. Table 3. Summary Of Second (Confirmatory) Mutation Test
  4. Comments On Second Mutation Test:
  5. Table 4. Summary Of Third (Confirmatory) Mutation Test (Optional):
  6. Comments On Third Mutation Test (Optional):
Overall Assessment And Summary
1. Assessment Of Study
2. Summary
References

GENETIC TOXICOLOGY STUDIES:

IN VITRO BACTERIAL REVERSE MUTATION (AMES) TESTS¹

Date of Submission:

Title of Petition or Notification:

Name and Address of Petitioner or Notifier:

I. IDENTIFICATION OF TEST

A. Test File Location (Volume and Page Number(s)):
B. Test Title/Report Number:
C. Name of Author or Director of Test:
D. Name and Address of Testing Laboratory:
E. Date of Test Report:
F. Dates Test Conducted:
G. Objective of Test:
H. Comments:

II. GOOD LABORATORY PRACTICE²

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

Chemical Name:
CAS Registry Name:
CAS Registry Number:
Structure: ³
Purity:
Impurities:
Physical Description:
Stability:
Comments:

B. TEST SUBSTANCE AS ADMINISTERED⁴

Batch/Lot Number:
Vehicle Used:
Tested for Concentration?
Tested for Stability?
Problems with Storage?

C. EXPERIMENTAL METHODOLOGY⁵

1. Bacterial Strains Used⁶ :

a. Salmonella Typhimurium:

TA98	TA100	TA1535	TA1537
TA97	TA97a	TA1538	TA102
Other S. Typhimurium strains (specify):

b. Escherichia Coli:

	WP2(uvrA)
	Other E. coli strains (specify):

c. Were the Strains:

Checked for appropriate genetic markers?⁷		Yes		No or not stated
Properly maintained?		Yes		No or not stated

2. In Vitro Metabolic Activation System (s9 Mix):

a Composition: ⁸

COMPONENT	CONCENTRATION OR AMOUNT (Unit)	COMMENT



S9 Fraction (Details on induction, see below)

b. 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:

3. control agents:

a.             Negative Control:
b.             Solvent/Final Concentration:
c.              Positive Controls Without Metabolic Activation⁹ :

TESTER STRAINS¹⁰	CHEMICAL	DOSE (mg/PLATE)

d. Positive Controls with Metabolic Activation:

TESTER STRAINS	CHEMICAL	DOSE (mg/PLATE)

e. Comments on Positive Controls:

4. Test Compound Concentrations Used:

a. Preliminary Cytotoxicity/Range-Finding Test:¹¹

CONDITION	TEST COMPOUND	CONCENTRATION/SOLVENT
NON-ACTIVATED (-S9)
ACTIVATED (+S9)

b. Mutagenicity Test(s)¹² :

CONDITION		TEST COMPOUND	CONCENTRATION/SOLVENT
INITIAL	- S9
	+S9
Second Confirmatory	- S9
	+S9
Third Confirmatory	- S9
	+S9

5. Test Procedure Used:

Standard plate incorporation test
Preincubation test	Time:	Temperature:
Other (describe)

6. Protocol¹³ :

7. Statistical Analysis¹⁴ :

8. Evaluation Criteria¹⁵ :

III. RESULTS

A. PRELIMINARY CYTOTOXICITY (RANGE-FINDING) TEST ¹⁶

1. Table 1. Summary of Preliminary Cytotoxicity (Range-Finding)Test

STRAIN	S9	DOSE RANGE (mg/PLATE)
TA100	-
TA100	-	Solvent control
TA100	-	Positive control
TA100	+
TA100	+	Solvent control
TA100	+	Positive control
WP2 (uvrA)	-
WP2 (uvrA)	-	Solvent control
WP2 (uvrA)	-	Positive control
WP2 (uvrA)	+
WP2 (uvrA)	+	Solvent control
WP2 (uvrA)	+	Positive control

2. Comments on Cytotoxicity (Range-Finding) Test¹⁸ :

B. MUTATION TESTS¹⁹

1. Table 2. Summary of Initial Mutation Test

STRAIN	S9	DOSE RANGE (mg/PLATE)
TA98	-
TA98	-	Solvent control
TA98	-	Positive control
TA98	+
TA98	+	Solvent control
TA98	+	Positive control
TA100	-
TA100	-	Solvent control
TA100	-	Positive control
TA100	+
TA100	+	Solvent control
TA100	+	Positive control
TA1535	-
TA1535	-	Solvent control
TA1535	-	Positive control
TA1535	+
TA1535	+	Solvent control
TA1535	+	Positive control
TA1537	-
TA1537	-	Solvent control
TA1537	-	Positive control
TA1537	+
TA1537	+	Solvent control
TA1537	+	Positive control
WP2 (uvrA)	-
WP2 (uvrA)	-	Solvent control
WP2 (uvrA)	-	Positive control
WP2 (uvrA)	+
WP2 (uvrA)	+	Solvent control
WP2 (uvrA)	+	Positive control

2. Comments on Initial Mutation Test:²¹

3. Table 3. Summary of Second (Confirmatory) Mutation Test

STRAIN	S9	DOSE RANGE (mg/PLATE)	HIGHEST MEAN NUMBER REVERTANTS /PLATE (AT DOSE?)	BACKGROUND LAWN²²	DOSE RESPONSE?
TA98	-
TA98	-	Solvent control
TA98	-	Positive control
TA98	+
TA98	+	Solvent control
TA98	+	Positive control
TA100	-
TA100	-	Solvent control
TA100	-	Positive control
TA100	+
TA100	+	Solvent control
TA100	+	Positive control
TA1535	-
TA1535	-	Solvent control
TA1535	-	Positive control
TA1535	+
TA1535	+	Solvent control
TA1535	+	Positive control
TA1537	-
TA1537	-	Solvent control
TA1537	-	Positive control
TA1537	+
TA1537	+	Solvent control
TA1537	+	Positive control
WP2 (uvrA)	-
WP2 (uvrA)	-	Solvent control
WP2 (uvrA)	-	Positive control
WP2 (uvrA)	+
WP2 (uvrA)	+	Solvent control
WP2 (uvrA)	+	Positive control

4. Comments on Second Mutation Test:²³

5. Table 4. Summary of Third (Confirmatory) Mutation Test (Optional)²⁴ :

STRAIN	S9	DOSE RANGE (mg/PLATE)
TA98	-
TA98	-	Solvent control
TA98	-	Positive control
TA98	+
TA98	+	Solvent control
TA98	+	Positive control
TA100	-
TA100	-	Solvent control
TA100	-	Positive control
TA100	+
TA100	+	Solvent control
TA100	+	Positive control
TA1535	-
TA1535	-	Solvent control
TA1535	-	Positive control
TA1535	+
TA1535	+	Solvent control
TA1535	+	Positive control
TA1537	-
TA1537	-	Solvent control
TA1537	-	Positive control
TA1537	+
TA1537	+	Solvent control
TA1537	+	Positive control
WP2 (uvrA)	-
WP2 (uvrA)	-	Solvent control
WP2 (uvrA)	-	Positive control
WP2 (uvrA)	+
WP2 (uvrA)	+	Solvent control
WP2 (uvrA)	+	Positive control

6. Comments on Third Mutation Test (Optional):

V. OVERALL ASSESSMENT AND SUMMARY

A. ASSESSMENT OF STUDY
B. SUMMARY

VI. REFERENCES

End Notes

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

¹FDA's recommendations for conducting a bacterial reverse mutation test are contained in Redbook 2000, IV.C.1.a. Bacterial Reverse Mutation Test http://www.cfsan.fda.gov/~redbook/redivc1a.html. This template is for the S. typhimurium Test (Ames Test), the most commonly used reverse mutation test. In addition to the S. typhimurium TA strains, an E. coli strain WP2 or its variants are often included in the test. The TA strains detect a reversion event from Histidine dependence to Histidine independence while the WP2 strains do the same for Tryptophan dependence.

²Indicate 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 test, 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. 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'.

⁴Indicate 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 testings for concentration and stability were done. How was the test substance in vehicle, etc., stored before being used in the test? Note anything that was not normal or routine.

⁵Provide adequate details so the information can be used to help prepare a 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.

⁶Put an X in the box to the left of each strain used and type in any other strain used.

⁷e.g. rfa mutations, R factor

⁸List the components and concentrations of cofactor solutions as well as the amount of S9 fraction in S9 mix. If the S9 mix was purchased, provide details.

⁹Enter all strains treated with a given positive control at the same dose. If a given positive control agent was used at different doses in different strains, add a separate row for each dose. 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.

¹⁰Frequently 2-Aminoanthracene (2-Anthramine) is used as the positive control without activation in all tester strains. If this is the case, just enter "All Strains", otherwise enter the individual strains treated with a given positive control at the same dose.

¹¹If no preliminary cytotoxicity test was performed, enter "not performed."

¹²If more than one mutagenicity test was conducted, list the concentrations used in each test.

¹³Give a brief description of the experimental protocol including tester strain preparation, media used, cell numbers, number of plates/dose/activation condition, incubation times and colony counting method.

¹⁴Briefly describe the method used. Usually in the Salmonella test, the criterion for a positive response is given as a fold-increase over the solvent control (two- or three-fold increase depending on the strain) and no statistical analysis is used. In that case, just enter "not conducted."

¹⁵Enter the criteria for an acceptable test and for a positive and negative response. Were the revertant colonies counted with an automatic counter or manually?

¹⁶TA100 and WP2 (uvrA) are frequently used in the cytotoxicity test; however, if other strains were used, modify the table as required using the standard Word commands.

¹⁷e.g. normal, thin, absent

¹⁸Briefly describe the results of the cytotoxicity (range-finding) test and the basis for the selection of concentrations used in the mutation tests. For example, "in the absence of any cytotoxic effect, the limit dose of 5 mg/plate was used as the upper dose" or "the upper dose was limited by cytotoxicity or solubility".

¹⁹The five most commonly used tester strains are entered in the table as defaults; however, the table should be modified to reflect the strains actually used in a given test.

²⁰Enter the description of the background lawn: e.g. normal, thin, absent.

²¹Briefly summarize the results of the mutation test with the goal of clarifying or expanding on the data in the table or adding something pertinent not included in the table. For example, comments such as the following could be included:

Cytotoxicity as based on a reduction in the background number of revertants per plate was seen in strain(s) at dose(s) of X - Y.
A precipitate was seen at concentrations of X and above.
A two-fold or greater increase in the number of revertants/plate was seen in strain(s) at X mg/plate.
A mutagenic effect was seen only in strain(s) indicating a frameshift method of action.
The positive and solvent control values were appropriate for the respective strains.
Based on results of the first mutation test, test material concentrations of X - Y were chosen for the second mutation test.

Essentially, this summary is a statement of conclusions drawn from the test.

²²Enter the description of the background lawn: e.g. normal, thin, absent.

²³Briefly summarize the second test as you did the first test. Do the results of the second test agree or disagree with those of the first test?

²⁴Delete the table and sections III.B.5-6 if not needed.

²⁵Enter the description of the background lawn: e.g. normal, thin, absent.

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Introduction to the Template for in vitro Bacterial Reverse Mutation (Ames) Test

Table Of Contents

I. IDENTIFICATION OF TEST

A. Test File Location (Volume and Page Number(s)): B. Test Title/Report Number: C. Name of Author or Director of Test: D. Name and Address of Testing Laboratory: E. Date of Test Report: F. Dates Test Conducted: G. Objective of Test: H. Comments:

II. GOOD LABORATORY PRACTICE2

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: