Template for Genetic Toxicity Study: in vitro Mammalian Chromosomal Aberration Test

Introduction to the Template for Genetic Toxicity Study: in vitro Mammalian Chromosomal Aberration Test

April 2005

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TABLE OF CONTENTS

1. IDENTIFICATION OF TEST
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. MOLECULAR WEIGHT
      6. SOURCE/BATCH/LOT NO
      7. PURITY
      8. IMPURITIES
      9. PHYSICAL DESCRIPTION
      10. STABILITY
      11. STORAGE CONDITIONS
   2. TEST SUBSTANCE AS ADMINISTERED
      1. vehicle used
      2. tested adequately for concentration?
      3. tested for stability?
      4. tested for homogeneity?
      5. problems with storage?
   3. EXPERIMENTAL METHODOLOGY
      1. test cells in culture
      2. in vitro metabolic activation system (s9 mix)
      3. control agents
      4. test agent
      5. analytical determination (if provided)
   4. TEST PROTOCOL
      1. preliminary cytotoxicity test
      2. cytogenetic test
      3. evaluation criteria
      4. statistical methods
4. RESULTS
   1. PRELIMINARY CYTOTOXICITY TEST
   2. CYTOGENETIC TEST
      1. summary of the cytogenetic test
      2. comments
5. OVERALL ASSESSMENT AND SUMMARY
   1. ASSESSMENT OF TEST
   2. EXECUTIVE SUMMARY
6. REFERENCES

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GENETIC TOXICITY STUDY: IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST [1] 

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. TEST AUTHOR OR DIRECTOR:

D. NAME AND ADDRESS OF TESTING LABORATORY:

E. DATE OF TEST REPORT:

F. DATES TEST 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/TEST SUBSTANCE:

III. MATERIALS AND METHODS

A. TEST SUBSTANCE

1. CHEMICAL NAME:

2. CAS REGISTRY NAME:

3. CAS REGISTRY NUMBERr:

4. STRUCTURE: [3] 

5. MOLECULAR WEIGHT:

6. SOURCE/BATCH/LOT NO:

7. PURITY:

8. IMPURITIES:

9. PHYSICAL DESCRIPTION:

10. STABILITY:

11. STORAGE CONDITIONSS:

B. TEST SUBSTANCE AS ADMINISTERED [4] 

1. VEHICLE USED:

2. TESTED ADEQUATELY FOR CONCENTRATION?

3. TESTED FOR STABILITY?

4. TESTED FOR HOMOGENEITY?

5. PROBLEMS WITH STORAGE?

C.EXPERIMENTAL METHODOLOGY [5]

1. TEST CELLS IN CULTURE: [6] 

A. TYPE OF CELLS:

B. MAINTENANCE OF CELL CULTURES:

IF AN ESTABLISHED CELL LINE OR STRAIN WAS USED:	YES	NO	NOT STATED
Was it checked for stability in modal chromosome number?
Periodically checked for Mycoplasma contamination?

C. CELL CULTURE MEDIA: [7] 

D. COMMENTS:

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 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, sex, and age of the animal induced.

Strain:

Sex:

Age:

C. COMMENTS:

3. CONTROL AGENTS: [9] 

A. SUMMARY:

NEGATIVE (IF NOT VEHICLE):	FINAL VOLUME (UNIT):
SOLVENT/VEHICLE:	FINAL VOLUME (UNIT):
POSITIVE: WITHOUT ACTIVATION: WITH ACTIVATION:	FINAL CONCENTRATION(S) (UNIT): FINAL CONCENTRATION(S) (UNIT):

B. COMMENTS:

4. TEST AGENT:

A. WAS THE HIGHEST CONCENTRATION TESTED BASED ON SOLUBILITY CHARACTERISTICS OR WAS IT THE LIMIT CONCENTRATION FOR THIS TEST METHOD (E.G., 5000 µ G/ML)?

B. ANY TEST AGENT INDUCED PH AND/OR OSMOLALITY CHANGES OF TREATMENT MEDIUM?

C. SUMMARY:

PRELIMINARY CYTOTOXICITY TEST: WITHOUT ACTIVATION: WITH ACTIVATION:	CONCENTRATION(S) (UNIT): CONCENTRATION(S) (UNIT):
CYTOGENETIC TEST: WITHOUT ACTIVATION: WITH ACTIVATION:	CONCENTRATION (UNIT): CONCENTRATION (UNIT):

D. COMMENT:

5. ANALYTICAL DETERMINATION (IF PROVIDED): [10] 

D. TEST PROTOCOL

1. PRELIMINARY CYTOTOXICITY TEST: [11]

2. CYTOGENETIC TEST: [12] 

A. EXPOSURE TIME(S) IN HOURS: [13] 

	TEST MATERIAL	SOLVENT CONTROL	POSITIVE CONTROLS
NON-ACTIVATED
ACTIVATED

COMMENTS:

B. CELL HARVEST TIME(S) (POST-EXPOSURE) IN HOURS:

	TEST MATERIAL	SOLVENT CONTROL	POSITIVE CONTROLS
NON-ACTIVATED
ACTIVATED

COMMENTS:

C. SPINDLE INHIBITION:

INHIBITOR/CONCENTRATION:

WHEN ADMINISTERED (HOURS BEFORE CELL HARVEST):

D. CELL HARVEST AND SLIDE PREPARATION: [14]  

E. METAPHASE ANALYSIS: [15]  

	YES	NO	NOT STATED
SLIDES CODED PRIOR TO ANALYSIS?
SCORED FOR STRUCTURAL ABERRATIONS?
SCORED FOR NUMERICAL ABERRATIONS?
NUMBER OF METAPHASE CELLS SCORED PER CONCENTRATION:

F. COMMENTS:

3. EVALUATION CRITERIA: [16] 

4. STATISTICAL METHODS: [17] 

IV. RESULTS

A. PRELIMINARY CYTOTOXICITY TEST [18] 

B. CYTOGENETIC TEST [19] 

(The following table is an example only and should be modified as required or completely replaced if that approach would be more convenient)

1. SUMMARY OF THE CYTOGENETIC TEST:

WITHOUT S9-MIX (X HOUR TREATMENT, Y HOUR RECOVERY)

TREATMENT	NUMBER OF CELLS WITH STRUCTURAL ABERRATIONS						% ABERRANT CELLS		% CELLS WITH NUMERICAL ABERRATIONS	CYTX (%)
	CTB	CSB	CTE	CSE	OTHER	GAPS	+ GAPS	- GAPS
SOLVENT										100
TEST AGENT
X µ G/ML
Y µ G/ML
Z µ G/ML
POSITIVE CONTROL

WITH S9-MIX (X HOUR TREATMENT, Y HOUR RECOVERY)

TREATMENT	NUMBER OF CELLS WITH STRUCTURAL ABERRATIONS						% ABERRANT CELLS		% CELLS WITH NUMERICAL ABERRATIONS	CYTX (%)
	CTB	CSB	CTE	CSE	OTHER	GAPS	+ GAPS	- GAPS
SOLVENT										100
TEST AGENT
X µ G/ML
Y µ G/ML
Z µ G/ML
POSITIVE CONTROL

Data Summarized from (study No., Table x, page y)

CTB = chromatid break

CSB = chromosome break

CTE = chromatid exchange

CSE = chromosome exchange

CYTX = cytotoxicity measured by degree of confluency, viable cell counts, plating efficiency or mitotic index. Value expressed as % of control.

2. COMMENTS:

V. OVERALL ASSESSMENT AND SUMMARY [20] 

A. ASSESSMENT OF TEST

B. EXECUTIVE SUMMARY [21] 

In a mammalian cell chromosomal aberration test, (specify cell line or type eg. CHO or V79 cells, human lymphocytes etc. cells/cell cultures) were exposed to (chemical name, batch/lot No.) in (solvent) at concentrations of 0, x, x, x Hg/mL with and without mammalian metabolic activation (usually S9-mix) for (specify duration of exposure with and without activation). Cells were harvested x hours post-exposure. The S9-fraction was obtained from (specify inducer) induced (specify source and tissue, eg. male Sprague-Dawley rat liver).

(Test agent) was tested to an upper concentration limited by (excessive cytotoxicity or solubility constraints or to the limit concentration of 5000 Hg/mL). (Provide brief justification such as reduction in mitotic index to x% at y Hg/mL or a precipitate seen in the culture medium at y Hg/mL and above). (Present a brief summary of results). The solvent and positive controls induced (did not induce) the appropriate response. There was (no) significant increase in the percentage of metaphase cells with structural chromosomal damage over the solvent control levels. (If significant, indicate whether a dose-response relationship was present.) There was (no) significant increase in the percentage of metaphase cells with numerical chromosomal damage over the solvent control levels. (If significant, indicate whether a dose-response relationship was present.)

VI. REFERENCES

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End Notes

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

[1] FDA recommendations for conducting an in vitro Mammalian Chromosomal Aberration Test are contained in Redbook 2000 (revised November, 2003): http://www.cfsan.fda.gov/~redbook/redivc1b.html. Although similar guidelines are available for this test such as the OECD Test Guideline 473, this FDA template refers to the FDA guidance only.

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

[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 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). Also note how testings for concentration and stability were done. Was the test substance in vehicle, etc., stored before being used in the test? Note anything that was not normal or routine.

[5] Provide adequate details so the information can be used to help prepare a report. Use the comments to indicate additional information about the experimental methodology. To modify the tables, refer to the instructions in the 'help' section of your software program.

[6] Any of a variety of primary cells, cell strains or cell lines may be used in this test. Identify the cells used with respect to type, tissue of origin, source, ATCC designation if available and any other pertinent information provided. If human lymphocytes were used, describe the donor's sex, health, smoking status, whether whole blood cultures or isolated lymphocytes were used, the mitogen used and any other information provided that helps characterize the cell.

[7] Describe composition of maintenance, treatment and any other media used in the study including supplements and additives.

[8] 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.

[9] A negative control (completely untreated) is not required and usually not included in this test. Just enter "none" in this case. A vehicle/solvent and positive controls should always be included.

[10] Summarize the results if test material solutions were analyzed for stability, homogeneity, concentration, etc. For example "Test material solutions (give concentrations) were analyzed using HPLC with UV detection and found to be within 5% of the nominal values". "Test material solutions were stable for the duration of the study".

[11] Describe the method used to determine cytotoxicity, for example, the cells were exposed to the test material for x hours and cytotoxicity determined by mitotic index, trypan blue exclusion, cell cycle kinetics, cloning efficiency, monolayer confluency etc. at y hours post-exposure.

[12] Briefly describe cell culture preparation and culture conditions, including such factors as the number of cells seeded, CO2 concentration, temperature, humidity and any other pertinent information provided.

[13] Cells may be exposed for more than one time period under non-activated conditions. For example, the cells can be exposed for 3 hrs, followed by 21 hrs of post-exposure incubation, and then harvested. The cells can also be exposed for 24 hrs continuously and then harvested. Under activated conditions, only a short exposure time of 3-6 hrs is feasible.

[14] Describe the cell harvest method, the hypotonic treatment used to swell the nuclei, the fixation procedure and briefly describe the slide preparation details.

[15] Provide the requested information and put an "x" in the "yes" or "no" boxes as appropriate.

[16] Briefly describe the criteria for an acceptable test and for a positive and negative response.

[17] Briefly state which data (e.g., frequency of damaged metaphase cells excluding gaps; frequency of polyploidy metaphase cells) were analyzed and what statistical method(s) (e.g., one-tailed, two-tailed, Cochran-Armitage trend test for dose response, Fisher's exact test for pairwise comparisons) were used.

[18] If a preliminary cytotoxicity test was conducted, report the results including dose range, evidence of test material precipitation, cytotoxicity observed, pH and/or osmolality changes of treatment medium, if given, and the justification for dose selection for the cytogenetic test. Results may be presented in a summary table as appropriate.

[19] Report the remarkable results of the cytogenetic test(s), including cytotoxicity in the test material-treated and positive control-treated cells along with statistical evaluation, if conducted. For unremarkable/negative results, provide a narrative.

[20] Briefly summarize the study and provide an assessment including deficiencies and overall conclusion.

[21] The executive summary should be a brief, concise, stand alone summary describing the test system, the test protocol and the results. The skeleton structure provided for the executive summary in the template is intended as a very basic guide and can be modified as required.

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