Genotoxicology |
| Genotoxicology (mutagenicity) tests evaluate the ability of a material to cause mutation or chromosomal damage. Any materials intended for pharmaceutical or topical use should be evaluated for mutagenic properties. Unpolymerized materials, additives, trace monomers or oligomers and biologics or biodegradative products can also all be potential mutagens. Guidelines issued by the International Conference on Harmonization of technical requirements for registration of pharmaceuticals (ICH) and the Organization for Economic Cooperation and Development (OECD) outline tests for Genotoxicology, carcinogenicity and reproductive toxicity. The ICH and OECD guidelines for genetic toxicity require three tests: one for gene mutation in bacteria (e.g., Bacterial Reverse Mutation Assay), one in vitro test in mammalian cells for either chromosomal damage or gene mutation (e.g., Chromosomal Aberration Assay or Mouse Lymphoma Assay) and one in vivo test for DNA damage (e.g., In Vivo Mouse Micronucleus Test). In this section |
| Bacterial Reverse Mutation Back to Top |
The Bacterial Reverse Mutation test system is commonly employed as an initial screen for mutagenic activity in general and point mutation activity in particular. An extensive database has demonstrated that many chemicals that are positive in these systems also exhibit mutagenic activity in other mammalian test systems. SAMPLE REQUIREMENTS
(Unless otherwise noted, these are recommended amounts for Bacterial Reverse Mutation testing.)
All samples should be submitted sterile.
Solids: 1 gram
Liquids: 10 ml
(Contact lab regarding concentration.)
SHIPPING TEMPERATURE
Per client’s discretion 30314 5 S. typhimurium
30315 4 S. typhimurium +
1 E. coli | The Ames mutagenicity screen is performed on liquid chemicals or on solutions of solid materials. To perform this test, five tester strains of bacteria (five Salmonella typhimurium or four Salmonella typhimurium and one Escherichia coli), in which specific mutations can be induced, are exposed to five concentrations of the test material and appropriate controls in the absence metabolic activation. This test may only be used as a screen. |
Ames Mutagenicity Screen Turnaround Time:
25 days |
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30316 5 S. typhimurium
30317 4 S. typhimurium +
1 E. coli
30318 5 S. typhimurium +
1 E. coli | The Bacterial Reverse Mutation test is performed using OECD test method 471. Tester strains of bacteria (five Salmonella typhimurium or four Salmonella typhimurium and one Escherichia coli or five Salmonella typhimurium and one Escherichia coli) are exposed to preparations of the test material in the presence and absence of a exogenous metabolic activation system. A dose range assay using 6 concentrations of the test article is performed prior to the definitive assay. Based on the results of the dose range assay, three dose levels of the test article will be used in the definitive assay along with positive and negative controls. |
Bacterial Mutagenicity Test Turnaround Time:
25 days |
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| 30345 | The pre-incubation Ames assay follows OECD test method 471. Tester strains of bacteria (five Salmonella typhimurium) are exposed to preparations of the test material in the presence and absence of an exogenous metabolic activation system before the addition of a soft agar. A dose range assay using 6 concentrations of the test article is performed prior to the definitive assay. Based on the results of the dose range assay, three dose levels of the test article will be used in the definitive assay along with positive and negative controls. |
Pre-incubation Ames Assay
Turnaround Time:
25 days
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| 30334 | Salmonella typhimurium strains TA98 and TA100 are exposed to eight concentrations of the test material in the presence and absence of an exogenous metabolic activation system in a typical plate incorporation assay. This assay provides a quantitative initial screen of the test material and only requires 200 mg of material. |
Abbreviated Ames Assay Turnaround Time:
25 days |
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| 30332 | The Ames Multiwell screen utilizes much less test article than a standard Ames assay (200 mg) while still acting as a good predictor of test article performance in the standard assay. Because this assay utilizes three sensitive strains over a wide dose range (5 doses), this assay is ideal for early research and development processes.
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Ames Multi-well Screen
Turnaround Time:
25 days |
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| 30319 | Test materials yielding positive results in the Bacterial Mutagenicity test should be repeated in a 3 point dose range test to confirm mutagenic effects. Typically, this dose range is similar to those chosen in the original assay.
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Ames Mutagenicity
Confirmation Test Turnaround Time:
25 days |
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| In Vitro Assays Back to Top |
SAMPLE REQUIREMENTS
(Unless otherwise noted, these are recommended amounts for In Vitro Genotoxicology testing.)
All samples should be submitted sterile.
Solids: 1 gram
Liquids: 10 ml
(Contact lab regarding concentration.)
SHIPPING TEMPERATURE
Per client’s discretion
| 30325 | This assay is designed to determine whether a test material is clastogenic, i.e. whether it has the capacity to break chromosomes. Clastogenicity is an important endpoint because it is through breakage and inappropriate rejoining that certain oncogenes, such as myc, can be activated and certain tumor suppressor genes, such as those causing retinoblastoma, can be inactivated. Mammalian cells are exposed to the test material and blocked in metaphase using a spindle poison. Visualization of the chromosomes is performed microscopically after hypotonic swelling, fixation and staining. Agents found to be capable of inducing chromosome breakage have a high probability of being carcinogens and have the potential for inducing inheritable chromosome defects. This test is performed according to OECD guideline 473. |
In Vitro Chromosomal
Aberration Assay
Turnaround Time:
10 weeks
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| 30335 | This assay is designed to determine whether a test material is clastogenic, i.e. whether it has the capacity to break chromosomes. Clastogenicity is an important endpoint because it is through breakage and inappropriate rejoining that certain oncogenes, such as myc, can be activated and certain tumor suppressor genes, such as those causing retinoblastoma, can be inactivated. Mammalian cells are exposed to the test material and blocked in metaphase using a spindle poison. Visualization of the chromosomes is performed microscopically after hypotonic swelling, fixation and staining. Agents found to be capable of inducing chromosome breakage have a high probability of being carcinogens and have the potential for inducing inheritable chromosome defects. In the initial assay, the cell line is exposed to varying concentrations of the test article in the presence and absence of a metabolic activation system. The independent repeat assay extends treatment and collection time of the cells, to provide additional data on delayed mutagenicity response that can be caused by toxic materials. |
In Vitro Chromosomal
Aberration Assay -
Universal Protocol with Independent Repeat
Turnaround Time:
15 weeks
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| 30321 | Mouse Lymphoma cells are used to determine whether a test material has the capacity to induce either point mutations or clastogenic (chromosomal breakage) events in a cultured mammalian cell line. In this case, the target gene, thymidine kinase (TK), is a dispensable enzyme. TK is involved in a salvage pathway for thymidine derived from DNA metabolism. Because the cells can synthesize thymidine de novo, loss of the TK does not render the cell non-viable. Mutants can be selected and mutant frequencies derived by including a thymidine analogue (trifluorothymidine, TFT) in the culture medium of cells after exposure to the test material. Normal cells with intact TK incorporate the analogue and die; mutant cells (which lack TK activity) survive, form colonies and are quantified. The mouse lymphoma cells will be exposed to eight concentrations of the test material in the presence and absence of a metabolic activation system. Four doses are chosen based on toxicity, to be carried through on to cloning. This test is performed according to OECD guideline 476. |
In Vitro Mouse Lymphoma
Assay
Turnaround Time:
8 weeks
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| 30322 | Sister chromatid exchanges (SCE) occur as a normal feature of cell division in mammalian cells. They are widely thought to represent the interchange of DNA replication products at apparently homologous loci and involve DNA breakage and reunion. Although SCE are readily observed experimentally, the mechanisms that mediate SCE are not understood. Particular types of DNA-damaging agents (e.g. bifunctional alkylating agents) are very efficient SCE inducers, while others (such as ionizing radiation) are very poor inducers. SCE are visualized by allowing Chinese Hamster Ovary cells (CHO) to replicate twice in the presence of the nucleotide analog, 5-bromo-2-deoxyuridine (BrdUrd) and the test article. Through the use of special staining techniques, the two chromatid arms (sister chromatids) display differential staining, permitting enumeration of SCE. Multiple dose levels of the test article will be tested in the presence and absence of a metabolic activation system. |
In Vitro Sister Chromatid Exchange Assay
Turnaround Time:
12 weeks
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| 30323 | This assay is designed to determine whether a test material damages DNA in a fashion that provokes "long patch" DNA repair. While DNA repair is obviously beneficial to the cells which carry it out, no repair process is completely error free and mutations can be expected to accompany DNA repair processes. Furthermore, to be effective, all DNA repair must be completed before a cell enters "S" phase of the cell cycle. Failure to complete repair in this time frame would lead to the replication of DNA on a damaged template which, in turn, results in mutations and chromosomal rearrangements. Primary rat hepatocytes are exposed to ten concentrations of the test materials in the presence of tritiated thymidine. Labeled thymidine is incorporated in "patches" where damaged DNA has been excised and new DNA resynthesized. Cell are fixed on slides and evaluated autoradiographically for the presence and quantity of repair sites. |
In Vitro Unscheduled DNA Synthesis (UDS) Assay
Turnaround Time:
12 weeks
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| In Vivo Assays Back to Top |
SAMPLE REQUIREMENTS
(Unless otherwise noted, these are recommended amounts for In Vivo Genotoxicology testing.)
All samples should be submitted sterile.
Solids: 1 gram
Liquids: 35 ml
(Contact lab regarding concentration.)
SHIPPING TEMPERATURE
Per client’s discretion
| 30324 | This test is used for the detection of damage induced to the chromosomes or mitotic apparatus of erythroblasts by analysis of erythrocytes from bone marrow in treated mice. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded. Any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualization of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosomal damage. Three concentrations of the test article will be administered to both male and female mice. Cells will be collected for analysis at 24 and 48 hours after dosing for test material and negative control groups. Positive controls will also be administered to both male and female mice, but collection will occur only at 24 hours. A dose range finding study (Protocol 30513) should be performed prior the definitive study. This protocol is performed according to OECD guideline 474. |
In Vivo Mouse Micronucleus
Test
Turnaround Time:
10 weeks
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| 30513 | A range of at least five doses is tested in a modified limit test. Five male and five female mice are tested at the top dose. Three males per group are tested at each of the four lower doses. Mice are evaluated for overt toxicity and for detrimental effects on erythropoiesis (blood cell cycle). The range finding assay should be performed prior to Protocol 30324 to determine the most effective dose range to be used in the definitive assay; it does not constitute a regulatory compliant assay for mutagenicity determination on its own. |
In Vivo Mouse Micronucleus Range Finding Test
Turnaround Time:
4 weeks
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| 30347 | This test is used for the detection of damage induced to the chromosomes or mitotic apparatus of erythroblasts by analysis of erythrocytes from bone marrow in treated mice. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded. Any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualization of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosomal damage. A single, maximum dose of 2000 mg/kg will be tested in this assay. The Sponsor must provide complete and accurate information justifying the use of this maximum dose. Furthermore, this test can only be used for test articles that are not toxic. Should this assay demonstrate test article toxicity, a dose range finding study (Protocol 30513) and a definitive assay (Protocol 30324) must be performed to satisfy OECD
guideline 474. |
In Vivo Mouse Micronucleus
Limit Assay
Turnaround Time:
8 weeks
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