Successfully Navigate the Waters of Genetic Toxicology Testing Requirements

As for other compounds positive in an Ames test, an in vivo mutation assay should be performed.

S9 = externally added metabolic activating system
*For indirect acting mutagens (requiring metabolic activation), adequate exposure to metabolite(s) should be demonstrated.
Reference ICH (2017)

For in vivo cytogenetics assays the minimum number of cells required to be scored are specified in the OECD TGs and are same, regardless of species. But the number is much lower for chromosome aberration (200 cells/animal) as compared to micronucleus (4000 cells/animal scored microscopically, but generally 20,000 cells/animal when scored by flow cytometry).

Yes, the Enhanced Ames Test was validated earlier this year and is conducted at two of our sites. The assay is done in the preincubation format with 30% rat and hamster S9s and without S9. Tester strains include S. typhimurium TA98, TA100, TA1535, and TA1537, and E. coli WP2uvrA pKM101.

Both are equally accepted by regulators.

Analysis in peripheral blood can be performed by flow cytometry, which is quicker, less expensive, highly reproducible, with many more cells scored (generally 20,000), which makes it more robust. Also, there is more flexibility in the timing of blood collection when the MN endpoint is incorporated into a repeat dose toxicology study.

Choosing bone marrow is often a matter of personal comfort or historical preference. However, if mode of action (MOA) information is required (via FISH or CREST), this is the only option available.

We do recommend human cells (e.g., HPBL or TK6) over transformed rodent cell lines (e.g., CHO, V79, L5178Y), but the preference is more related to the p53 status of the cells. p53-deficient cells such as CHO, V79, and L5178Y are generally more prone to irrelevant positive responses than the p53-proficient HPBL or TK6 cells. We also recommend TK6 over HPBL since we see less day-to-day (and donor-to-donor) variability in the TK6 cell line relative to primary HPBL cultures.

One other note: “false” positive is now considered a misnomer since results generally are reproducible over and over in a particular test cell type. However, those positive results may be irrelevant as related to cancer and human health, hence the false vs. irrelevant distinction.

We advocate for the in vitro MN over the CAb using a cell line, specifically TK6. The MN assay has a more straightforward plus-minus scoring and does not require any specialized cytogenetics training. Additionally, the MN assay is quicker and less expensive to conduct. Using a cell line such as TK6 instead of HPBL can reduce the day-to-day variability and that which can come from different individual donors. Also, TK6 cells are p53-proficient (as are HPBLs) which reduces the incidents of irrelevant positive results.

You may be confusing this with the mammalian cell cytogenetics assays and the mouse lymphoma assay, which have specific short and long treatment requirements.

For the Ames test, there is no 4- vs 24-hr treatment. The general requirement is to incubate the bacteria with the test material for two to three days.

However, one common variation is the preincubation method where the bacteria are first incubated with the test material in suspension, in a very small volume for 20 or 30 minutes, after which top agar is added and the mixture is poured onto the plates. Incubation then continues for the same two or three days.

Another variation is the “treat and plate” or “treat and wash” method, which is essentially a preincubation where the test material is washed out by centrifugation after the initial preincubation step. It is generally employed when a biological test material contains histidine or tryptophan (both of which can interfere with the assay).

The preincubation method is generally considered to be more sensitive (due to the higher initial treatment concentration) and is specifically required to be used for the enhanced Ames test for nitrosamines. Otherwise, all versions are acceptable under the appropriate circumstances.

Assuming it is a metabolite produced by human microsomes or hepatocytes, it will depend on the degree of metabolism that occurs in the in vitro test system with the standard induced rat liver S9. If a/the metabolite is produced at ≥30% of the parent API, independent evaluation of that metabolite is generally not required. Otherwise, the metabolite(s) will have to be evaluated independently.

The same is true for in vivo studies. If not produced at the required level, the metabolite will need to be independently evaluated in vivo.

MultiFlow data have been submitted to the FDA as a follow-up to positive TK6 MN results. If the result shows an aneugenic MOA, the comet assay generally would not be required. A safety margin argument should be acceptable to proceed into the clinic. However, a comet assay also would be required if the MOA is clastogenic.

The MultiFlow assay is generally conducted non-GLP, owing to the “black box” data analyses performed at Litron. However, all other aspects of the assay could be GLP-compliant (taking an exception for the analyses done at Litron).

Pros: 3Rs-friendly since fewer animals are used; reduced cost as compared to a standalone in vivo MN. Cons: the study design might not be appropriate for MN assessment; if an in vitro cytogenetics assay is positive, the 28-day dose levels will likely be inappropriate, and a standalone study should be performed.

We find TK6 cells are:

• p53-proficient (leading to fewer irrelevant positive responses) and produce more consistent results (less day-to-day, and no donor-to-donor, variability)

There is no need for any further work, as the 2 negative in vivo endpoints are sufficient to overrule or negate the positive in vitro result.

We have validated and routinely use N-nitroso-dimethylamine (NDMA) and 1-cyclopentyl-4-nitrosopiperazine (CPNP) as the two nitrosamine-specific positive controls in the EAT. We also include our standard 2-aminoanthracene (2AA). All are evaluated in all five tester strains with 30% induced rat and hamster S9.

I believe you mean enhanced Ames test for nitrosamines. We have validated and routinely use N-nitroso-dimethylamine (NDMA) and 1-cyclopentyl-4-nitrosopiperazine (CPNP) as the two nitrosamine-specific positive controls in the EAT. We also include our standard 2-aminoanthracene (2AA). All are evaluated in all five tester strains with 30% induced rat and hamster S9.

Blood can be taken as early as Day 4 of the repeat dose study for MN evaluation and then the animals can be sacrificed 3 hours after the last dose for comet evaluation. However, MN + comet studies are generally performed as a standalone study, not piggybacked onto a longer-term study.

If following up a positive in vitro result, we think one would have to test up to the MTD or MFD or limit dose using an acute dose regimen. That also would be the recommended and conservative approach if following ICH S2(R1) Option 2.

The best study design is the one giving the highest exposure. Generally, the MTD using 1 - 3 daily doses produce a higher exposure than a repeat dosing often performed at lower levels.

One can use a single dose with two harvest times at 24 and 48 hours after dosing (for bone marrow, slightly later if peripheral blood). However, if analyzing bone marrow this would require a terminal sac, using more animals and being more expensive. Alternatively, one can use a single harvest time at 24 hours after two or more doses for bone marrow (slightly later if peripheral blood). However, under ICH S2(R1) Option2, if the Ames and in vitro mammalian cell assays are negative, the MN endpoint can be integrated into a multiple dose toxicology study and the dose levels are considered appropriate provided the toxicology study meets the criteria for an adequate study to support human clinical trials. In that case, there is no requirement to test up to the MTD.

This is certainly true for aneugens since they have a defined threshold and mechanistic data to back up that assessment. While the NOGEL approach has been well established in recent years, recognition by the regulatory agencies has been painfully slow (if at all). It likely would require overwhelming evidence to make one’s cases (see, for example, https://www.sciencedirect.com/science/article/abs/pii/S0378427409001878 and https://doi.org/10.1016/j.mrgentox.2011.06.005)

The frequency varies by tissue, but the best source for this information would be the OECD Detailed Review Paper on Transgenic Rodent Mutation Assays, Series on Testing and Assessment, Number 103 summarized in Table 4.7:

Table 4-7 continued

^a Shaded boxes indicate positive carcinogenicity reported by rodent bioassay
b Values are percentages (number of chemicals that are positive in a TGR assay in this tissues that are target tissues are carcinogenicity/number of chemicals studied using the TGR assay that also induce tumors in the specified tissue [shaded].

At present the short answer is no, as there is no regulatory guideline for this assay. However, regulators are always interested in looking at all available data, GLP or not, with a guideline or not, if it helps tell a story and explain results.

We recommend using the MultiFlow assay as a rapid, low-cost screen, or as a follow-up test to determine mode-of-action when a positive response is obtained in the in vitro micronucleus test.

It is highly specific based on compound or therapeutic class, but in general most positives MN responses are clastogenic in nature. However, the reverse would be true (i.e., mostly aneugenic) for certain classes, like kinase inhibitors.

TK6 is recommended over CHO because TK6 is p53-proficient, which reduces incidence of irrelevant positive responses. That said, there are some clients who love and only use CHO and are very happy. If no problems have been encountered, one tends to stick with what one knows.