Applications of Threshold of Toxicological Concern (TTC) In Pharmaceuticals

TTC concept and Insilco system: Introduction and Outcome

Threshold of Toxicological Concern (TTC) and Insilco system are very helpful in reducing -extensive toxicity assessments for Genotoxic impurities during pharmaceutical development. Control of Genotoxic impurities is challenging for Pharmaceutical professionals. It needs knowledge, skills and experience. In this post, you will learn the sources of Genotoxic chemicals, the classification of Genotoxic chemicals, the TTC concept, types standard TTC, staged TTC, advantages of TTC, exceptions to the TTC and Insilco system. This post will clear all your doubts about the TTC concept.

Sources of Genotoxic impurities

The following are the major sources of Genotoxic impurities in APIs (Active pharmaceutical ingredients):

• Purchasing materials like Key starting materials (KSM) and starting materials (SM)
• Reagents and solvents used in the process of Active pharmaceutical ingredients and the process of purchasing materials used in the proces.
• Intermediates
• Related substances or isomers
•  Byproducts
• Degradants of API or degradants of Intermediates or degradants of Impurities and
• Excipients (In case of dosages forms)

Impurities classification

Impurities have been classified in the following five classes:

Class-1: Impurities known to be Genotoxic (Mutagenic) and Carcinogenic

This group includes known animal carcinogens with reliable data for a genotoxic mechanism, and human carcinogens. The genotoxic nature of the impurity is demonstrated using published data on the chemical structure. Impurities of this class having compound specific limit.

Class-2: Impurities known to be genotoxic (mutagenic), but with unknown carcinogenic potential

This group includes impurities with demonstrated mutagenicity based on testing of the impurity in conventional genotoxicity tests. Impurities of this class is controlled at threshold limit called TTC (Threshold of toxicological concern)

Class-3: Impurities that have an alerting structure unrelated to the structure of the API, and of unknown genotoxic (mutagenic) potential

This group includes impurities with functional moieties that can be linked to genotoxicity based on structure. However, these moieties have not been tested as isolated compounds and are identified based on chemistry and using knowledge-based expert systems for structure activity relationships (SAR). Impurities of this class is controlled at threshold limit called TTC.

Class-4: Impurities with an alerting structure related to the API and impurities that contain an alerting functional moiety that is shared with the structure of the API

Impurities of this class are treated similar as API.

Class-5: No alerting structure or indication of Genotoxic potential

Although genotoxic impurities are classified, the major challenge is to design strategies to control these genotoxins. The major challenges were regarding Class 2 and Class 3 inaccuracies. Now the question is; Can these genotoxins show genotoxicity at all levels? If yes then what is the solution and if not, at what levels will these Genotoxins be controlled? We are going to discuss the same in the TTC concept section.

TTC Concept

“No Food or color additive could be approved if found to cause cancer in Man or Animals-Delaney Rule-1958 (James Delaney)”. 

This Clause is a provision of the 1958 Food Additive Amendment to the Food, Drug and Cosmetic Act of 1938. But with the increasing sensitivity of Analytical tools, it has been observed that many chemicals which might be carcinogenic beyond a certain level were used as preservative  below certain level.  The FDA was the first agency who confronted this problem with Diethylstilbestrol (see below structure) :

Diethylstilbestrol was being used to increase the shelf life of meat in the meat industries. It addressed the issue by using quantitative risk assessment, declaring that if a carcinogenic food additive was present at a concentration of less than 1 part in 1,000,000, the risk was negligible.

This standard became known as the “exceedingly low /de minimis” exception to the Delaney Rule and was used throughout the FDA and other agencies. Using this concept threshold of Genotoxins was proposed.

A TTC-based acceptable intake of a mutagenic impurity of 1.5 µg per person per day is considered to be associated with a negligible risk (theoretical excess cancer risk of <1 in 100000 over a lifetime of exposure) and can in general be used for most pharmaceuticals as a default to derive an acceptable limit for control. This approach would usually be used for mutagenic impurities present in pharmaceuticals for long-term treatment (> 10 years) and where no carcinogenicity data are available (Classes 2 and 3).

This concept is called TTC (threshold of toxicological concern) concept.

1.5 μg/day is being considered acceptable where compound specific tox data are not available. This is also called registration limit .

Types of TTC

• Standard TTC and
• Staged TTC

Standard TTC

In standard TTC, permitted level in Active substance is calculated by following formula:

GTI (in ppm)= 1.5µg/Maximum daily dose (in gram)

Permitted level in active substance (in ppm) = TTC/ Maximum daily Dose (in gram)

Case studies

Rilpivirine, Max dose = 25mg

Permitted GI in Rilpivirine(in ppm) = 1.5/ 0.025 (in gram) = 60 ppm

Staged TTC

Since the TTC value is calculated for lifetime exposure and consequently, it was decided to allow higher levels for treatments of shorter duration. This concept is called Staged TTC. It is accepted to the clinical development phase.
EMEA limits based on 70-year exposure assumptions:

• Based on duration of exposure
• Dose and Impurity Concentration Limit – relation

Lifetime exposure allowed will be 38.33 mg by the following is the calculation:

1.5x (70 years x 365 days) = 38.33mg

The above allowable daily intake for Staged TTC may be calculated by following formulae:

• For less 1 month: 38.33/30 = 120µg/person/day

TTC Advantages

The following are the advantages of the TTC:

• TTC (Threshold of toxicological concern) is an approach aimed at reducing  extensive toxicity evaluations
• Genotoxins without sufficient data must be limited at TTC (1.5 μg/day)
• Genotoxins with more than TTC level must be justified toxicologically
• Staged TTC is most used in the clinical trials

Exception of TTC

Following high-potency Carcinogens are excluded from the TTC approach:

• Aflatoxin-like-
• Nitroso amines
• Azoxy-compounds

The above structural groups were identified to be of such high potency that intakes even below the TTC would theoretically be associated with a potential for a significant carcinogenic risk. This group of high-potency mutagenic carcinogens is referred to as the cohort of concern.

Exception of TTC; A TTC value higher than 1.5 μg/day may be acceptable under the following conditions:

A TTC value higher than 1.5 μg/day may be acceptable under the following conditions:

• Short-term exposure
• Treatment of a life-threatening condition
• When life expectancy is less than 5 years
• Where the impurity is a known substance and human exposure will be much greater from other sources (e.g., food).
• Genotoxic impurities that are also significant metabolites may be assessed based on the acceptability of the metabolites

In silico system for Identification of Genotoxins

Several systems are available for Genotoxic evaluation and out of them the following systems are widely used in Pharmaceutical industries:

• DEREK (Deductive Estimation of Risk from Existing Knowledge)
• MCase (Multi Computer Automated Structure Evaluation)
• CPDB (Carcinogenic Potency Database)

Advantages of in silico predictions

The following are the advantages of the in silico predictions:

• Predict for compounds not yet synthesized or purified, by using (expected) chemical structure
• Utilize available information from literature for known compounds
•  Quick indication whether compound may be toxic and needs to be tested in vitro
• Save on expensive in vitro and animal testing

Information required for Insilco predictions

Following informations are required for Insilco predictions:

• ROS of all  purchasing materials (e.g. KSM, SM….) of  each vendor with reagents, chemicals and solvents
• ROS of API with reagents, chemicals and solvents
• Intermediates structure
• Structure of side reaction products
• Impurity profile peaks that have reached ICH ID threshold
• Isomers structures
• Potential impurities structures
• Degradants structures

Challenges with Insilco predictions

• Most of the Industries utilize DEREK where a few Industries have their own databases. Now question is; Is the regulatory authority use the same system? The answer is no. Authorities may use multiple systems or system as per their choice.
• Regulatory authorities have some concern over the robustness of In-silico tools.
• It has been observed many times that different versions of the same system predict unimaginable results. This put scientists in a difficult situation

Conclusion

I spite of various challenges, TTC concept and In-silico approach are very helpful during drug development. Hope this post has clear all your doubts related to TTC concept and In-silico system. Write your learning and questions related to this post in the comment section.

Interview questions on TTC concept

What are the different sources of Genotoxic impurities?

The different sources of Genotoxic impurities are raw materials like SM. KSM, chemicals,, solvent and reagents used in the process. Genotoxic impurities may form in the process also

What is the TTC concept of toxicology ?

A TTC based acceptable intake of a mutagenic impurity of 1.5 µg per person per day is considered to be associated with a negligible risk (theoretical excess cancer risk of <1 in 100000 over a lifetime of exposure) and can in general be used for most pharmaceuticals as a default to derive an acceptable limit for control

What is TTC in Genotoxic impurities?

1.5 µg per person per day

What is the TTC value for the Cramer class?

The following are the TTC value for cramer classes I, II and III

What are the advantages of TTC?

TTC is an approach aimed at reducing  extensive toxicity evaluations

What are the exception of TTC?

A TTC value higher than 1.5 μg/day may be acceptable in conditions like When life expectancy is less than 5 years, short term exposure etc.

What is Insilco system and what are its advantages?

Insilco system is used to evaluate genotoxic nature of a molecule based on its structure. It reduces the drug development cost.

What are the challenges with the Insilco system?

There are several systems are available to predict genotoxic nature of a chemicals and for some molecules they don’t provide uniform result. It has been also observed many times that different versions of the same system predict unimaginable results. This put scientists in a difficult situation

What is the cohort of concern?

The structural groups Aflatoxin-like, Nitroso amines and Azoxy-compounds shows carcinogenic nature even below the TTC level. This group of high-potency mutagenic carcinogens is referred to as the cohort of concern.

References

• https://en.wikipedia.org/wiki/James_J._Delaney
• https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5708
• https://database.ich.org/sites/default/files/ICH_M7%28R2%29_Guideline_Step4_2023_0216_0.pdf
• https://www.ema.europa.eu/en/ich-m7-assessment-control-dna-reactive-mutagenic-impurities-pharmaceuticals-limit-potential-carcinogenic-risk-scientific-guideline
• https://www.teva-api.com/knowledge-center/control-of-genotoxic-impurities-as-a-critical-quality-attribute/

Abbreviations:

• TTC: Threshold of toxicological concern
• GI: Genotoxic impurities
• KSM: Kay starting material
• SM: Starting material