IVT - In Vitro Toxicity Testing - 2002


  1. To compile and review information on in vitro toxicity testing and apply learnings to further biological research.
  2. To organise and conduct periodically proficiency testing of tobacco and tobacco related products.

The In Vitro Toxicity Testing of Tobacco Smoke Task Force was established in 2002 to ensure that CORESTA provides leadership on assessing toxicity evaluation. The first phase was stated as follows:

  • To prepare a report covering the rationale and strategy for conducting in vitro toxicity testing of cigarette smoke
  • To identify key procedures based upon internationally recognized guidelines, adapted to accommodate the nature and unique properties of tobacco smoke

The Task Force work for this mandate focused on “cigarette smoke condensate” (CSC) - the total particulate matter (TPM) from mainstream smoke collected on a Cambridge filter pad and extracted in dimethylsulfoxide. The Task Force achieved these objectives, and the Task Force recommendations have been designed to enable meaningful data to be produced using in vitro assays. However, interpretation of the biological significance of the in vitro data can only be done by considering available chemical and biological data, and placing the data in the context of the product. Thus, the Task Force report does not provide interpretational guidance concerning the biological significance of in vitro results.

The Task Force recommended a test battery composed of the following assays:

  1. A bacterial mutagenicity assay: The Task Force recommends the Ames Salmonella mutagenicity assay.
  2. A mammalian cell assay for cytogenetics / mutation: The Task Force recommends the micronucleus assay, the chromosome aberration assay or the L5178Y mouse lymphoma assay.
  3. A cytotoxicity assay conducted with an appropriate mammalian cell line.  The Task Force recommends the neutral red cytotoxicity assay.

The group concluded that these assays, when properly conducted, provide a scientifically-sound foundation for assessing the in vitro toxicity of tobacco smoke TPM. Key procedures of those assays including sample preparation and data analysis were also determined. In 2019, this report was reviewed and updated.  Both CORESTA Technical reports are available on the website.

  • The Rationale and Strategy for Conducting In Vitro Toxicology of Tobacco Smoke (June 2004)
  • Rationale and Strategy for In Vitro Toxicity Testing of Combustible Tobacco Products (September 2019)

Beginning 2004, the group conducted an inter-laboratory study on Ames, NRU, and Micronucleus assays of cigarette smoke condensate (total particulate matter in dimethyl sulfoxide) from four kinds of cigarettes. 13 laboratories participated in this study. The test cigarettes used for this studies were the following: 100% single grade U.S. Burley, 100% single grade Brazilian flue-cured, 1:1 mixture of the above two grades, and 2R4F Kentucky Reference cigarettes.  Cigarettes were mailed to each participating laboratory, with each laboratory using their own protocols for sample preparation and assays. This study was an ambitious undertaking in a relatively short period of time, with a large number of variables. The Ames assay showed the most concordance, while data from both the Neutral Red Update assay and the in vitro micronucleus assays exhibited much greater variability. The CORESTA Technical report is available on the website.

  • Report on Inter-laboratory Study of the In Vitro Toxicity of Particulate Matter from Four Cigarettes (Ames, Neutral Red Cytotoxicity and Micronucleus Assays) (July 2007)

In 2007, the group conducted a preliminary inter-laboratory study to compare the whole smoke exposure systems under development in a number of laboratories of Task Force members.  The group agreed to evaluate the contribution ratio of vapour and particulate phases to the cytotoxicity of smoke with NRU assay, using the same 4 test cigarettes described above.  Seven laboratories participated, each using their own exposure platform.  All laboratories were able to measure the whole smoke cytotoxicity of the test cigarettes provided, and in general, the rankings between the samples were similar.  The Technical report is available on the CORESTA website.

  • In Vitro Exposure of Cells to Smoke at the Air Liquid Interface (July 2007)

Since the initial objectives of the Task Force had been achieved, the Task Force recommended disbanding in 2010. In 2011, the second phase of the In vitro Task Force was established with new objective as follows:

  • To conduct a proficiency testing programme to evaluate cigarette smoke using a common experimental protocol and the Task Force’s recommended test battery”

Proficiency tests have been carried out on the Ames, NRU, and micronucleus assays involving 12, 10, and 11 laboratories respectively.  Major differences between the first and second phase of the Task Force’s work were:

  1. The group conducted these assays with cigarette smoke condensates (CSCs) prepared at one laboratory instead of individually collecting CSCs.
  2. The group defined an agreed-upon protocol which defined minimum requirements to be followed. The Study Design was approved by a Study Manager at each participating laboratory.
  3. A QA auditor reviewed data for each Study against the Study Plan to determine if the assays had been carried out as agreed.

Generally, the results showed significant potential to discriminate TPMs derived from different kinds of test cigarettes, and a certain concordance between laboratories. The reports are available on the CORESTA website.

The group has also actively discussed whole smoke exposure test methods. Technologies are advancing for the measurement of dosimetry, including photometers, optical tar, carbon monoxide, deposited mass assessed by Quartz Crystal Microbalances (QCM), smoke markers using wet chemistry techniques, computer fluid dynamics. These technologies potentially allow cross-platform comparisons and alignment of cross-platform doses. Intensive discussion is held among participants and the group is considering conducting exploratory proficiency trials using some of these dosimetry tools.

All these studies require highly skilled people in state of the art laboratories, with CORESTA as the forum to gather robust data to enable scientific debates and support regulatory decisions.

In 2015, the Task Force was changed to a Sub-Group with the following new objectives:

  1. To conduct a proficiency testing programme to evaluate cigarette smoke using common experimental protocols and theTask Force’s recommended test battery.
  2. To compile and review information on in vitro whole smoke methodology.
  3. To critique and review published papers and other available information on tobacco-related toxicity and suggest suitable work for further biological research and/or proficiency tests.

In 2016, the Sub-Group amended its name ("of Tobacco Smoke" was removed) and further amended its objectives, as follows, in view of harmonisation with other groups and to widen its scope:

  1. To compile and review information on in vitro toxicity testing and apply learnings to further biological research.
  2. To organise and conduct periodically proficiency testing of tobacco and tobacco related products.

The Sub-Group has since published the following Technical Reports:

  • Neutral Red Uptake Assay Proficiency Studies (November 2015 and January 2021)
  • In vitro Micronucleus Assay Proficiency Study 2011 (March 2016)
  • Ames Assay Proficiency Study (March 2016)
  • In vitro Micronucleus Assay Inter-Laboratory Proficiency Study (November 2019)
  • Neutral Red Uptake Assay Inter-Laboratory Proficiency Study (January 2021)
  • Mouse Lymphoma Assay Inter-Laboratory Proficiency Study (January 2021)
  • Ames Assay Inter-Laboratory Proficiency Study 2019 (February 2021)
  • A Review of Aerosol Exposure Systems Relative to the Analysis of Cytotoxicity (July 2021)

The Sub-Group has also published the following external publication:


Scientists who contribute to the group can be contacted through the CORESTA Secretariat.



Updated August 2021