Short-Term Biological Tests

In order to develop a cigarette that was less prone to cause cancer, BAT scientists needed an assay procedure to test out various possible product modifications. Mouse skin painting was BAT's standard test for carcinogens, but it required up to two years to get the results of a single experiment. This built-in delay and expense made rapid progress impossible. Accordingly, Sir Charles Ellis suggested that BAT develop a battery of short-term tests for use in-house. In January 1964, coinciding with the publication of the US Surgeon General's report, Sir Charles wrote Richard P. Dobson at BAT headquarters, recommending that BAT develop a set of short-term toxicological tests for the use of B&W {1103.02}.

Sir Charles suggested that Battelle, the contract research organization that did other work for BAT on both nicotine pharmacology (Project Hippo) and carcinogenicity of tobacco smoke (Project Janus), or some other organization be engaged to set up the ciliastasis test. He wanted to use it in assessing cigarette brands selected by B&W. In addition, he wanted to develop two other tests from among the five suggested, or to develop other sorts of short-term bioassays. The proposed testing program seems to have been mainly for the benefit of B&W. It is unclear why the work was to be done at Battelle rather than in-house, except that Battelle was shortly to do the company's mouse skin–painting work to

evaluate carcinogenic potency of cigarette smoke under the Janus project. Having the toxicology work done in an overseas, outside laboratory also provided B&W a buffer against discovery in legal proceedings (see also the section headed "Specific Strategies to Avoid Discovery" in chapter 7).

In his letter Sir Charles speculates that having such a battery of tests available would be of similar importance to conventional quality control:

If this project were to prove feasible it might lay the basis for a continuing health monitoring service on our cigarettes analogous to the Quality Control practised for physical characteristics. The work would be quite distinct from the researches into the relation between smoking and lung cancer which is carried out on a co-operative basis by T.R.C. [at Harrogate]. {1103.02, p. 3}

A week later, in Louisville, Tom Wade of R&D at B&W wrote an analysis of this memorandum for Ed Finch, B&W's president {1103.01}. A copy was hand-carried to Mr. Dobson of BAT when he was in New York on the following day. Wade endorses the concept of rapid tests without commenting on the technical feasibility of the specific proposals made by Sir Charles. Wade also blurs the distinction between tests to look for carcinogenic potential and tests for immediate toxicity. He concludes, "Naturally the whole purpose back of this is to get a reasonably rapid method to determine differences" {1103.01}.

The documents do not include further information on this attempt to establish short-term biological testing within BAT's research establishment. Nearly three years later, though, in October 1966, an internal progress report on a research project at the Southampton lab demonstrated that there was by then active work on the development of shortterm assays. In this case the goal was the development of a short-term test that would predict carcinogenic activity {1107.01}.

By November 1968, as a memo from Dr. R. A. Sanford, technical manager of research at B&W, to J. W. Burgard (with copies to the company president, general counsel, and the director of research and development) indicates, the contract lab operated by Battelle in Frankfurt, Germany, had achieved some success with a short-term test that measured hyperplasia in mouse skin as a predictor of malignant transformation {1111.01}. The test required only eight days to perform. It measured thickening of the skin on the backs of mice exposed to the test materials. This test had shown agreement with the mouse skin–painting test in most of the following situations:

The addition of certain PCL's, Celanese synthetic materials, or certain additives to the blend reduces biological activity rating.

Filters containing charcoal, cellulose acetate, PEI (polyehtyleneimine), or paper do not affect tumorigenic results.

With the exception of one result, increasing puff volume progressively from 10 to 50 ml reduces activity.

U. S. cigarettes are less active [i.e., less capable of inducing cancerous changes in the mice] than English varieties. The addition of either burely or up to 50% CRS [cut, rolled stems] to flue-cured tobacco reduces activity. {1111.01, p. 1}

Dr. Sanford suggested that some experimental versions of Viceroy be submitted for testing in this system and that it be used in connection with an upcoming project called Project Hilton {1111.01}. Project Hilton was to be an inhalation study under the management of Battelle, with the goal of looking at the short-term toxicity of tobacco smoke in animals.

By the middle of December, Dr. Sanford had received clearance to send samples to Europe for the hyperplasia test {1112.04}. He sent current versions of Kool and Viceroy, the same brands with 28 percent WTS (water-treated system), and Life filters and Life filters without PEI (polyethyleneimine) but with a Viceroy tobacco rod. The mention of Life filters without PEI suggests that the normal filter for Life contained this additive, an ingredient that was known to increase the proportion of "extractable nicotine" in cigarette smoke {1205.03}. It had not, however, reduced tumorigenic activity of smoke condensate {1111.01}. By 1968, then, B&W had at its disposal a short-term bioassay that correlated reasonably well with the standard test for carcinogenesis, mouse skin painting.

Notes on the 1974 Duck Key, Florida, research conference refer tersely to a short-term bioassay test that the BAT lab in Germany had developed.

BAT (Germany) NMFI test is proving of significant value in rapid prediction of mouse skin activity. We propose not to make this test available to competitors at this time since it might be of considerable commercial advantage [emphasis in original]. {1125.01, p. 2}

This assay was the subject of a research report from the BAT affiliate in Canada three years later. A 1977 research report from Imperial Tobacco Limited (Canada) details the possible use of a test called "the nitromethane fraction index (NMFI)" as an indicator of "biological activity" {1129.01}. The NMFI test was designed to be an inexpensive and quick test that predicted carcinogenicity in mouse skin. The test involved the extraction of cigarette smoke in a way known to concentrate polycyclic aromatic hydrocarbons; this smoke fraction was then mixed with egg albumin, and the resulting degree of binding by fluorescence

was determined. The degree of binding in turn showed a correlation with the biological activity of the smoke.

The test had been developed at the BAT lab in Germany and also was in use at Southampton. The test was found to be reproducible and to correlate well with the results of mouse skin–painting carcinogenicity tests conducted under the Janus program. Despite some reservations, the report recommends the routine inclusion of the NFMI test among the tests done "in future projects where there is a possibility of a change in the biological activity of the smoke" {1129.01, p. 4}. The report emphasizes that the NMFI test is for internal use only; it is not to be shared with other companies. In this context, the term "biological activity" is used as a euphemism for "carcinogenic activity." The development of this test is one more example of how the company knew its products caused cancer, and tried to do something about it, while publicly denying this fact.