On September 6, 2002, the draft report on the toxicology and carcinogenesis studies of trimethylolpropane triacrylate received public review by the National Toxicology Program’s Board of Scientific Counselor’s Technical Reports Review Subcommittee. The review meeting was held at the National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Dr. C.J. Portier, NIEHS, introduced the review by explaining that, as a result of consultation with the committee prior to the meeting, the original plan to evaluate the conclusions concerning possible carcinogenic hazards of the two multifunctional acrylates trimethylolpropane triacrylate and pentaerythritol triacrylate had changed. The revised purpose of the review would be to evaluate the design and appropriate methods of analysis for these transgenic mouse studies. Further questions to be addressed were whether the Levels of Evidence of Carcinogenic Activity categories currently used for standard 2-year cancer bioassays would be applicable and what reporting format would be appropriate.

Dr. J.R. Bucher, NIEHS, outlined the agenda of presentations and described a variety of design and interpretation issues related to the use of genetically modified mouse models for cancer assessment. These included choice of animal model, group size, study duration, route of exposure, dose selection, use of positive or negative controls, extent of pathology evaluation, and appropriate statistical analysis method. Other fundamental questions were whether these should be considered carcinogenicity studies or promotion studies and what sort of interpretive conclusions could be drawn.

Dr. J.E. French, NIEHS, described the Tg.AC transgenic mouse model, the construction of the v-Ha-ras trans-gene, and the use of the model as a squamous epithelium reporter phenotype. Dr. D.B. Dunson, NIEHS, described a generalized Poisson system to analyze the incidence, multiplicity, and onset times for the skin papillomas that are the primary endpoint of the Tg.AC model.

Dr. R.S. Chhabra, NIEHS, described the study nomination and uses of trimethylolpropane triacrylate and pentaerythritol triacrylate, the results of the traditional 2-week and 3-month toxicity studies, and the protocol and results for the 6-month transgenic mouse study of trimethylolpropane triacrylate. Effects observed were hyperplasia, hyperkerotosis, inflammation, and squamous cell papilloma of the skin at the site of chemical application in males and females, plus skin carcinoma in females, and myelodysplasia (a hematopoietic disorder) in males and females. In the standard mouse bioassay system the tumor response would have been judged “clear evidence of carcinogenic activity.” Dr. J.R. Hailey, NIEHS, described the histologic characterization of the skin lesions observed and contrasted the more severe inflammatory responses to chemical exposure in the skin of B6C3F₁ mice to the milder inflammatory response in Tg.AC mice.

Dr. Elwell, the first principal reviewer, agreed that the squamous cell neoplasms observed in the trimethylolpropane triacrylate study could be considered a positive response. He asked for more information on the response to the positive control (TPA) and expressed concern that the papilloma response was observed only at doses that also caused skin inflammation.

Dr. Storer, the second principal reviewer, suggested that a different form of conclusion other than “evidence of carcinogenic activity” would be more appropriate to describe results in the Tg.AC model. He also argued that it was unclear that the model would give equivalent responses as the classic skin promotion model. He inquired if a time sequence of histologic observations might help distinguish between two mechanisms for tumor formation, particularly in the forestomach: systemic exposure to inflammatory cytokines as a consequence of skin irritation or direct oral exposure to the chemical from grooming.

Dr. Piegorsch, the third principal reviewer, felt that the Dunson statistical model was reasonable, noting it may be specific for the Tg.AC system.

Dr. Chhabra noted that the systemic effect was seen only in the high dose female group in the trimethylolpropane triacrylate study. Dr. French added that the dose regimen was determined operationally based on TPA doses that provided a robust response without being overtly toxic. In response to Dr. Storer’s question about time progression of tumorigenesis, Dr. Chhabra noted that the papillomas formed quickly, in a matter of a few weeks, and

Dr. French added that the papillomas kept developing with chemical administration, so there was no acclimation or adaptation to exposure. Dr. Hailey said that the hematopoietic proliferation was thought to be associated with the myeloid rather than the erythroid component and thus more likely attributable to the inflammatory response rather than systemic exposure. Dr. Storer asked if one could infer that the zeta-globin promoter construct of the transgene was responsive to the inflammatory cytokines. Dr. French answered that, while that was a possibility, the proliferation more likely was a generalized response of the hematopoietic system.

Dr. Thrall said that use of complete blood count would have helped discern whether myelodysplasia, a preleukemic condition, or just an inflammatory response, occurred. Dr. Hailey agreed.

Dr. Walker inquired if there would be a qualitative difference in interpretation of response if some gene other than ras (for example, green fluorescent protein) were joined to and activated by the zeta-globin promoter. That is whether cancer or some other gene expression was the endpoint of the model. Dr. French replied that there were two contexts for the expression of the gene in the Tg.AC model, that it was correctly turned on at day 12 of the embryogenesis, and that other regulatory control regions were also being brought into play.

Dr. Ho also questioned whether Tg.AC could be termed a cancer model and inquired if functional genomics or chromosomal characterizations had been done for the observed neoplasms. Dr. French replied that the primary focus had been on the downstream events for ras expression: p53 mutation or inactivation. About 30% of the metaphase cells showed trisomy at chromosome 15, but changes in chromosome number did not seem a prerequisite for expression.

Dr. Vore asked about the relationship between inflammation or wounding and papilloma formation. Dr. French cited examples of studies where both effects were observed and other studies where either inflammation or papilloma formation occurred without the other. Dr. Chhabra added that one of the dose groups in the companion pentaerythritol triacrylate study was another such example.

Dr. Boekelheide asked if any difference in responses had been observed between sexes, and if the available surface changed once papillomas began forming.

Dr. French noted that in a study of benzene the magnitude of response was greater in males. Whether that was due to a hormonal difference or because of different animal housing conditions was speculative. He also felt that any additional dosing after papillomas had begun forming was superfluous because the process was irreversible.

Dr. J. Van Miller, representing the American Chemistry Council (ACC) discussed the general class of chemicals known as specialty acrylates and methacrylates (SAM), the use of monomeric forms of SAM in producing cross-linked polymers, and a series of industry studies on two representative chemicals from this group (triethylene glycol diacrylate and the corresponding methacrylate). He noted that skin irritation is characteristic of SAM, but they are not carcinogenic in the bioassays conducted by the ACC. He suggested that the skin tumors observed in the present Tg.AC studies may have been driven by irritation and urged that conclusions about carcinogenicity be withdrawn until the mechanism of papilloma formation was clarified. Dr. J. Allen, representing the ACC, also addressed the severe dermal toxicity in the trimethylolpropane triacrylate and pentaerythritol triacrylate studies and suggested that the observed tumors resulted from nonspecific skin toxicity.

Because of the general similarity of findings between the two acrylate studies, the panel agreed to forego a formal presentation on the companion pentaerythritol triacrylate study. Dr. Walker inquired about the seeming low purities of pentaerythritol triacrylate cited in the report. Dr. Chhabra explained that these were highly reactive materials and that those measures were just of the monomer, whereas the technical grade material consisted of a mixture including oligomers and other monofunctional acrylates.

Dr. Drinkwater then turned the discussion to the general question of the use of transgenic models by the NTP, noting that suggestions have ranged from use as a preliminary screen to complete replacement of the conventional bioassay. Dr. Ho expressed optimism that the shorter time involved in the transgenic assays would enable rapid decisions about which tests would be most appropriate for a given chemical. Dr. Storer differentiated between reporter models, such as the Tg.AC model, and oncogene or tumor suppressor gene models such as p53 or ras-H2. He felt the latter might merit conclusions about carcinogenicity, whereas systems such as the Tg.AC model would more appropriately be used as part of larger summaries of a collection of studies. Dr. Piegorsch noted that in an NTP evaluation of various strategies for identifying carcinogens, the strongest concordance came from a combination of data from p53 and traditional rat bioassays and genotoxicity. Responding to Dr. Drinkwater’s suggestion, Dr. Storer agreed the Tg.AC studies might fit better in a different type of Report than the Technical Report series.

Drs. Walker and Elwell inquired about how positive and negative results from transgenic studies would be used in decisions about whether to perform additional testing. Dr. Klaunig concurred with the notion of using the transgenic models in a triage approach for testing and also emphasized the need to understand the mechanism for tumor formation in such models. Regarding the type of interpretive conclusion that can be drawn from transgenic models, Dr. Roberts noted that many of the transgenic models might not be predictive for carcinogenicity per se. Dr. Storer felt that while the p53 or ras-H2 models more closely approximated the normal tumorigenic processes, the Tg.AC model was more questionable in that regard. Dr. Walker concurred.

No vote was taken on the conclusion statements in the draft reports.