An expert consortium review of the EC-commissioned report "alternative (Non-Animal) methods for cosmetics testing: current status and future prospects - 2010".

The European cosmetics legislation foresees a review in 2011 and possible postponement of the 2013 marketing ban to enforce the testing ban for systemic and repeated-dose animal tests. For this purpose, a 119-page report commissioned by the European Commission was published recently. Here, a group of 17 independent experts from the US, Europe, and Japan was brought together to evaluate the report. The expert panel strongly endorsed the report and its conclusions. A number of important options not considered were identified; these do not, however, affect the overall conclusions regarding the current lack of availability of a full replacement, especially for the areas of repeated dose toxicity, carcinogenicity testing, and reproductive toxicity, though a roadmap for change is emerging. However, some of these options may provide adequate data for replacement of some animal studies in the near future pending validation. Various recommendations expand the original report. The reviewers agree with the report that there is greater promise in the short term for the areas of sensitization and toxicokinetics. Additional opportunities lie in more global collaborations and the inclusion of other industry sectors.

Toxicity testing in the 21st century: a vision and a strategy.

With the release of the landmark report Toxicity Testing in the 21st Century: A Vision and a Strategy, the U.S. National Academy of Sciences, in 2007, precipitated a major change in the way toxicity testing is conducted. It envisions increased efficiency in toxicity testing and decreased animal usage by transitioning from current expensive and lengthy in vivo testing with qualitative endpoints to in vitro toxicity pathway assays on human cells or cell lines using robotic high-throughput screening with mechanistic quantitative parameters. Risk assessment in the exposed human population would focus on avoiding significant perturbations in these toxicity pathways. Computational systems biology models would be implemented to determine the dose-response models of perturbations of pathway function. Extrapolation of in vitro results to in vivo human blood and tissue concentrations would be based on pharmacokinetic models for the given exposure condition. This practice would enhance human relevance of test results, and would cover several test agents, compared to traditional toxicological testing strategies. As all the tools that are necessary to implement the vision are currently available or in an advanced stage of development, the key prerequisites to achieving this paradigm shift are a commitment to change in the scientific community, which could be facilitated by a broad discussion of the vision, and obtaining necessary resources to enhance current knowledge of pathway perturbations and pathway assays in humans and to implement computational systems biology models. Implementation of these strategies would result in a new toxicity testing paradigm firmly based on human biology.

Raloxifene and selective cell cycle specific agents: a case for the inclusion of raloxifene in current breast cancer treatment therapies.

BACKGROUND: Breast cancer patients are at increased risk of osteoporosis. Contributing factors include age and/or chemotherapy. The selective estrogen modulator, raloxifene (RAL), effective in the prevention of breast cancer and approved for the treatment and prevention of osteoporosis, may prove beneficial in current breast cancer treatment modules. The purpose of this study was to evaluate RAL in combination with 5-fluorouracil (5-FU) and trimetrexate (TMX) to determine the most effective sequence in which to administer these cell cycle specific agents while taking into consideration the cellular mechanism of action. The goal was to maintain cytotoxicity to breast cancer cells and capitalize on the selective estrogen receptor modulatory effects of RAL. MATERIALS AND METHODS: MCF-7 cells were exposed to (i) TMX, 5-FU or RAL alone, or (ii) RAL 24 h prior to 5-FU followed 2 h later by TMX, or (iii) 5-FU 2 h prior to TMX followed 24 h later by RAL. The cell viability was determined using the Quick Cell Proliferation Assay. RESULTS: The growth rate of MCF- 7 cells exposed to early RAL was 68.25 +/- 4.11% that of the control, however, late RAL exposure produced a growth of 34.75 +/- 4.79% that of the control. Late RAL maintained the cytotoxicity of the regimen. The findings were further supported by cell flow cytometry and Western blot analysis data. CONCLUSION: RAL given prior to 5-FU/TMX significantly compromised cytotoxicity to breast cancer cells.

Sequence-dependent administration of raloxifene and 5-fluorouracil/pemetrexed protects against pemetrexed cytotoxicity in human bone marrow.

BACKGROUND: Pemetrexed (Alimta) is a new-generation multitargeted antifolate that inhibits several key enzymes in the de novo pathways of pyrimidine and purine biosynthesis, including thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). Alimta has demonstrated antitumor activity in a broad array of human malignancies, e.g. breast, non-small cell lung cancer, malignant pleural mesothelioma and pancreatic, colorectal, gastric, bladder, head and neck cancer, and is currently in phase III clinical trials. It has been reported that a dose of 600 mg/m2 of pemetrexed showed toxicity to bone marrow and the gastrointestinal system. The aim of this investigation was to evaluate raloxifene (RAL) in combination with 5-fluorouracil (5-FU)/pemetrered multitargeted antifolate (MTA) to determine the most effective regimens and cellular mechanism of action to mitigate pemetrexed cytotoxicity in human bone marrow cells. MATERIALS AND METHODS: In order to determine the sequence-dependent interaction between MTA, 5-FU and RAL on proliferation, cell viability was carried out using the Quick Cell Proliferation Assay by exposing the HS-5 and MCF-7 cells to (i) MTA, 5-FU and RAL alone, or (ii) RAL 24 h prior to 5-FU followed 2 h later by MTA, or (iii) 5-FU 2 h prior to MTA followed 24 h later by RAL. RESULTS: The growth rate in MCF-7 in early RAL was 69 +/- 8.65% and late RAL was 36 +/- 4.6% of the control whereas in bone marrow early RAL was 78 +/- 8.65% and late RAL was 52 +/- 5.49% of the control. The late RAL exhibits significant protection against MTA cytotoxicity in bone marrow. The findings were further supported by cell flow cytometry, apoptosis and Western blot analysis data. CONCLUSION: This study suggests that sequence-dependent administration of RAL (5FU/MTA/RAL), in combination with 5-FU/MTA, protects against MTA toxicity in human bone marrow while maintaining the maximum inhibitory effect of pemetrexed in breast cancer.

Sequence-dependent administration of 5-fluorouracil maintains methotrexate antineoplastic activity in human estrogen-negative breast cancer and protects against methotrexate cytotoxicity in human bone marrow.

BACKGROUND: Breast cancer is a leading cause of morbidity and mortality in women in developed countries and in increasingly developing countries. In general, estrogen receptor (ER)-positive breast cancers have a better prognosis and are often more responsive to anti-estrogen therapy. Unfortunately, ER-negative breast cancers are more aggressive and unresponsive to anti-estrogens. The aim of this investigation was to evaluate the 5-fluorouracil (5-FU) and methotrexate (MTX) combination to determine the most effective regimen considering the mechanism of action in treating ER-negative human breast cancer cells and at the same time mitigating methotrexate cytotoxicity in human bone marrow cells. MATERIALS AND METHODS: In order to determine the sequence-dependent interaction between MTX and 5-FU on proliferation, cell viability was carried out using the Quick Cell Proliferation Assay by exposing the human estrogen negative (MDA-MB-436 and Hs-578T) and bone marrow (HS-5) cells to: (i) MTX and 5-FU alon; (ii) MTX 2 h prior to 5-FU (MTX/5-FU; (iii) 5-FU 2 h prior to MTX (5-FU/MTX). RESULTS: The growth rate in MDA-MB-436 was 23.5 +/- 3.98%, in Hs-578T 30 +/- 5.9% and HS-5 32 +/- 3.1% of the control for MTX/5-FU. Whereas the growth rate in MDA-MB-436 was 28.5 +/- 4.1%, in Hs-578T 34.7 +/- 3.5% and HS-5 68.6 +/- 6.3% of the control for 5-FU/MTX combinations. The later combination exhibits significant protection against MTX cytotoxicity in bone marrow and at same time maintained maximum cytotoxicity in estrogen negative breast cancer cell lines. The findings were further supported by cell flow cytometry, apoptosis and Western blot analysis data. CONCLUSION: The combination of 5-FU/MTX effectively maintains the maximum inhibitory effect of MTX in ER-negative breast cancer and protects against MTX cytotoxicity in human bone marrow.

TestSmart and toxic ignorance.

There are a number of national and international efforts designed to screen chemicals for toxicity. Although the emphasis in terms of the specific chemicals is different, e.g. endocrine disruptors, children's health, High Production Volume (HPV), the European Registration, Evaluation and Authorisation of Chemicals (REACH) programme, the purpose is the same. Each is intended to evaluate the potential toxicity of chemicals to humans and, in some cases, to the environment. How best can these tasks be accomplished? The first need is to provide a realistic prioritisation of which chemicals need to be evaluated. Once identified, a defined decision-tree approach with an emphasis on short-term in vitro assays and new genomic technologies offers the greatest promise. The more practical matter of screening the chemicals would be by using a tiered decision-tree approach. Common features of the approach would be the use of three tiers. The first tier would be a screening/prioritisation tier, the second would provide an initial characterisation of toxicity, and the third would discern mode of action/biological activity. The intent of this approach is to provide a concept that will allow decisions to be made as to which chemicals need to be tested, provide some idea as to their toxicity and finally mode of action, and at the same time, taking into account the Three Rs, reduction, refinement and replacement. Thus, specific batteries of tests are not discussed, as these would need to be tailored to the specific chemicals of concern, e.g. endocrine disruptors, HPV. Neither are regulatory requirements factored into the concept, but the data that would be gathered should consider the possibility of eventual submission of the data obtained by in vitro and other non-traditional approaches by regulatory authorities.