Contribution of toxicological pathology to occupational health: lung carcinogenicity of fibrous and particulate substances in rats.

In this review, we focus on the rat pulmonary carcinogenicity of two solid substances, fibrous multi-walled carbon nanotube (MWCNT) and particulate indium tin oxide (ITO). Inhalation exposure to MWNT-7, a type of MWCNTs, and ITO induced lung carcinogenicity in both male and female rats. Toxicity to the alveolar epithelium is induced by macrophages undergoing frustrated phagocytosis or frustrated degradation of engulfed particles (referred to as frustrated macrophages). Melted macrophage contents contribute significantly to development of hyperplasia of the alveolar epithelium, which eventually results in the induction of lung carcinoma. MWNT-7 and ITO induce secondary genotoxicity; consequently, a no-observed-adverse-effect level can be applied to these materials rather than benchmark doses that are used for non-threshold carcinogens. Thus, establishing occupational exposure limit values for MWNT-7 and ITO based on the existence of a carcinogenic threshold is reasonable.

Carcinogenicity and chronic toxicity of butyl methacrylate in rats and mice by a two-year inhalation study.

We conducted a two-year inhalation study of butyl methacrylate using F344/DuCrlCrlj rats and B6D2F1/Crl mice. Rats were exposed to 0, 30, 125 and 500 ppm (v/v) and mice were exposed to 0, 8, 30 and 125 ppm (v/v) using whole-body inhalation chambers. Non-neoplastic lesions developed in the nasal cavities of both rats and mice, but neoplastic lesions were not found. There was also a positive trend in the incidence of large granular lymphocytic (LGL) leukemia in the spleen of male rats. No changes were observed in female rats. Overall, there is some evidence of carcinogenicity in male rats, but there is no evidence of carcinogenicity in female rats. In male mice, there was a positive trend by Peto's test in the incidence of hepatocellular adenomas, and the incidence of hepatocellular adenomas and hepatocellular carcinomas combined was significantly increased compared to the controls by Fisher's exact test in the 30 ppm exposed male group. In female mice, the incidence of hemangiosarcoma in all organs combined showed a positive trend by Peto's test. Therefore, there is some evidence of carcinogenicity in male mice, and there is equivocal evidence of carcinogenicity in female mice.

PET With 11C-Methyl-l-Methionine as a Predictor of Consequential Outcomes at the Time of Discontinuing Temozolomide-Adjuvant Chemotherapy in Patients With Residual IDH-Mutant Lower-Grade Glioma.

PURPOSE: The aim of this study was to clarify whether PET with 11C-methyl-l-methionine (11C-met PET) can predict consequential outcomes at the time of discontinuing temozolomide (TMZ)-adjuvant chemotherapy in patients with residual isocitrate dehydrogenase gene (IDH)-mutant lower-grade glioma. PATIENTS AND METHODS: Among 30 patients showing residual lesions of IDH-mutant lower-grade glioma, we compared the tumor-to-normal brain tissue ratio of standardized uptake values (SUVT/N) from 11C-met PET at the time of discontinuing TMZ-adjuvant chemotherapy with putative predictive factors including age, Karnofsky Performance Scale, number of courses of adjuvant therapy, residual tumor size, and promotor methylation status of O6-methylguanine-DNA methyl-transferase gene (MGMT). For each factor, progression-free survival (PFS) was compared between groups divided by cutoff values, determined to predict tumor relapse using receiver operating characteristic curves for each factor. Univariate and multivariate analyses were conducted using log-rank testing and Cox regression analysis, respectively. In addition, PFS was compared between patients grouped by combined findings from multiple predictors identified from univariate and multivariate analyses. RESULTS: Univariate and multivariate analyses identified SUVT/N from 11C-met PET and MGMT methylation status as independent predictors of outcomes after TMZ discontinuation. When comparing 3 groups assigned by the combination of MGMT and SUVT/N findings, PFS differed significantly among groups. CONCLUSIONS: The present study suggested that 11C-met PET at the time of discontinuing TMZ-adjuvant chemotherapy allows prediction of outcomes at least comparable to MGMT methylation status in patients with residual IDH-mutant lower-grade glioma. Further, 11C-met PET allows more precise prediction of outcomes by assessment in combination with MGMT findings.

Impacts on Histological Features and 11C-Methyl-L-methionine Uptake After "One-Shot" Administration with Bevacizumab Before Surgery in Newly Diagnosed Glioblastoma.

BACKGROUND: Bevacizumab (BEV), an antiangiogenic agent, induces dramatic normalization of the tumor vasculature in glioblastoma. This study aimed to clarify how one-time administration of BEV changes histological features in glioblastoma and how histological changes affect the uptake of 11C-methyl-L-methionine (11C-met) as an amino-acid tracer. MATERIALS AND METHODS: Subjects were 18 patients with newly diagnosed glioblastoma who were assigned to two groups: BEV group, single intravenous administration of BEV before surgical tumor removal; and control group, surgical tumor removal alone. After surgery, we compared the densities of tumor cells and microvessels, and microvascular structures including vascular pericytes and L-type amino acid transporter-1 (LAT1) between the BEV and control groups. Correlations between 11C-met uptake on positron emission tomography before surgery, microvascular density, and LAT1 expression were assessed in each group. RESULTS: BEV induced significant reductions in microvascular density, while tumor cell density and proliferation were retained in the BEV group. Percentages of vessels with pericytes and vascular endothelium with LAT1 expression were lower in the BEV group than in controls. Uptake of 11C-met correlated significantly with microvascular density in the BEV group but not with LAT1expression. CONCLUSIONS: The present study showed that even one course of BEV administration induced reductions in microvessels, vascular pericytes, and LAT1 expression in glioblastomas. One course of BEV therapy also reduced 11C-met uptake, which might have been largely attributed to reductions in microvessels rather than reductions in LAT1 expression.

Comparisons Between PET With 11C-Methyl-L-Methionine and Arterial Spin Labeling Perfusion Imaging in Recurrent Glioblastomas Treated With Bevacizumab.

PURPOSE: The aim of this study was to clarify whether arterial spin labeling (ASL) perfusion imaging can assess biological effects from bevacizumab (BEV) therapy as reliably as PET with C-methyl-L-methionine (C-met-PET). MATERIALS AND METHODS: Twenty-four patients with recurrent glioblastoma were examined using both ASL and C-met-PET before and 4 and 8 weeks after starting BEV treatment. Tumor-to-normal brain (T/N) ratios, fluctuations in T/N ratio, and tumor volumes were compared between ASL and C-met-PET. Accuracy of predicting patient with long progression-free survival (PFS) was assessed for T/N ratios and fluctuations for ASL and C-met-PET in each phase and in each period using receiver operating characteristic curves. Between 2 groups of patients assigned by cutoff values from receiver operating characteristic curves, PFS was compared in each phase or in each period. RESULTS: T/N ratios, fluctuations in ratio, and tumor volumes correlated significantly between ASL and C-met-PET at all time points and all periods. Arterial spin labeling was eligible as a predictor for long PFS only in assessment of fluctuations in T/N ratio. However, the most accurate predictors for long PFS were T/N ratio from C-met-PET at 8 weeks and the fluctuation from baseline to 4 weeks in T/N ratio from C-met-PET. CONCLUSIONS: Blood flows on ASL correlated with accumulations of C-met on PET in recurrent glioblastoma under BEV treatment. Although C-met-PET offered superior accuracy for predicting patients with long PFS from time points, ASL offered reliable prediction of long PFS, provided that fluctuations in T/N ratio between consecutive scans are assessed.

Carcinogenicity of multi-walled carbon nanotubes: challenging issue on hazard assessment.

OBJECTIVES: This report reviews the carcinogenicity of multi-walled carbon nanotubes (MWCNTs) in experimental animals, concentrating on MWNT-7, a straight fibrous MWCNT. METHODS: MWCNTs were administered to mice and rats by intraperitoneal injection, intrascrotal injection, subcutaneous injection, intratracheal instillation and inhalation. RESULTS: Intraperitoneal injection of MWNT-7 induced peritoneal mesothelioma in mice and rats. Intrascrotal injection induced peritoneal mesothelioma in rats. Intratracheal instillation of MWCNT-N (another straight fibrous MWCNT) induced both lung carcinoma and pleural mesothelioma in rats. In the whole body inhalation studies, in mice MWNT-7 promoted methylcholanthrene-initiated lung carcinogenesis. In rats, inhalation of MWNT-7 induced lung carcinoma and lung burdens of MWNT-7 increased with increasing concentration of airborne MWNT-7 and increasing duration of exposure. CONCLUSIONS: Straight, fibrous MWCNTs exerted carcinogenicity in experimental animals. Phagocytosis of MWCNT fibers by macrophages was very likely to be a principle factor in MWCNT lung carcinogenesis. Using no-observed-adverse-effect level-based approach, we calculated that the occupational exposure limit (OEL) of MWNT-7 for cancer protection is 0.15 µg/m3 for a human worker. Further studies on the effects of the shape and size of MWCNT fibers and mode of action on the carcinogenicity are required.

Development of Correction for Signal-to-Noise Ratio Using a T2* With Improved Phase Method.

OBJECTIVE: This study aims to improve the signal-to-noise ratio (SNR) of the phase image focusing T2 and to develop an improved phase (iPhase) image acquired high SNR. METHODS: The iPhase images of phantom and brain were acquired with multi-echo spoiled gradient-echo. The phantom component was a gadopentetate dimeglumine (Gd-DTPA) solution made of different concentrations (0.1, 0.5, and 1 wt%) and Gd-DTPA (0.1, 0.5, and 1 wt%) with agar (1.0 wt%). We applied the iPhase image to susceptibility weighed image (SWI) and evaluated SNR of SWI. RESULTS: In phantom study, SNRs of conventional SWI at each sample were 19.8, 15.7, 7.4, 20.0, 17.4, and 27.3, respectively. Signal-to-noise ratios of SWI derived from iPhase method were 29.5, 33.7, 21.7, 28.5, 24.3, and 14.7, respectively. Then, the SNR showed an improvement of 196% at maximum (for the Gd-DTPA 1 wt% sample). In healthy volunteer study, SWI derived from iPhase method had the good contrast between white matter and gray matter. CONCLUSIONS: The iPhase image was able to improve the phase SNR. Moreover, iPhase method makes it possible to obtain a high SNR image when applying to SWI.

In vitro chromosomal aberrations induced by various shapes of multi-walled carbon nanotubes (MWCNTs).

OBJECTIVES: IARC has classified one type of multi-walled carbon nanotubes (MWCNTs), MWNT-7, as possibly carcinogenic to humans (Group 2B); however, other types of MWCNT were categorized as not classifiable as to their carcinogenicity to humans (Group 3). In vitro chromosomal aberration assays of MWNT-7 showed polyploid formation but not structural abnormalities. This study investigated the influence of the shape and size of MWCNT on in vitro induction of chromosomal aberrations. METHODS: Microscopic analysis and viable cell counting were used to assay for chromosomal aberrations and cytotoxicity induced in a Chinese hamster lung cell line (CHL/IU) exposed to different MWCNTs. RESULTS: Using scanning electron microscopy, seven MWCNTs were classified into three types: straight fibrous, curved fibrous, and tangled. The straight fibrous MWCNTs were the strongest inducers of polyploidy and the most cytotoxic among the three types of MWCNTs. The curved fibrous MWCNTs induced more polyploidy than the tangled MWCNTs, and the cytotoxicity of both types seemed to be a reflection of their induction of polyploidy. None of the seven MWCNTs induced structural chromosomal aberrations. CONCLUSION: The non-clastogenicity of the MWCNTs indicates that the MWCNTs may not interact directly with DNA. Since the straight fibrous MWCNTs, which exhibit a structure similar to asbestos, were the strongest inducers of polyploidy, MWCNT shape may be an important factor in induction of polyploidy. We hypothesize that CHL/IU cells endocytosed MWCNTs and formed endosomes with shapes corresponding to those of the endocytosed MWCNTs, and that the long axis diameter of the endosome is important in the capability of MWCNTs to induce polyploidy.

MRI and 11C-methyl-L-methionine PET Differentiate Bevacizumab True Responders After Initiating Therapy for Recurrent Glioblastoma.

PURPOSE: Normalization of tumor vasculature after administering bevacizumab (BEV) makes assessment of therapeutic response using MRI difficult. The aim of this study was to clarify whether PET with C-methyl-L-methionine (MET-PET) would supplement MRI assessing of response after initiating BEV in glioblastoma. METHODS: Twenty patients with recurrent glioblastoma were treated with biweekly BEV plus temozolomide. Both MRI and MET-PET were performed before treatment (baseline) and at 4 and 8 weeks after starting treatment. Results on MRI (response or nonresponse) were compared with those on MET-PET, with response defined as a tumor-to-normal brain ratio of SUV (SUVT/N) of less than 1.6. Progression-free survival (PFS) was compared between responders and nonresponders on MRI alone and MET-PET alone. Progression-free survival was also compared between patients showing response on both MRI and MET-PET and patients showing response on MRI but nonresponse on MET-PET at each time point. RESULTS: PFS was significantly longer in responders than nonresponders on both MRI at 4 and 8 weeks and MET-PET at 8 weeks, whereas MET-PET at 4 weeks provided no information regarding outcomes. Combined assessment with MRI and MET-PET at 4 weeks was not provide predictive of PFS, whereas patients showing response on both modalities (true responders) at 8 weeks exhibited significantly longer PFS than did patients showing response on MRI but nonresponse on MET-PET (pseudoresponders). CONCLUSIONS: Combined assessment with MRI and MET-PET at 8 weeks can differentiate true responders who are predicted to show more favorable prognosis from pseudoresponders.

High-uptake areas on positron emission tomography with the hypoxic radiotracer (18)F-FRP170 in glioblastomas include regions retaining proliferative activity under hypoxia.

OBJECTIVE: The aim was to evaluate the proliferative activity of high-uptake areas on positron emission tomography (PET) with the hypoxic cell radiotracer, 1-(2-[(18)F]fluoro-1-[hydroxymethyl]ethoxy)methyl-2-nitroimidazole (FRP170). METHODS: Thirteen patients with glioblastoma underwent FRP170 PET before tumor resection. During surgery, tumor specimens were stereotaxically obtained from regions corresponding to high (high-uptake areas, HUAs) and relatively low (low-uptake areas, LUAs) accumulation of FRP170. We compared immunohistochemical staining for Ki-67 and hypoxia-inducible factor (HIF)-1α between HUA and LUA. RESULTS: HIF-1α index was significantly higher in HUAs than in LUAs. In contrast, mean Ki-67 indices did not differ significantly between HUAs and LUAs. CONCLUSIONS: Findings for HIF-1α index clearly indicated that HUAs on FRP170 PET represented hypoxic regions in glioblastoma. However, findings of Ki-67 index suggest that HUAs on FRP170 PET include regions retaining proliferative activity regardless of tissue hypoxia.

The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks.

BACKGROUND: The toxicity of multi-walled carbon nanotubes (MWCNT) may be related to the immune system. The objective of this study was to obtain information for immunotoxic mechanisms of MWCNT in situ. METHODS: Using whole-body inhalation, male and female rats were exposed to 0, 0.2, 1 or 5 mg MWCNT/m³ for 13 weeks. Thereafter, spleens were recovered from the rats. Real-time PCR was done to assess expression of TNFα, IL-1ß, IL-6, IL-10, MCP-1 and MIP-1α mRNA in the splenic macrophages; splenic T-lymphocytes were examined for IL-2 and TGF-ß1 mRNA expression. RESULTS: The relative expression of IL-1ß mRNA in the cells from female rats exposed to 5 mg MWCNT/m³ was significantly higher than that in control cells. For IL-6 and IL-10, cells from rats in the 0.2 and 5 mg MWCNT/m³ had significantly higher mRNA expressions than did cells from controls. Expression of IL-1ß, IL-6 and TNFα genes in cells from males in all exposure groups were higher than in control cells. Expression of MIP-1α in the cells from female 5-mg group was significantly higher than that in cells in the control. Only IL-2 was expression reduced, i.e. cells from male and female rats in all MWCNT groups had significantly lower mRNA expressions than control cells. CONCLUSIONS: Systemic inflammation would likely occur in rats (or other hosts) exposed to MWCNT via inhalation due to increases in the expression of inflammatory cytokines in splenic macrophages. Moreover, decreases in IL-2 expression in T-lymphocytes may be critical to the potential reductions in anti-tumor responses in MWCNT-exposed hosts.

Development of an automatic ROI setting program for input function determination in 99mTc-ECD non-invasive cerebral blood flow quantification.

Non-invasive quantitative measurements are useful for clinical study as these are simple and pain-free procedures. A new non-invasive semi-automatic quantitative measurement method, the improved brain uptake ratio (IBUR) method using (99m)Tc-ECD SPECT, has recently been reported. If an automatic ROI setting algorithm could be developed to determine the input function for the IBUR method, analysis of regional cerebral blood flow (rCBF) can be completed within a few min without recourse to complex techniques, through a fully automatic rCBF analysis program. The purpose of this study was to develop an automatic input function determination program for (99m)Tc-ECD non-invasive cerebral blood flow quantification and to confirm the feasibility of use of this program. The images of 15 consecutive patients who underwent both (99m)Tc-ECD chest RI angiography and SPECT examinations were used for development of the automatic arterial input function program. The images of 69 consecutive patients were used for validation of the program. The coincidence ratio between the ROI automatic method and the manual setting method was 98%. The mean difference in the ROI location was ±6.4 mm in the X direction and ±8.6 mm in the Y direction. Individual rCBF values obtained using these independent techniques were also reasonably well correlated (r = 0.95). The total time for the IBUR analysis using the automatic method is 2-3 min as compared to 20-30 min for the current analysis method. This technique improves the throughput of nuclear medical examinations.

Development of a new multi-walled carbon nanotube (MWCNT) aerosol generation and exposure system and confirmation of suitability for conducting a single-exposure inhalation study of MWCNT in rats.

Because the primary route of human exposure to multi-walled carbon nanotube (MWCNT) is via inhalation, a new dry MWCNT aerosol generation and exposure system for whole-body inhalation exposure using a cyclone and sieve has been developed. The system was tested for operational performance at 0.2, 1 and 5 mg/m(3). Additionally, it was examined whether this system can be employed in animal whole-body inhalation studies by exposing rats to MWCNT aerosol for 6 h at 5 mg/m(3). The system could consistently provide aerosols with a similar particle size distribution and configuration at all the target exposure concentrations. Almost all MWCNTs were fibrous, and the presence of many well-dispersed, nano-sized particles was confirmed. Additionally, the animal study revealed that large amounts of MWCNTs were inhaled into the lung, resulting in an inflammatory response, with increased LDH and albumin levels, and granulomatous change. Therefore, the aerosol generation and exposure system appears useful for MWCNT inhalation studies using rats.

Standardized uptake value in high uptake area on positron emission tomography with 18F-FRP170 as a hypoxic cell tracer correlates with intratumoral oxygen pressure in glioblastoma.

PURPOSE: The aim of this study was to clarify the reliability of positron emission tomography (PET) using a new hypoxic cell tracer, 1-(2-[(18)F]fluoro-1-[hydroxymethyl]ethoxy)methyl-2-nitroimidazole ((18)F-FRP170). PROCEDURES: Twelve patients with glioblastoma underwent (18)F-FRP170 PET before tumor resection. Mean standardized uptake value (SUV) and normalized SUV were calculated at regions within a tumor showing high (high-uptake area) and relatively low (low-uptake area) accumulations of (18)F-FRP170. In these areas, intratumoral oxygen pressure (tpO2) was measured using microelectrodes during tumor resection. RESULTS: Mean tpO2 was significantly lower in the high-uptake area than in the low-uptake area. A significant negative correlation was evident between normalized SUV and tpO2 in the high-uptake area. CONCLUSION: The present findings suggest that high accumulation on (18)F-FRP170 PET represents viable hypoxic tissues in glioblastoma.

Validation of novel calibration scheme with traceable point-like (22)Na sources on six types of PET scanners.

OBJECTIVE: To improve the reliability and convenience of the calibration procedure of positron emission tomography (PET) scanners, we have been developing a novel calibration path based on traceable point-like sources. When using (22)Na sources, special care should be taken to avoid the effects of 1.275-MeV γ rays accompanying ß (+) decays. The purpose of this study is to validate this new calibration scheme with traceable point-like (22)Na sources on various types of PET scanners. METHOD: Traceable point-like (22)Na sources with a spherical absorber design that assures uniform angular distribution of the emitted annihilation photons were used. The tested PET scanners included a clinical whole-body PET scanner, four types of clinical PET/CT scanners from different manufacturers, and a small-animal PET scanner. The region of interest (ROI) diameter dependence of ROI values was represented with a fitting function, which was assumed to consist of a recovery part due to spatial resolution and a quadratic background part originating from the scattered γ rays. RESULTS: The observed ROI radius dependence was well represented with the assumed fitting function (R (2) > 0.994). The calibration factors determined using the point-like sources were consistent with those by the standard cross-calibration method within an uncertainty of ±4 %, which was reasonable considering the uncertainty in the standard cross-calibration method. CONCLUSION: This novel calibration scheme based on the use of traceable (22)Na point-like sources was successfully validated for six types of commercial PET scanners.

Hepatotumorigenicity of ethyl tertiary-butyl ether with 2-year inhalation exposure in F344 rats.

Carcinogenicity of ethyl tertiary-butyl ether (ETBE) was examined with inhalation exposure using F344/DuCrlCrlj rats. Groups of 50 male and 50 female rats, 6 week old at commencement, were exposed to ETBE at 0, 500, 1,500 or 5,000 ppm (v/v) in whole-body inhalation chambers for 6 h/day, 5 days/week for 104 weeks. A significant increase in the incidence of hepatocellular adenomas was indicated in males exposed at 5,000 ppm, but not in females at any concentration. In addition, significantly increased incidences of eosinophilic and basophilic cell foci were observed in male rats at 5,000 ppm. Regarding non-neoplastic lesions, rat-specific changes were observed in kidney, with an increase in the severity of chronic progressive nephropathy in both sexes at 5,000 ppm. Increased incidences of urothelial hyperplasia of the pelvis were observed at 1,500 ppm and above, and mineral deposition was apparent in the renal papilla at 5,000 ppm in males. There were no treatment-related histopathological changes observed in any other organs or tissues in either sex. The present 2-year inhalation study demonstrated hepatotumorigenicity of ETBE in male, but not in female rats.

Improvement of the (99m)Tc-ECD brain uptake ratio (BUR) method for measurement of cerebral blood flow.

OBJECTIVE: The brain uptake ratio (BUR) method for the (99m)Tc-ECD SPECT, a non-invasive measurement method of rCBF, has been used in clinical practice in Japan, because it is simple to use. However, the accuracy of this method is limited, as it has problems in the determination of input function and the regression equation. The purpose of this study is to improve the BUR method by reconstructing the determination process of the input function and regression equation based on measurement of the rCBF by H (2) (15) O PET. METHOD: The input function was obtained by setting the region of interest on the ascending aorta instead of the aortic arch. The 3DSRT algorithm was used to obtain the anatomically standardized rCBF, and developed a semi-automatic analyzing software using C++ in order to stabilize the repeatability of the improved BUR (IBUR) method. The regression equation for the IBUR method was obtained by the H (2) (15) O PET rCBFs in 15 patients with the arterial blood sampling method. All the measurements in this study were performed with the patient in the resting state. RESULT: A good correlation was observed between the rCBF values measured by H (2) (15) O PET and the regional BURs measured by the IBUR method (r = 0.86, p < 0.0001). The rCBF values were calculated for only 5 min using a semi-automatic analyzing software. CONCLUSION: The BUR method was improved by changing the location of the input function from the aortic arch to the ascending aorta based on arterial blood flow dynamics, and reconstructing regression equation based on the rCBF values obtained using H (2) (15) O PET. This finding indicates the potential clinical usefulness of this method.

Application of the improved BALB/c 3T3 cell transformation assay to the examination of the initiating and promoting activities of chemicals: the second interlaboratory collaborative study by the non-genotoxic carcinogen study group of Japan.

The Non-genotoxic Carcinogen Study Group in the Environmental Mutagen Society of Japan organised the second step of the inter-laboratory collaborative study on one-stage and two-stage cell transformation assays employing BALB/c 3T3 cells, with the objective of confirming whether the respective laboratories could independently produce results relevant to initiation or promotion. The method was modified to use a medium consisting of DMEM/F12 supplemented with 2% fetal bovine serum and a mixture of insulin, transferrin, ethanolamine and sodium selenite, at the stationary phase of cell growth. Seventeen laboratories collaborated in this study, and each chemical was tested by three to five laboratories. Comparison between the one-stage and two-stage assays revealed that the latter method would be beneficial in the screening of chemicals. In the test for initiating activity with the two-stage assay (post-treated with 0.1microg/ml 12-O-tetradecanoylphorbol-13-acetate), the relevant test laboratories all obtained positive results for benzo[a]pyrene and methylmethane sulphonate, and negative results for phenanthrene. Of those laboratories assigned phenacetin for the initiation phase, two returned positive results and two returned negative results, where the latter laboratories tested up to one dose lower than the maximum dose used by the former laboratories. In the exploration of promoting activity with the twostage assay (pretreated with 0.2microg/ml 3-methylcholanthrene), the relevant test laboratories obtained positive results for mezerein, sodium orthovanadate and TGF-beta1, and negative results for anthralin, phenacetin and phorbol. Two results returned for phorbol 12,13-didecanoate were positive, but one result was negative - again, the maximum dose to achieve the latter result was lower than that which produced the former results. These results suggest that this modified assay method is relevant, reproducible and transferable, provided that dosing issues, such as the determination of the maximum dose, are adequately considered. The application of this two-stage assay for screening the initiating and promoting potential of chemicals is recommended for consideration by other research groups and regulatory authorities.