Analysis of historical negative control group data from the rat in vivo micronucleus assay.

A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results.

Derivation of point of departure (PoD) estimates in genetic toxicology studies and their potential applications in risk assessment.

Genetic toxicology data have traditionally been employed for qualitative, rather than quantitative evaluations of hazard. As a continuation of our earlier report that analyzed ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) dose-response data (Gollapudi et al., 2013), here we present analyses of 1-ethyl-1-nitrosourea (ENU) and 1-methyl-1-nitrosourea (MNU) dose-response data and additional approaches for the determination of genetic toxicity point-of-departure (PoD) metrics. We previously described methods to determine the no-observed-genotoxic-effect-level (NOGEL), the breakpoint-dose (BPD; previously named Td), and the benchmark dose (BMD10 ) for genetic toxicity endpoints. In this study we employed those methods, along with a new approach, to determine the non-linear slope-transition-dose (STD), and alternative methods to determine the BPD and BMD, for the analyses of nine ENU and 22 MNU datasets across a range of in vitro and in vivo endpoints. The NOGEL, BMDL10 and BMDL1SD PoD metrics could be readily calculated for most gene mutation and chromosomal damage studies; however, BPDs and STDs could not always be derived due to data limitations and constraints of the underlying statistical methods. The BMDL10 values were often lower than the other PoDs, and the distribution of BMDL10 values produced the lowest median PoD. Our observations indicate that, among the methods investigated in this study, the BMD approach is the preferred PoD for quantitatively describing genetic toxicology data. Once genetic toxicology PoDs are calculated via this approach, they can be used to derive reference doses and margin of exposure values that may be useful for evaluating human risk and regulatory decision making.

Quantitative approaches for assessing dose-response relationships in genetic toxicology studies.

Genetic toxicology studies are required for the safety assessment of chemicals. Data from these studies have historically been interpreted in a qualitative, dichotomous "yes" or "no" manner without analysis of dose-response relationships. This article is based upon the work of an international multi-sector group that examined how quantitative dose-response relationships for in vitro and in vivo genetic toxicology data might be used to improve human risk assessment. The group examined three quantitative approaches for analyzing dose-response curves and deriving point-of-departure (POD) metrics (i.e., the no-observed-genotoxic-effect-level (NOGEL), the threshold effect level (Td), and the benchmark dose (BMD)), using data for the induction of micronuclei and gene mutations by methyl methanesulfonate or ethyl methanesulfonate in vitro and in vivo. These results suggest that the POD descriptors obtained using the different approaches are within the same order of magnitude, with more variability observed for the in vivo assays. The different approaches were found to be complementary as each has advantages and limitations. The results further indicate that the lower confidence limit of a benchmark response rate of 10% (BMDL(10) ) could be considered a satisfactory POD when analyzing genotoxicity data using the BMD approach. The models described permit the identification of POD values that could be combined with mode of action analysis to determine whether exposure(s) below a particular level constitutes a significant human risk. Subsequent analyses will expand the number of substances and endpoints investigated, and continue to evaluate the utility of quantitative approaches for analysis of genetic toxicity dose-response data.

The cyclin-dependent kinase inhibitor p57(Kip2) is epigenetically regulated in carboplatin resistance and results in collateral sensitivity to the CDK inhibitor seliciclib in ovarian cancer.

BACKGROUND: Carboplatin remains a first-line agent in the management of epithelial ovarian cancer (EOC). Unfortunately, platinum-resistant disease ultimately occurs in most patients. Using a novel EOC cell line with acquired resistance to carboplatin: PEO1CarbR, genome-wide micro-array profiling identified the cyclin-dependent kinase inhibitor p57(Kip2) as specifically downregulated in carboplatin resistance. Presently, we describe confirmation of these preliminary data with a variety of approaches. METHODS: Cytotoxicity testing (MTT) and cell cycle blockade assessed drug responsiveness. Methylation specific PCR and pyrosequencing identified sites of promoter methylation in p57(Kip2). siRNA to p57(Kip2) was used to look at the changes in apoptosis of carboplatin treated EOC cells. EOC tissues (20 cases) were assessed for mRNA levels of p57(Kip2). RESULTS: Carboplatin resistance was reversed using 5-aza-cytidine in vitro. Promoter methylation sites and preferential sensitivity to seliciclib were seen in PEO1CarbR cells. Silencing p57(Kip)2 decreased the apoptotic response to the effects of platinum but produced sensitisation to seliciclib. EOC biopsies indicated an association of high levels of p57(Kip2)mRNA with complete responses to chemotherapy and improved outcome. CONCLUSION: We conclude that p57(Kip2) is a candidate biomarker of platinum sensitivity/resistance in EOC and such cases may show preferential response to the cyclin-dependent kinase inhibitor seliciclib.

Reduced adhesion formation following laparoscopic versus open colorectal surgery.

BACKGROUND: Adhesion formation is common after abdominal surgery. This study aimed to compare the extent of adhesion formation following laparoscopic and open colorectal surgery. METHODS: An observational study was undertaken to identify adhesions in patients undergoing laparoscopy after previous laparoscopic or open colectomy. Adhesions were scored according to a system validated for interobserver (median kappa = 0.80) and intraobserver (kappa = 0.82) agreement. The primary endpoint was the overall adhesion score (0-10); a secondary endpoint was the adhesion score at the main incision site (0-6). RESULTS: Forty-six patients were recruited (13 laparoscopic and 33 open colectomy). In most patients (n = 29), laparoscopy was performed for tumour staging before liver resection. The median (interquartile range) overall adhesion score was 7 (5-8) in the open group and 0 (0-3) in the laparoscopic group (P < 0.001). A similar difference was found for the main incision score: 6 (4-6) versus 0 (0-0) (P < 0.001). CONCLUSION: There may be a reduction in adhesion formation following laparoscopic compared with open colectomy, although the small sample size limits this conclusion.

Report of an Expert Panel on the reanalysis by of a 90-day study conducted by Monsanto in support of the safety of a genetically modified corn variety (MON 863).

MON 863, a genetically engineered corn variety that contains the gene for modified Bacillus thuringiensis Cry3Bb1 protein to protect against corn rootworm, was tested in a 90-day toxicity study as part of the process to gain regulatory approval. This study was reanalyzed by Séralini et al. who contended that the study showed possible hepatorenal effects of MON 863. An Expert Panel was convened to assess the original study results as analyzed by the Monsanto Company and the reanalysis conducted by Séralini et al. The Expert Panel concludes that the Séralini et al. reanalysis provided no evidence to indicate that MON 863 was associated with adverse effects in the 90-day rat study. In each case, statistical findings reported by both Monsanto and Séralini et al. were considered to be unrelated to treatment or of no biological or clinical importance because they failed to demonstrate a dose-response relationship, reproducibility over time, association with other relevant changes (e.g., histopathology), occurrence in both sexes, difference outside the normal range of variation, or biological plausibility with respect to cause-and-effect. The Séralini et al. reanalysis does not advance any new scientific data to indicate that MON 863 caused adverse effects in the 90-day rat study.

Indicators of bacterial infection in patients with acute exacerbation of chronic bronchitis for application in clinical trials of antibacterial drugs.

OBJECTIVES: This study examined the accuracy of: (a) patient symptoms; (b) microscopic examination of sputum purulence (>25 WBCs and <10 epithelial cells) and (c) microscopic examination of morphological bacterial cell types, in identifying bacterial infection in patients with an acute exacerbation of chronic bronchitis (AECB) for entry to clinical trials. METHODS: Subjects had a worsening of at least two symptoms from: dyspnoea, sputum volume, and sputum purulence (Anthonisen Type 1 or 2 exacerbation). Sputum samples were collected from all subjects. RESULTS: A total of 97 sputum samples were evaluated. Overall, 58 (60%) subjects were culture-positive; 22 of 29 (76%) subjects with Type 2 exacerbation had a bacterial pathogen isolated compared with 36 of 68 (53%) Type 1 subjects. This difference was not statistically significant. Microscopically purulent samples were found to be significantly more likely to be culture-positive than non-purulent samples. However, the sensitivity (60%) and specificity (67%); and the positive predictive value (73%) and negative predictive value (53%) observed, means that this is not an ideal predictive test for clinical trials. A semi-quantitative approach to Gram staining was identified as a potential indicator of bacterial infection. Sputum specimens with one bacterial cell type present at >10 cells per field, or more than one cell type present with at least one type at a concentration of >25 cells per field, had a high proportion (91%) of culture-positive specimens. CONCLUSIONS: Symptoms alone are a poor indicator of bacterial infection. A semi-quantitative examination of a Gram-stained sputum preparation was the best indicator of bacterial infection. This finding may have relevance in the design of clinical trials of antibacterial drugs in AECB.

Validation and reproducibility of a semi-quantitative Food Frequency Questionnaire for use in elderly Swiss women.

OBJECTIVE: The principal aim of this study was to develop a Swiss Food Frequency Questionnaire (FFQ) for the elderly population for use in a study to investigate the influence of nutritional factors on bone health. The secondary aim was to assess its validity and both short-term and long-term reproducibility. DESIGN: A 4-day weighed record (4 d WR) was applied to 51 randomly selected women of a mean age of 80.3 years. Subsequently, a detailed FFQ was developed, cross-validated against a further 44 4-d WR, and the short- (1 month, n = 15) and long-term (12 months, n = 14) reproducibility examined. SETTING: French speaking part of Switzerland. SUBJECTS: The subjects were randomly selected women recruited from the Swiss Evaluation of the Methods of Measurement of Osteoporotic Fracture cohort study. RESULTS: Mean energy intakes by 4-d WR and FFQ showed no significant difference [1564.9 kcal (SD 351.1); 1641.3 kcal (SD 523.2) respectively]. Mean crude nutrient intakes were also similar (with nonsignifcant P-values examining the differences in intake) and ranged from 0.13 (potassium) to 0.48 (magnesium). Similar results were found in the reproducibility studies. CONCLUSION: These findings provide evidence that this FFQ adequately estimates nutrient intakes and can be used to rank individuals within distributions of intake in specific populations.

Gleason scoring varies among pathologists and this affects clinical risk in patients with prostate cancer.

AIMS: To investigate whether our practice of specialist review of all diagnostic biopsies was necessary to prevent misgrading of referred prostate cancer patients, and whether this misclassification, if any, would have resulted in misclassification of clinical risk grouping (Seattle Risk Grouping [SRG]) and subsequent treatment strategy and prognosis. MATERIALS AND METHODS: Important prognostic indicators for prostate cancer include the presenting prostate-specific antigen (PSA), clinical stage and Gleason sum of the tumour. These three variables are incorporated into the SRG cohorts to establish treatment strategy. Patients with prostate cancer referred for brachytherapy had their prostate biopsies reviewed by a reference pathologist (PD) with a special interest in prostate cancer. We compared the agreement between the scoring of the referring pathologists with that of PD, and evaluated if any differences changed the SRG and therefore the clinical risk and treatment strategy for the patients. RESULTS: In only 52% (43/83) of cases, was there total agreement between the two sets of pathologists. The inter-rater agreement was statistically 'fair' (unweighted kappa statistic 0.27). In 90% (36/40) of cases with disagreement, PD assigned higher Gleason sums. In 40% (16/40) of cases with disagreement, the change in Gleason sum altered the SRG; in one out of 16 cases, the SRG was downgraded from 'intermediate' to 'low' risk disease; in six out of 16 cases, it was upgraded from 'low' to 'intermediate' risk, and, in nine out of 16, from 'intermediate' to 'high' risk. CONCLUSION: Our findings confirm previous reports of only limited correlation between pathologists in reporting Gleason sums. In this study, 19% (16/83) of cases had their grading changed to a level that altered clinical risk, almost always (94%; 15/16) to one that worsened prognosis. This would have significantly affected treatment strategy for these patients, and thus we recommend that all centres ensure accurate Gleason grading by the use of pathologists with special interests in prostate cancer.

Plasma antibody titres to heat shock proteins-60, -65 and-70: their relationship to coronary risk factors in dyslipidaemic patients and healthy individuals.

OBJECTIVE: To investigate the factors that may affect antibody titres to heat shock proteins (Hsp)-60, -65 and -70, and serum C-reactive protein (CRP) concentrations in patients with dyslipidaemia and other features of the metabolic syndrome as defined by ATPIII criteria. MATERIAL AND METHODS: The study comprised 237 dyslipidaemia patients and 135 healthy individuals recruited from amongst university and hospital employees. RESULTS: Compared to the healthy individuals, the dyslipidaemic patients had higher antibody titres to Hsp-60 (p<0.01), Hsp-65 (p<0.001) and Hsp-70 (p<0.05), and higher serum CRP concentrations (p<0.001). The best-fitting multifactorial models revealed that known coronary risk factors explained little of the variation in Hsp antibody titres: 3 % for Hsp-60, 1 % for Hsp-65 and 4 % for Hsp-70 amongst the dyslipidaemic subjects. The corresponding values for the subgroup with the metabolic syndrome were 8 %, 3 % and 1 %, respectively. In contrast, the best-fitting model explained 13.5 % of the variation in serum CRP concentrations among the dyslipidaemic patients, obesity being a major determinant; and 14 % in the subgroup with metabolic syndrome. CONCLUSIONS: The higher antibody titres to Hsp-60, -65, and -70 in the dyslipidaemic patients may be related to a heightened state of immunoactivation associated with atherosclerosis in this group. Our data indicate that antibody titres to these Hsps are not associated with the classical coronary risk factors, although serum high sensitivity (hs)CRP concentrations were significantly related to obesity.

Report and summary of the major conclusions from statistics in genotoxicity testing working group from the International Workshop on Genotoxicity Test Procedures (IWGTP), March 1999.

A working group of five statisticians experienced in the use of statistical methods in mutagenicity reviewed aspects of the statistical analysis of genotoxicity test procedures. Issues discussed included methods for integrating biological importance and statistical significance, the relationship of the experimental unit to the experimental design, and the impact of new developments in statistics and computing. Three major recommendations were made relating to the need for: (1) the effective use of statistical advice in designing interlaboratory and intralaboratory investigations; (2) the development of appropriate experimental designs for new assays; and (3) education and training in the use of statistical methodology in mutagenicity testing. Environ. Mol. Mutagen. 35:260-263, 2000 Published 2000 Wiley-Liss, Inc.

Dose-response and threshold-mediated mechanisms in mutagenesis: statistical models and study design.

The objective of this paper is to review the use, in mutagenesis, of various mathematical models to describe the dose-response relationship and to try to identify thresholds. It is often taken as axiomatic that genotoxic carcinogens could damage DNA at any level of exposure, leading to a mutation, and that this could ultimately result in tumour development. This has led to the assumption that for genotoxic chemicals, there is no discernible threshold. This assumption is increasingly being challenged in the case of aneugens. The distinction between 'absolute' and 'pragmatic' thresholds is made and the difficulties in determining 'absolute' thresholds using hypothesis testing approaches are described. The potential of approaches, based upon estimation rather than statistical significance for the characterization of dose-response relationships, is stressed. The achievement of a good fit of a mathematical model to experimental data is not proof that the mechanism supposedly underlying this model is operating. It has been argued, in the case of genotoxic chemicals, that any effects produced by a genotoxic chemical which augments that producing a background incidence in unexposed individuals will lead to a dose-response relationship that is non-thresholded and is linear at low doses. The assumptions underlying this presumption are explored in the context of the increasing knowledge of the mechanistic basis of mutagenicity and carcinogenicity. The possibility that exposure to low levels of genotoxic chemicals may induce and enhance defence and repair mechanisms is not easily incorporated into many of the existing mathematical models and should be an objective in the development of the next generation of biologically based dose-response (BB-DR) models. Studies aimed at detecting or characterizing non-linearities in the dose-response relationship need appropriate experimental designs with careful attention to the choice of biomarker, number and selection of dose levels, optimum allocation of experimental units and appropriate levels of replication within and repetition of experiments. The characterization of dose-response relationships with appropriate measures of uncertainty can help to identify 'pragmatic' thresholds based upon biologically relevant criteria which can help in the regulatory process.

Genetic variation in the metabolism of coumarin in mouse liver.

The metabolism of 50 microM [3-14C] coumarin to polar products separated by high performance liquid chromatography (HPLC) and covalently bound metabolites in liver microsomes was compared in a series of inbred strains of mice. Coumarin metabolism to total polar products was higher in female than male mice. In all strains, the coumarin 3,4-epoxidation pathway was the major route of metabolism with o-hydroxyphenylacetaldehyde (o-HPA) as the major metabolite. However, in females, there was a major strain difference in the degree of metabolism to coumarin 7-hydroxylase with DBA/2 and 129 having high 7-hydroxycoumarin formation, CBA/Ca having intermediate levels and the other strains low levels. The differences between the strains was much less pronounced in the male mice. There was also evidence for strain variation in metabolism in the quantities of a number of other coumarin metabolites as detected by HPLC analysis of incubate extracts. However, this variation was of a quantitative nature and relatively small. The metabolism of B6C3F1 hybrid mice, in which coumarin had been identified as carcinogenic in a long-term cancer bioassay, was qualitatively similar to that of the other genotypes. The DBA/2 mouse has been suggested as a model for the metabolism of coumarin in humans. The pattern of metabolism found in this strain is different from most other strains. However, the pattern found for all the mouse strains, including DBA/2, differed appreciably from the profiles for other species including humans in the extent of 7-hydroxylation.

Statistical analysis of adaptive response in sister chromatid exchanges in human lymphocytes after treatment with very low and extremely low doses of N-methyl-N'-nitro-N-nitrosoguanidine using a study design to control variability.

Previous studies have been interpreted as suggesting that low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) have an adaptive effect in the cultured lymphocytes of responsive donors (that is, the cells are protected against the mutagenic effects of a subsequent challenge with a higher concentration of MNNG). The objectives of the present study were to investigate, under stringent experimental conditions, whether a protective effect exists at very low and extremely low doses of MNNG (10(-8) and 10(-24) M, respectively). Peripheral blood lymphocytes from a donor considered responsive in a previous study were stimulated to divide and were cultured under standard conditions. Pre-adaptive treatments with dilutions of MNNG were added to the cultures repeatedly before a challenge treatment with MNNG. Bromodeoxyuridine was added at the same time as the challenge treatment and, following mitotic arrest, cells were differentially stained so that the number of sister chromatid exchanges (SCEs) could be counted. The study was designed to address potential criticisms of earlier studies which did not include replicate cultures. Samples of blood were divided into two identical batches for independent processing. Five replicate cultures were prepared for each combination of pre-adaptive and challenge treatments in each batch. The complete experiment was repeated to provide a further test of the consistency of results. Five replicates per treatment combination were chosen in an attempt to provide an experiment of adequate statistical power. Considerable precautions were taken to minimise the effect of factors outside experimental control on the results. Scoring was done by three scorers. In order to minimise inter-scorer variation, 240 cells were scored at each treatment observation (five cells per-scorer, three scorers per culture, four cultures per batch, two batches per experiment and two experiments). The study was designed in this way to take account of the sources of variability to ensure that any response obtained would exceed that obtainable by experimental variability alone. A high level of quality assurance monitoring was undertaken throughout the investigation. Two measures of SCE induction were used: (i) the mean frequency of SCEs; (iii) proportion of cells with at least 20 SCEs. In both experiments, the challenge concentration of MNNG significantly increased SCE frequency. There were, however, highly significant differences between the two experiments. The proportion of high frequency cells (HFCs) in Experiment 1 was increased significantly; the proportion of HFCs was also increased in Experiment 2, but the increase was not statistically significant. The pre-adaptive concentrations of MNNG included an extremely low dilution of 6.8 x 10(-24) M and a very low dilution of 6.8 x 10(-8) M in Experiment 1 and 1.4 x 10(-7) M in Experiment 2. The various pre-adaptive concentrations used had no consistent protective effect against the SCE-inducing capacity of the challenge concentration of MNNG of 6.8 x 10(-6) M. It is concluded that an adaptive response to the alkylating agent MNNG could not be demonstrated in cultured human lymphocytes. Neither a very low nor an extremely low dilution of MNNG elicited an adaptive response in terms of SCE induction (measured either as SCE frequency or as proportion of HFCs). This is in contradiction to previous reports published by us and other groups. This study was carefully designed with large numbers of replicates, a preliminary statistical power calculation, predefined comparisons and extensive quality assurance at each treatment administration. Despite these precautions the variability between scorers and between batches was much larger than anticipated. This resulted in some statistically significant differences, but these are likely to be false positives. Our findings indicate the need for such methodological refinement in human cell adaptive response studies.

Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro comet studies.

A wide range of experimental designs are used in investigations using the Comet assay. The statistical issues associated with this assay are however not particularly unusual or difficult. It is important however to recognize that the sample rather than the cell is the experimental unit. Statistical analyses based upon measures from the individual cells can lead to serious misinterpretation of results. Interpretation of the results of the assay should be related to identifying changes of biological importance rather than relying solely on the P values of specific statistical tests.

Statistical analysis of adaptive response in sister chromatid exchanges in human lymphocytes after treatment with very... to control variability

Previous studies have been interpreted as suggesting that low concentrations of N-methyl-N'-nitrosoguanidine (MNNG) have an adaptive effect in the cultured lymphocytes of responsive donors (that is, the cells are protected against the mutagenic effects of a subsequent challenge with a higher concentration of MNNG). The objectives of the present study were to investigate, under stringent experimental conditions, whether a protective effect...(AU)

Principal Component Analysis of Tissue Scores from Substances used in the COLIPA Eye Irritation Validation Study.

Principal component analyses (PCA) have been carried out on the tissue scores from Draize eye irritation tests on the 55 formulations and chemical ingredients included in the COLIPA Eye Irritation Validation Study. A PCA was carried out on the tissue scores 24, 48 and 72 hours after instillation of the substances. The first Principal Component (PC I) explained 77% of the total variation in the tissues scores and showed a high negative correlation (r=-0.971) with the scores used to derive the Modified Maximum Average Score (MMAS). The second component (PC II) explained 7% of the total variability and contrasted corneal and iris damage with conjunctival damage as in a similar analysis carried out previously on the ECETOC databank. The third component (PCIII), while only explaining about 3% of the variability, identified individuals treated with formulations that were observed to have low corneal opacity but large corneal area scores. This may represent some particular manner of scoring at the laboratory administering the Draize test or a specific effect of some formulations. A further PCA was carried out on tissue scores from observations at 1hr to 21 days. PC I in this analysis explained 62% of the variability and there was a high negative correlation with the sum of all the tissue scores, while PC II explained 14% of the variability and contrasted damage up to 72 hours with damage after 72 hours. A number of formulations were identified with relatively low MMAS scores but tissue damage that persisted. PCA analysis is thus shown to be a powerful method for exploring complex datasets and for identification of outliers and subgroups. It has shown that the MMAS score captures most of the information on tissue scores in the first 72 hours following exposure, and it is unlikely to be of any advantage in using individual tissue scores for comparisons with alternative tests. The relationship of the classifications schemes used by three alternative methods in the COLIPA study with the results of the PCA were investigated and the implications of the effect of persistence of tissue damage for various classifications schemes visualized.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

Quantitative risk assessment and the limitations of the linearized multistage model.

1. Quantifying carcinogenic risk is an important objective for assisting in the assessment and management of risks from chemical exposure. The most widely used of the many mathematical models proposed for extrapolation of carcinogenicity data from animal studies to low dose human exposures is the linearized multistage (LMS) model. This has, in effect, become the default approach for much of Quantitative Risk Assessment (QRA). The practical properties of this model have been investigated. 2. Analysis of stimulated data using the LMS model showed (i) that the Maximum Likelihood Estimate (MLE) of the low dose slope, q1, was unstable and extremely sensitive to small changes in the data; (ii) the 95% Upper Confidence Limit (UCL) estimate, q1*, preferred by the US Environmental Protection Agency (EPA) was insensitive with only small changes in values being obtained for large changes in the data; (iii) data sets where there was no statistical significance could give risk estimates similar to those obtained from data sets with clear dose-related effects; (iv) the size of the values of the Virtually Safe Dose (VSD) obtained did not necessarily relate to the biological interpretation of the data sets; (v) the value of q1* obtained was closely related to the top dose used in the study. 3. Limitations of the LMS model were illustrated by examples of its use in assessing the carcinogenicity of 2, 3, 7, 8-TCDD leading to the conclusion that the existing models are not suitable for routine use in the estimation of the risk from chemical carcinogens. The use of the LMS model has been justified in part by its original derivation from a mathematical model based upon a multistage model of carcinogenesis. However, estimates of the parameters of the model used to provide estimates of low dose risk to humans have no direct relationship to specific biological event in carcinogenesis. Further developments in mathematical models and increased understanding of the biological events underlying the carcinogenesis will lead to more biologically plausible QRA methods which would then justify serious consideration of QRA by regulatory authorities throughout the world.