Automated pipeline for classifying Aroclors in soil by gas chromatography/mass spectrometry using modulo compressed two-way data objects.

Seven polychlorinated biphenyls (PCBs) commercial mixtures, Aroclor 1016, 1221, 1232, 1242, 1248, 1254, and 1260, were analyzed by gas chromatography/mass spectrometry (GC/MS) combined with solid phase microextraction (SPME). Three pattern recognition methods: a fuzzy rule-building expert system (FuRES), partial least-squares discriminant analysis (PLS-DA), and a fuzzy optimal associative memory (FOAM) were used to build classification models. Modulo compression was introduced for data preprocessing to extract the characteristic features and compress the data size. Baseline correction and data normalization were also applied prior to data processing. Four GC/MS data set configurations were constructed and used to evaluate the classifiers and data pretreatments including two-way modulo compressed, two-way data, one-way total ion current and one-way total mass spectrum. The results indicate that modulo compression and baseline correction methods significantly improved the performance of the classifiers which resulted in improved classification rates for FuRES, PLS-DA, and FOAM classifiers. By using two-way modulo compressed data sets, the average classification rates with FuRES, PLS-DA, and FOAM were 100±0%, 94.6±0.7%, and 96.1±0.6% for 100 bootstrapped Latin partitions of the Aroclor standards. The classifiers were validated by application to Aroclor samples extracted from soil with no parametric changes except that the calibration set of standards and validation set of soil samples were individually mean centered. The classification rates for the GC/MS modulo 35 compressed data obtained from the Aroclor soil samples with FOAM, FuRES, and PLS-DA were 100%, 96.4%, and 78.6%, respectively. Therefore, a chemometric pipeline for SPME-GC/MS data coupled with chemometric analysis was devised as a fast authentication method for different Aroclors in soil.

Common classification schemes for PCB congeners and the gene expression of CYP17, CYP19, ESR1 and ESR2.

BACKGROUND: Reliable techniques to measure polychlorinated biphenyl (PCB) congeners make the clearer definition of their effects on human health possible. Given that PCBs are classified as endocrine disrupters, we sought to explore the expression of some key genes involved in sex steroid metabolism. OBJECTIVES: To examine common classification schemes of PCB congeners and determine whether exposure to groups classified by mechanism of action alter the gene expression (GE) of CYP17, CYP19, and ESR1 and ESR2. METHODS: GE and exposure to various classifications of lipid-adjusted PCB congeners were examined in 139 daughters of the Michigan Fisheaters' Cohort. Using mixed models analyses and adjusting for age, menopausal status, and current use of oral contraceptives and hormone replacement therapy, GE data were regressed on exposure to PCB congener groupings based on mechanism of action. RESULTS: Three novel findings are elucidated: first, that up-regulation of CYP19 expression is associated with exposure to PCB groupings containing dioxin-like, potentially anti-estrogenic, immunotoxic congeners, including PCB IUPAC #74, #105, #118, #138, #156, #157, #158, #167, and #170 from this cohort. Second, that exposure to similar congeners (PCB IUPAC #105, #156, #157, #158, and #167 in this cohort) but using a classification based solely on hormonal mechanisms of action is associated with increased expression of ESR2. Third, that increased expression of CYP17 is of borderline significance when associated with exposure to PCB IUPAC #118, #138, and #156. CONCLUSIONS: These findings are both counter-intuitive and intriguing. Rather than exhibiting anti-estrogenic effects alone, they suggest that these congeners up-regulate the major enzyme involved in estrogen synthesis and tend to confirm previous findings of links between AhR and ER signaling pathways. Replication of these findings, expansion of the number of genes examined, exploration of mixtures of environmental chemicals, and subsequent study of health outcomes in a larger cohort are future priorities.

Physical and chemical effects of ingested plastic debris on short-tailed shearwaters, Puffinus tenuirostris, in the North Pacific Ocean.

We investigated the plastics ingested by short-tailed shearwaters, Puffinus tenuirostris, that were accidentally caught during experimental fishing in the North Pacific Ocean in 2003 and 2005. The mean mass of plastics found in the stomach was 0.23 g per bird (n=99). Plastic mass did not correlate with body weight. Total PCB (sum of 24 congeners) concentrations in the abdominal adipose tissue of 12 birds ranged from 45 to 529 ng/g-lipid. Although total PCBs or higher-chlorinated congeners, the mass of ingested plastic correlated positively with concentrations of lower-chlorinated congeners. The effects of toxic chemicals present in plastic debris on bird physiology should be investigated.

Multivariate toxicity profiles and QSAR modeling of non-dioxin-like PCBs--an investigation of in vitro screening data from ultra-pure congeners.

The non-dioxin-like PCBs (NDL-PCBs) found in food and human samples have a complex spectrum of adverse effects, but lack a detailed risk assessment. The toxicity profiles of 21 carefully selected PCBs (19 NDL-PCBs) were identified by in vitro screening in 17 different assays on specific endpoints related to neurotoxicity, endocrine disruption and tumor promotion. To ensure that the test results were not affected by polychlorinated dioxins, dibenzofurans or DL-PCB contaminants, the NDL-PCB congeners were thoroughly purified before testing. Principal component analysis (PCA) was used to derive general toxicity profiles from the in vitro screening data. The toxicity profiles indicated different structure-activity relationships (SAR) and distinct mechanisms of action. The analysis also indicated that the NDL-PCBs could be divided into two groups. The first group included generally smaller, ortho-substituted congeners, comprising PCB 28, 47, 51, 52, 53, 95, 100, 101, 104 and 136, with PCB 95, 101 and 136 as generally being most active. The second group comprising PCB 19, 74, 118, 122, 128, 138, 153, 170, 180 and 190 had lower biological activity in many of the assays, except for three endocrine-related assays. The most abundant congeners, PCB 138, 153, 170, 180 and 190, cluster in the second group, and thereby show similar SAR. Two quantitative structure-activity relationship (QSAR) models could be developed that added information to the SAR and could aid in risk assessments of NDL-PCBs. The QSAR models predicted a number of congeners as active and among these e.g., PCB 18, 25, 45 and 49 have been found in food or human samples.

Three structurally different polychlorinated biphenyl congeners (Pcb 118, 153, and 126) affect hormone production and gene expression in the human H295R in vitro model.

Polychlorinated biphenyls (PCB) are ubiquitous environmental pollutants that have been linked to adverse health effects including endocrine disruption. This study compared the mono-ortho-substituted PCB 118 and di-ortho-substituted PCB 153 with the non-ortho-substituted PCB 126, for possible effects on steroid hormone production and on the expression of 10 genes encoding proteins involved in steroidogenesis. The H295R human adenocarcinoma cell line was used as an in vitro model. Cells were exposed for 48 h to solvent control (dimethyl sulfoxide, DMSO) or 6 different concentrations ranging from 40 pM to 4 muM of one of the three test compounds. All three congeners significantly increased the production of estradiol-17beta. PCB 118 produced a rise in progesterone and cortisol in a concentration-dependent manner, similar to PCB 126. Testosterone was significantly reduced in response to PCB 153 but not PCB 118 or PCB 126. All three congeners elevated aldosterone at the highest concentration tested. A significant increase was observed in CYP11B2 mRNA levels in cells exposed to the three congeners. In addition, PCB 126 upregulated CYP19, 3beta-HSD2, StAR, and HMGR mRNA levels at the highest concentration tested, and downregulated CYP21 at 40 nM. In conclusion, all three PCB congeners are capable of modulating steroidogenesis in H295R in a concentration-dependent manner, whereby the hormone profile following PCB 118 exposure resembles that of PCB 126. Where changes in gene expression profile are concerned, exposure to PCB 126 showed the greatest effects.

Spatial and temporal trends of PCBs (polychlorinated biphenyls) in mussel from Galician coast (1998-2008).

PCBs were determined in wild mussel samples collected in several points from Galician Rías (Rías de Ferrol, A Coruña, Muros, Vigo and Arousa), Spain, during the period 1998-2008. The concentration levels of ΣPCBs ranged from 0.62 to 107.5 ng g(-1), w.w. The isomer concentrations in the Mytilus galloprovincialis were in the order hexachlorobiphenyls>pentachlorobiphenyls>tetrachlorbiphenyls>trichlorobiphenyls. CBs 153, 138 and 101 congeners were the most abundant in these samples. Two biological parameters, fat content and condition index (CI), have also been investigated for these mussel samples. Univariate techniques confirm that levels of some compounds, CBs 28, 52, 138, 153, and 180, presented significant relation (p<0.05) with biological parameters. Multivariate techniques of data exploration such as Principal Component Analysis (PCA) showed that spatial trends of PCB levels appeared in the studied samples. Rias de Ferrol and A Coruña presented the highest levels of PCBs and Ría de Arousa the lowest levels of these compounds. In general, temporal trends (linear regressions) showed a decrease of PCB levels along the period 1998-2008.

Selection of non-dioxin-like PCBs for in vitro testing on the basis of environmental abundance and molecular structure.

The non-dioxin-like polychlorinated biphenyls (NDL-PCBs) constitute the major proportion of PCBs found in food and human tissues. It is important to improve our understanding of the toxicity, environmental and human risks associated with the NDL-PCBs, since their toxicology is incompletely characterized and a human health risk assessment is required. This paper discusses the selection of a training set of 20 tri- to hepta-chlorinated biphenyls, PCBs 19,28,47,51,52,53,74,95,100,101,104,118,122,128,136,138,153,170,180, and 190. Suggested for comprehensive screening using in vitro assays to identify critical mechanisms of toxicological action. The selected PCBs form a balanced basis for developing of quantitative structure-activity relationship (QSAR) models for prediction of physicochemical and toxicological properties of non-tested PCB congeners. Chemical and physical properties, environmental abundance and toxicological activities of the congeners were considered during the selection process. A complementary set of PCBs, a reference set, was selected using D-optimal onion design including PCBs 18,20,28,30,37,40,50,54,60,77,82,99,122,132,153,161,170,188,192, and 193. Congeners of this set are well suited for validation of QSAR models developed using the training set. For visualization of the chemical diversity of environmentally abundant PCBs and congeners of the training and reference sets, principal component analysis (PCA) was used. Statistical molecular design was used to verify the structural representation. As a reference structure for dioxin-like PCBs, PCB 126 was added in the training set. The selected set of NDL-PCBs is proposed for use in toxicological testing programs to provide rational basis for risk assessment of the NDL-PCBs.

Associations between prenatal exposure to polychlorinated biphenyls and neonatal thyroid-stimulating hormone levels in a Mexican-American population, Salinas Valley, California.

BACKGROUND: Studies have reported that prenatal exposure to polychlorinated biphenyls (PCBs) may alter neurodevelopment in both humans and animals. Furthermore, prenatal exposure to some PCB congeners and commercial mixtures has been shown to decrease free and total thyroxine (T(4)) blood levels in animals. Because thyroid hormones (TH) are essential for normal neurologic development, it has been suggested that the deleterious neurodevelopmental effect of PCBs may occur through TH disruption. PCBs may in turn affect TH levels by inducing the microsomal enzyme uridinediphosphate glucuronosyltransferase (UDP-GT), which is involved in TH elimination. OBJECTIVES: Our goals were to group PCB congeners based on their potential to induce microsomal enzymes in animals, and to examine the relationship between neonatal TSH levels and prenatal exposure to PCB congeners grouped according to their structure and potential mechanisms of action. METHODS: We measured the concentration of 34 PCB congeners in serum samples collected from 285 pregnant women and the thyroid-stimulating hormone (TSH) levels in their children's blood collected shortly after birth. RESULTS: We found no association between the sum of PCB congeners, the toxic equivalents, or structure-based groupings (mono- or di-ortho substituted congeners), and TSH blood concentration. However, we found a positive association between the sum of congeners suspected to be UDP-GT inducers (more specifically cytochrome P450 2B inducers) in animals and neonatal TSH levels. In individual congener analyses, PCBs 99, 138, 153, 180, 183, 187, 194, and 199 were positively associated with neonatal TSH levels after adjustment for covariates. PCBs 194 and 199 remained significant after adjustment for multiple hypothesis testing. CONCLUSIONS: Our results support grouping PCB congeners based on their potential mechanism of action of enzyme induction when investigating associations with TH. Findings also suggest that PCBs affect TH homeostasis even at the low background level of exposure found in the CHAMA-COS (Center for the Health Assessment of Mothers and Children of Salinas) population.

Development of a gas chromatography/ion trap mass spectrometry based method for the quantification of polybrominated diphenyl ethers and polychlorinated biphenyls in adipose tissue.

A combined gas chromatographic mass spectrometric (GC/MS/MS) method for the determination of seven polybrominated diphenyl ethers (PBDEs) and seven marker polychlorinated biphenyls (PCBs) in adipose tissue has been developed. Adipose tissue was melted and filtered through anhydrous sodium sulphate to obtain pure fat. Clean-up was performed using a glass column containing acidified silica, deactivated alumina and anhydrous sodium sulphate. Polybrominated biphenyl (PBB) 155 and Mirex were added as internal standards for PBDEs and PCBs, respectively. Injection standards, PBB 103 and PCB 143, for PBDEs and PCBs, respectively, were added before analysis with GC/MS/MS. The developed GC/MS/MS method has the advantage of being more selective than single MS methods because matrix effects are largely eliminated. Validation of this method was conducted according to Commission Decision 2002/657/EC. Decision limits for PBDEs and PCBs ranged from 0.06-0.15 ng g(-1) and from 0.35-1.22 ng g(-1), respectively. Detection capabilities were all between 0.23-0.55 ng g(-1) for PBDEs and between 0.98-2.29 ng g(-1) for PCBs. Precision, recovery, bias and selectivity were tested, with satisfactory results.

A reassessment of the nomenclature of polychlorinated biphenyl (PCB) metabolites.

Polychlorinated biphenyls (PCBs) are a widespread class of persistent organic chemicals that accumulate in the environment and humans and are associated with a broad spectrum of health effects. PCB biotransformation has been shown to lead to two classes of PCB metabolites that are present as contaminant residues in the tissues of selected biota: hydroxylated (HO) and methyl sulfone (MeSO2) PCBs. Although these two types of metabolites are related structures, different rules for abbreviation of both classes have emerged. It is important that a standardized nomenclature for the notation of PCB metabolites be universally agreed upon. We suggest that the full chemical name of the PCB metabolite and a shorthand notation should be adopted using the International Union of Pure and Applied Chemistry's chemical name/original Ballschmiter and Zell number of the parent congener, followed by the assignment of the phenyl ring position number of the MeSO2- or HO-substituent. This nomenclature provides a clear, unequivocal set of rules in naming and abbreviating the PCB metabolite structure. Furthermore, this unified PCB metabolite nomenclature approach can be extended to the naming and abbreviation of potential metabolites of structurally analogous contaminants such as HO-polybrominated biphenyls and HO-polybrominated diphenyl ethers.

Potential human cancer risks from exposure to PCBs: a tale of two evaluations.

In 1999 the Agency for Toxic Substances and Disease Registry (ATSDR) released a Draft Toxicological Profile for Polychlorinated Biphenyls (PCBs). In reviewing the potential human carcinogenicity of PCBs, ATSDR (1999) concluded that "The weight of evidence does not support a causal association for PCBs and human cancer at this time." Just 1 year later, in an updated Toxicological Profile for Polychlorinated Biphenyls (PCBs), the conclusions of another analysis (ATSDR, 2000) on whether exposure to PCBs might represent a carcinogenic risk to humans had dramatically changed to "Overall, the human studies provide some evidence that PCBs are carcinogenic" and "some of these studies provide meaningful evidence that PCBs are carcinogenic in humans." Because this is a substantially different conclusion than that reached only one year previously, it raises a number of questions that must be considered particularly since "weight of evidence" has a precise meaning in the context of evaluating a body of epidemiological data. The present review addresses the additional scientific data that became available between the ATSDR 1999 and 2000 evaluations that was of a magnitude to offset the weight of evidence from numerous epidemiological studies that exposure to PCBs was not causally associated with human cancer to a conclusion only 1 year later that there was now "meaningful evidence" that PCBs posed a carcinogenic risk to humans. Also of interest are the criteria upon which this conclusion is based and the distinction between "weight of evidence" and the newer descriptors of "some evidence" and "meaningful evidence." However, as shown in this review, only one relevant study was published between the ATSDR 1999 and 2000 evaluations and the results of this study were unequivocally supportive of the 1999 conclusion. Because of the continuing controversy surrounding this issue, in this review, all relevant epidemiological data on PCBs are summarized and subjected to another weight of evidence evaluation. This critical review is based on the most recent guidelines (U.S. EPA, 1999a, 2003) for conducting weight-of-evidence evaluations on a body of epidemiological data. Applying a weight-of-evidence evaluation to the PCB epidemiological studies can only lead to the conclusion that there is no causal relationship between PCB exposure and any form of cancer, thereby confirming the conclusions of ATSDR (1999). Also considered is the methodology and logic used by ATSDR (2000) that resulted in overturning the weight of evidence conclusions concerning the human carcinogenicity of PCBs in ATSDR (1999). This issue may have public health and policy implications. It seems appropriate that unbiased evaluations of a body of data, even of controversial issues such as the potential human carcinogenicity of PCBs, be conducted in a transparent manner following applicable guidelines. The dramatic differences between the conclusions of ATSDR (1999) and ATSDR (2000) do not appear to be consistent with this process.

Case-control study of PCBs, other organochlorines and breast cancer in Eastern Slovakia.

This case-control study was designed to investigate association between polychlorinated biphenyls (PCBs) and risk of breast cancer in an area of high environmental exposure in the Michalovce district of eastern Slovakia. Incident breast cancer cases from the Michalovce district diagnosed between May 1997 and May 1999 were recruited through the Oncology Department of the District Hospital. A total of 15 individual PCB congeners, 2,2'-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE), 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT), and hexachlorobenzene (HCB) were measured in the serum of 24 breast cancer patients and 88 population controls in 1998-1999. The median levels of total PCBs were similar in cases (2586 ng/g of lipid) and controls (2682 ng/g of lipid). Higher serum levels (highest vs. lowest tertile) of total PCBs (odds ratio (OR)=0.42, 95% CI 0.10-1.82, p-for trend=0.31), group 1 congeners (OR=0.37, 95% CI 0.10-1.43, P-for trend=0.02), group 2 congeners (OR=0.32, 95% CI 0.07-1.56, P-for trend=0.60), and group 3 congeners (OR=0.49, 95% CI 0.12-2.04, P-for trend=0.51) were inversely associated with risk of breast cancer. Higher serum levels of DDE (OR=3.04, 95% CI 0.65-14.3, P-for trend=0.10) were positively associated with risk of breast cancer, while there was no association for DDT (OR=1.19, 95% CI 0.27-5.23, P-for trend=0.68), and an inverse association for HCB (OR=0.45, 95% CI 0.06-3.19, P-for trend=0.67). While generally not statistically significant, PCB and HCB levels were inversely associated with risk of breast cancer in this highly exposed population. DDE, but not DDT, was positively associated with risk.

Breast adipose tissue concentrations of polychlorinated biphenyls and other organochlorines and breast cancer risk.

Numerous studies have examined the relationship between organochlorines and breast cancer, but the results are not consistent. In most studies, organochlorines were measured in serum, but levels in breast adipose tissue are higher and represent cumulative internal exposure at the target site for breast cancer. Therefore, a hospital-based case-control study was conducted in Ontario, Canada to evaluate the association between breast cancer risk and breast adipose tissue concentrations of several organochlorines. Women scheduled for excision biopsy of the breast were enrolled and completed a questionnaire. The biopsy tissue of 217 cases and 213 benign controls frequency matched by study site and age in 5-year groups was analyzed for 14 polychlorinated biphenyl (PCB) congeners, total PCBs, and 10 other organochlorines, including p,p'-1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene. Multiple logistic regression was used to assess the magnitude of risk. While adjusting for age, menopausal status, and other factors, odds ratios (ORs) were above 1.0 for almost all organochlorines except five pesticide residues. The ORs were above two in the highest concentration categories of PCB congeners 105 and 118, and the ORs for these PCBs increased linearly across categories (Ps for trend < or =0.01). Differences by menopausal status are noted especially for PCBs 105 and 118, with risks higher among premenopausal women, and for PCBs 170 and 180, with risks higher among postmenopausal women. Clear associations with breast cancer risk were demonstrated in this study for some PCBs measured in breast adipose tissue.

An evaluation of proposed frameworks for grouping polychlorinated biphenyl (PCB) congener data into meaningful analytic units.

BACKGROUND: Polychlorinated biphenyls (PCBs) have been associated with a variety of health outcomes. Enhanced laboratory techniques can provide a relatively large number of individual PCB congeners for investigation. However, to date there are no established frameworks for grouping a large number of PCB congeners into meaningful analytic units. METHODS: In a case-control study of serum PCB levels on breast cancer risk, measured levels of 56 PCB congener peaks were available for analysis. We considered several approaches for grouping these compounds based on 1) chlorination, 2) factor analysis, 3) enzyme induction, 4) enzyme induction and occurrence, and 5) enzyme induction, occurrence, and other toxicological aspects. The utility of a framework was based on the mechanism of biologic actions within each framework, lack of collinearity among congener groups, and frequency of detection of PCB congener groups in measured serum levels of 192 healthy postmenopausal women. RESULTS: Most participants had detectable levels for the proposed PCB congeners groups, using degree of chlorination as a grouping framework. In addition, the previously proposed grouping approach based on enzyme induction, occurrence, and other toxicological aspects was an applicable alternative to the crude approach of grouping by degree of chlorination. Grouping these congeners with respect to P450 enzyme induction activity, and the previously proposed framework based on enzyme induction and occurrence, did not fit these data as well, because only a small proportion of participants had detectable levels for the congener groups with the greatest toxicological potential. Statistical grouping did not result in an interpretable and meaningful clustering of these exposures. CONCLUSIONS: In these data, grouping with respect to degree of chlorination and the previously proposed framework based on enzyme induction, occurrence, and other toxicological aspects were the most useful approaches to reducing a large number of PCB congeners into meaningful analytic units. Factors affecting the utility of the proposed grouping frameworks are discussed.

Organochlorine exposure and the risk of endometriosis.

OBJECTIVE: To compare mean concentrations of organochlorine in women with a new diagnosis of endometriosis and in controls. DESIGN: Case-control study. SETTING: Women attending an institutional clinic of reproductive endocrinology. PATIENT(S): Cases and controls were selected among women who underwent laparoscopy for chronic pelvic pain, infertility, or tubal fulguration between January 1994 and December 1994. Eighty-six women with endometriosis and 70 controls, matched for the indication for laparoscopy, were recruited. MAIN OUTCOME MEASURE(S): Mean organochlorine plasma concentrations of 14 polychlorinated biphenyl congeners and 11 chlorinated pesticides were compared between the cases and controls. Analysis of covariance was used to adjust means for confounding variables, and odds ratios were estimated by logistic regression. RESULT(S): Crude geometric mean concentrations did not differ significantly between cases and controls for any of the organochlorine compounds. Similarly, crude or adjusted means of the sum of polychlorinated biphenyl congeners, the sum of chlordanes, or the sum of dichlorodiphenyltrichloroethanes did not differ between the groups. There was no significant linear trend in the adjusted odds ratios for endometriosis as organochlorine concentrations increased. CONCLUSION(S): These results suggest that exposure to polychlorinated biphenyls and chlorinated pesticides during adulthood is not associated with endometriosis in the general population.

Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay.

Polychlorinated biphenyls (PCBs) are stable pollutants, which can be found in almost every compartment of terrestrial and aquatic ecosystems. They are very lipophilic and therefore have the potency of accumulating in the fat stores of animals. The mechanisms by which PCBs exert their adverse effects are still unclear. It is known that PCBs induce some important biotransformation enzymes, but their mutagenic properties are still controversial. The DNA breakage and clastogenic potency of a planar PCB77 (3,3',4,4'-tetrachlorobiphenyl) was determined in vivo in fish, using the single cell gel electrophoresis or comet assay and the micronucleus test, on erythrocytes of the brown trout exposed for 3, 9 and 14 days to initial PCB concentrations of 780 and 918 pg/ml, dissolved in the water. Blood was taken by a caudal puncture and the erythrocytes were either deposited in an agarose gel (0.6%) for the comet assay or smeared directly on slides for the micronucleus test. Five fish were studied per treatment and 50 and 2000 erythrocytes per concentration and per animal were analysed for the comet assay and the micronucleus test respectively. Ethyl methanesulphonate (EMS) at a concentration of 25 mg/l water was used as a positive control. Although EMS induced a statistically significant increase of single strand breaks in the comet assay, in neither of the two tests used, were mutagenic effects due to PCB exposure observed.