Splenic Vein Thrombosis Following Pancreas Transplantation: Identification of Factors That Support Conservative Management.

Prophylaxis for graft portal/splenic venous thrombosis following pancreas transplant varies between institutions. Similarly, treatment of venous thrombosis ranges from early re-exploration to conservative management with anticoagulation. We wished to determine the prevalence of graft splenic vein (SV) thrombosis, as well as the clinical significance of non-occlusive thrombus observed on routine imaging. Records of 112 pancreas transplant recipients over a 5-year period at a single center were reviewed. Venous thrombosis was defined as absence of flow or presence of thrombus identified in any part of the graft SV on ultrasound. Thirty patients (27%) had some degree of thrombus or absence of flow in the SV on postoperative ultrasound. There were 5 graft losses in this group. Four were due to venous thrombosis, and occurred within 20 days of transplant. All patients with non-occlusive partial SV thrombus but normal arterial signal on Doppler ultrasound were successfully treated with IV heparin followed by warfarin for 3-6 months, and remained insulin independent. Findings of arterial signal abnormalities, such as absence or reversal of diastolic flow within the graft, require urgent operative intervention since this finding can be associated with more extensive thrombus that may lead to graft loss.

Pancreas-After-Islet Transplantation in Nonuremic Type 1 Diabetes: A Strategy for Restoring Durable Insulin Independence.

Islet transplantation offers a minimally invasive approach for ß cell replacement in diabetic patients with hypoglycemic unawareness. Attempts at insulin independence may require multiple islet reinfusions from distinct donors, increasing the risk of allogeneic sensitization. Currently, solid organ pancreas transplant is the only remaining surgical option following failed islet transplantation in the United States; however, the immunologic impact of repeated exposure to donor antigens on subsequent pancreas transplantation is unclear. We describe a case series of seven patients undergoing solid organ pancreas transplant following islet graft failure with long-term follow-up of pancreatic graft survival and renal function. Despite highly variable panel reactive antibody levels prior to pancreas transplant (mean 27 ± 35%), all seven patients achieved stable and durable insulin independence with a mean follow-up of 6.7 years. Mean hemoglobin A1c values improved significantly from postislet, prepancreas levels (mean 8.1 ± 1.5%) to postpancreas levels (mean 5.3 ± 0.1%; p = 0.0022). Three patients experienced acute rejection episodes that were successfully managed with thymoglobulin and methylprednisolone, and none of these preuremic type 1 diabetic recipients developed stage 4 or 5 chronic kidney disease postoperatively. These results support pancreas-after-islet transplantation with aggressive immunosuppression and protocol biopsies as a viable strategy to restore insulin independence after islet graft failure.

Cosmetics Europe multi-laboratory pre-validation of the EpiOcular™ reconstituted human tissue test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation's EpiOcular™ Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular™ used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [ ≤60%=irritant (I) (GHS categories 2 and 1); >60%=no classification (NC)]. Test substance single exposure time was 30 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.

Transmission of anaplastic large cell lymphoma via organ donation after cardiac death.

Recently, donation after cardiac death (DCD) has been encouraged in order to expand the donor pool. We present a case of anaplastic T-cell lymphoma transmitted to four recipients of solid organ transplants from a DCD donor suspected of having bacterial meningitis. On brain biopsy, the donor was found to have anaplastic central nervous system T-cell lymphoma, and the recipient of the donor's pancreas, liver and kidneys were found to have involvement of T-cell lymphoma. The transplanted kidneys and pancreas were excised from the respective recipients, and the kidney and pancreas recipients responded well to chemotherapy. The liver recipient underwent three cycles of chemotherapy, but later died due to complications of severe tumor burden. We recommend transplanting organs from donors with suspected bacterial meningitis only after identification of the infectious organism. In cases of lymphoma transmission, excision of the graft may be the only chance at long-term survival.

An investigation of new toxicity test method performance in validation studies: 1. Toxicity test methods that have predictive capacity no greater than chance.

An approach commonly used to measure new toxicity test method (NTM) performance in validation studies is to divide toxicity results into positive and negative classifications, and the identify true positive (TP), true negative (TN), false positive (FP) and false negative (FN) results. After this step is completed, the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are calculated. Although these statistics are widely used and often the only statistics used to assess the performance of toxicity test methods, there is little specific guidance in the validation literature on what values for these statistics indicate adequate performance. The purpose of this study was to begin developing data-based answers to this question by characterizing the CPS obtained from an NTM whose data have a completely random association with a reference test method (RTM). Determining the CPS of this worst-case scenario is useful because it provides a lower baseline from which the performance of an NTM can be judged in future validation studies. It also provides an indication of relationships in the CPS that help identify random or near-random relationships in the data. The results from this study of randomly associated tests show that the values obtained for the statistics vary significantly depending on the cut-offs chosen, that high values can be obtained for individual statistics, and that the different measures cannot be considered independently when evaluating the performance of an NTM. When the association between results of an NTM and RTM is random the sum of the complementary pairs of statistics (sensitivity + specificity, NPV + PPV) is approximately 1, and the prevalence (i.e., the proportion of toxic chemicals in the population of chemicals) and PPV are equal. Given that combinations of high sensitivity-low specificity or low specificity-high sensitivity (i.e., the sum of the sensitivity and specificity equal to approximately 1) indicate lack of predictive capacity, an NTM having these performance characteristics should be considered no better for predicting toxicity than by chance alone.

An investigation of new toxicity test method performance in validation studies: 2. Comparison of three measures of toxicity test performance.

An area that requires further research is how best to measure test method performance in validation studies and how to set criteria that should be used to judge the adequacy of this performance. The studies reported here were designed to begin an investigation of these questions. Computer simulations were used to generate data sets similar to those that might be obtained from a large validation study. These data were then analysed using three procedures including determination of the 95% prediction interval (PI), calculation of Pearson's correlation coefficient and calculation of the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The results of this work suggest that of the three approaches examined, quantitative measurements with calculation of the 95% PI provide the most information to allow discrimination between the performance of several different NTMs. The results also suggest that dividing data sets into positive and negative toxicity classifications followed by the calculation of CPS leads to considerable information loss. This loss of information may be so significant that it is not possible in certain circumstances to distinguish between NTMs that are adequate and those that are not.

An investigation of new toxicity test method performance in validation studies: 3. Sensitivity and specificity are not independent of prevalence or distribution of toxicity.

Often, the only measures of toxicity test performance provided in validation studies are the contingent probability statistics (CPS) sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity and specificity are generally used in preference to NPV and PPV since NPV and PPV are assumed to vary with changes in prevalence while sensitivity and specificity are assumed to be independent of changes in prevalence. The purpose of the studies reported here was to test whether or not sensitivity and specificity are actually independent of changes in prevalence. Results derived from these studies indicate that sensitivity and specificity vary significantly depending on the prevalence of toxic substances in the set of chemicals being tested. This means sensitivity and specificity should not always be considered constant indicators of toxicity test performance.

Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay.

The isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay were evaluated for their ability to predict the eye irritation potential of a range of hair shampoo formulations, some containing a novel non-surfactant ingredient known to be an ocular irritant. The additional endpoints of corneal swelling and histological examination were incorporated into the standard BCOP protocol. Historic Draize data were available for several of the formulations and served as a reference. The standard BCOP assay (without histology) failed to distinguish between shampoos of low and high irritant potential, when exposure times of 10 and 60 min were employed (for undiluted and 10% dilution of the shampoos, respectively) and the in vitro score classified the majority of formulations as mild. The incorporation of the histological endpoint to the BCOP protocol allowed discrimination between formulations of differing irritancy, and should be included to augment the standard BCOP protocol. Corneal swelling values did not, however, correlate with the irritant potential of the shampoos tested. The IRE which includes the endpoints of corneal swelling and histopathological scoring produced classifications of irritancy that were fairly consistent with in vivo data and distinguished between the high and low irritant potential shampoos.

A prevalidation study on in vitro tests for acute skin irritation. results and evaluation by the Management Team.

A prevalidation study on in vitro tests for acute skin irritation was conducted during 1999 and 2000. The overall objective of validation in this area, of which this prevalidation study is an initial stage, is to identify tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union (EU) risk phrases ("R38"; no classification) and the harmonised OECD criteria ("Irritant"; no label). This prevalidation study specifically addressed aspects of: protocol refinement (phase I), protocol transfer (phase II), and protocol performance (phase III), in accordance with the prevalidation scheme defined by the European Centre for the Validation of Alternative Methods (ECVAM). The tests evaluated were: EpiDerm (phases I, II and III), EPISKIN (phases I, II and III), PREDISKIN (phases I and II, and additional protocol refinement), the non-perfused pig ear method (phases I and II, and additional protocol refinement), and the mouse skin integrity function test (SIFT; phases I and II). Modified, standardised test protocols and well-defined prediction models were available for each of the tests at the end of phase I. The results of phase I (intralaboratory reproducibility) were sufficiently promising for all of the tests to progress to phase II. Protocol transfer between the Lead Laboratory and Laboratory 2 was undertaken for all five tests during phase II, and additional refinements were made to the test protocols. For EpiDerm, EPISKIN and the SIFT, the intralaboratory and interlaboratory reproducibilities were acceptable; however, better standardisation of certain aspects of the test protocols was needed prior to commencing phase III. Neither PREDISKIN nor the pig ear test performed sufficiently well in phase II to progress to phase III. The PREDISKIN protocol was overly sensitive, resulting in the prediction of all the NI chemicals as I. The variability in the pig ear test results was too great, indicating that the test would show limited predictive ability. In additional studies (a repeat of phase I), further modification of the PREDISKIN protocol and a change in the prediction model considerably improved the ability of the test to distinguish I from NI chemicals. However, attempts to improve the intralaboratory reproducibility of the pig ear test were unsuccessful. In phase III an initial assessment of the reproducibility and predictive ability, in three independent laboratories per test, was undertaken for the EpiDerm and EPISKIN tests (the SIFT was a late inclusion in the prevalidation study, and is being evaluated in a separate phase III study). A set of 20 coded chemicals (10 I, 10 NI) were tested with the final, refined, test protocols. The intralaboratory reproducibility was acceptable for both EpiDerm and EPISKIN. The interlaboratory reproducibility was considered to be acceptable for EPISKIN; however, for EpiDerm, analysis of variance (ANOVA) indicated that there was a statistically significant laboratory effect on the overall variability, suggesting that the interlaboratory transferability of the test needs to be improved. The EpiDerm test had an overall accuracy of 58%, with an over-prediction rate of 37% and an under-prediction rate of 47%. The EPISKIN test had an overall accuracy of 58%, showing an under-prediction rate of 23% and an over-prediction rate of 60%. It is concluded that, as yet, none of the tests evaluated in this prevalidation study are ready for inclusion in a formal validation study on in vitro tests for acute skin irritation. Overall protocol performance of the SIFT is currently being evaluated in a phase III study. Further studies are also in progress to improve the test protocols and prediction models for EpiDerm and EPISKIN.

Assessment of the chorioallantoic membrane vascular assay (CAMVA) in the COLIPA in vitro eye irritation validation study.

The chorioallantoic membrane vascular assay (CAMVA) is an alternative to the Draize rabbit eye irritation method. The CAMVA employs the vascularized membrane of a fertile hen's egg to assess eye irritation potential. This irritation potential is a function of alterations in the vasculature following the administration of test material. Because of the history of use of the CAMVA it was selected as one of the methods for a validation study organized and sponsored by COLIPA. For this validation study mathematical prediction models (PMs) were developed to convert the CAMVA results into predicted Draize eye irritation scores known as a modified maximum average Draize score (MMAS). These predicted scores were statistically compared with the observed scores to assess the relevance of the CAMVA. The assay was conducted on the same set of test materials by two independent laboratories. These two sets of data were compared to assess the interlaboratory reproducibility of the assay. The results of this validation study of the CAMVA show that for test materials with MMASs in the 0 to 5 range or the 55 to 110 range, the CAMVA did not give a good prediction. The predictions were better for samples of mild to moderate irritation (MMAS 5-55). The difficulty in predicting at the low end of the irritation scale appears to be due to the biological variability of the test system and the subjective nature of the CAMVA evaluation. For those samples with an MMAS above 55, the CAMVA appeared to be limited in demonstrating the more severe response. This may be due to the fact that the PMs were developed using historical data sets of test materials with MMASs below this range. Two approaches for improving the CAMVA for eye irritation prediction are (1) to decrease the variability at the low end by reducing the subjectivity in the scoring and (2) to develop better prediction models using more data in the range of severe irritants.

Assessment of the Cytosensor Microphysiometer Assay in the COLIPA In Vitro Eye Irritation Validation Study.

The Cytosensor(TM) microphysiometer assay and its associated prediction model were evaluated in the COLIPA ocular irritation validation study for cosmetic ingredients and formulations. Test materials were prepared in low-buffer medium and exposed to L929 cells grown in transwells. The metabolic rate of the cell population was measured after each dose and the dose inducing a 50% decrease in the rate (MRD(50)) was determined and used to predict the ocular irritation potential. Only 29 of the 55 materials could be tested because of solubility limitations. The irritancy potential of many chemical classes was underpredicted by the assay, particularly acids, bases and organics. The use of the assay for surfactants and surfactant-based formulations showed promise, confirming the use of the method for these types of materials, although some revision of the prediction model would be necessary. Excellent interlaboratory reproducibility for the MRD(50) values across all test materials was observed.

Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination.

The analysis of the gel electrophoresis banding patterns and relative migration distances for the individual isoforms of intracellular enzymes, such as lactate dehydrogenase, purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase, is used routinely in the biopharmaceutical industry for confirmation of cell line species of origin. In the present study, the sensitivity of the technique (AuthentiKit, Innovative Chemistry, Marshfield, MA) for determining interspecies cell line cross-contamination was examined. Extracts were prepared from a CHO-K1 line (AA8, Chinese hamster), MRC-5 (human) cells, and L929 (mouse) cells and from several proportional mixtures of the various binary combinations of cells. The isoenzymes were analyzed according to standard procedures for the technique. Contamination of MRC-5 cells with CHO-K1 or with L929 cells was clearly detectable with each enzyme analyzed. Similarly, the contamination of L929 or CHO-K1 cells with MRC-5 cells was readily apparent with each enzyme. On the other hand, contamination of CHO-K1 cells with L929 cells was only detected with lactate dehydrogenase analysis, and contamination of L929 cells with CHO-K1 cells was not detected with any of the four enzymes examined. For the latter case, the analysis of an additional enzyme (peptidase B) was required. The results indicate that interspecies cross-contamination should be detectable with isoenzyme analysis if the contaminating cells represent at least 10% of the total cell population.

In vitro alternatives for ocular irritation.

The necessity of using animals to test whether new chemicals and products are eye irritants has been questioned with increasing frequency and fervor over the last 20 years. During this time many new nonanimal methods have been proposed as reliable alternatives to the traditional rabbit (Draize) test. To date, however, none of these nonanimal (in vitro) tests have become universally accepted as a complete replacement for the Draize test. To understand why a complete replacement has not been found, one has to first understand the reasonably complex structure of the eye, the standard Draize scoring scale--which is based on a qualitative evaluation of three different tissues--the differences between human and rabbit eyes, the intrinsic variability of the animal test, and the details of the different in vitro tests that have been proposed as replacements. The in vitro tests vary from relatively simple assays using single cells to more sophisticated assays that use discarded animal tissue or artificially constructed human tissue. It is clear that appropriately designed in vitro tests will eventually give more useful mechanistic information about ocular injury from which we can more comfortably predict the risk of human eye irritation from new products and ingredients.

IRAG working group 4. Cell cytotoxicity assays. Interagency Regulatory Alternatives Group.

Twenty-seven data sets from 12 cellular cytotoxicity assays, intended to predict ocular irritation, were submitted to the Interagency Regulatory Alternatives Group (IRAG) for review. These data consisted of paired in vivo (Draize) and in vitro responses to individual chemicals and formulations. In vivo data consisted of individual tissue scores so that the predictive value of the in vitro assay could be assessed for each tissue response normally measured in the standard Draize assay. Data were compiled and evaluated according to the IRAG Guidelines Document. The Pearson's linear correlation coefficient was used as the first step in assessing the relationship between the in vitro and in vivo responses. The majority of the data sets represented the study of surfactant-based materials. In many cases, there was good correlation between the in vitro scores and the in vivo tissue responses. Most pronounced were the particularly good correlations between the in vitro scores and conjunctival redness scores across most of the assays. Based on the data submitted, a number of the cell cytotoxicity assays show considerable promise as screens for ocular irritancy. None of the submitters recommended that their cell cytotoxicity assay be used as a sole replacement for in vivo assessment. For almost all of these assays, the materials being tested should be water-soluble/miscible. The toxicity of products with reserve acidity or alkalinity or with high reactivity may be underestimated. A given user may prefer certain assays depending on the types of materials to be tested, the expected range of toxicities and the resources available. The cell cytotoxicity assays can serve as a valuable component of a tiered or battery testing program. As with any assay, a sufficient number of replicate values, concurrent positive and negative controls, and a strict adherence to assay acceptance criteria are essential to produce credible data.

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.

Evaluation of the mutagenicity of an N-nitroso contaminant of the sunscreen Padimate O: N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO).

The nitrosamine contaminant, N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO), of the major sunscreen ingredient Padimate O (4-N,N'-dimethylamino-benzoic acid, 2-ethylhexyl ester) was synthesized and tested for mutagenicity in the Salmonella typhimurium and mouse lymphoma L5178Y TK +/- assays. In contrast to the previously reported positive responses in S. typhimurium tester strains TA100 and TA1535 [Loeppky et al., 1991], there were no increases in the number of revertants with strains TA98, TA100, TA1535, and TA1538 in either the Salmonella plate incorporation [Ames et al., 1975] or preincubation [Yahagi et al., 1977] assays. Additional testing with Salmonella, following the modified preincubation procedure [Rogan, 1990] that gave the initial positive response, was also negative. Data from the mouse lymphoma assays were also uniformly negative. During synthesis of NPABAO, small amounts of 4-N,N'-dimethylamino-3-nitrobenzoic acid, 2-ethylhexyl ester (DMANBAO) can be formed. To determine whether the reported positive mutagenicity response of NPABAO could be the result of trace amounts of DMANBAO in the NPABAO, that compound was also synthesized and tested for mutagenicity with Salmonella. Positive responses were obtained with tester strains TA98 and TA 1538 but not with TA100 and TA1535, indicating that DMANBAO was not responsible for the increase in revertants originally reported.

Evaluation of the human epidermal keratinocyte neutral red release and neutral red uptake assay using the first 10 MEIC test materials.

Two methodologies used in vitro to estimate cytotoxicity in cell culture systems were compared: these were the neutral red uptake assay (NRU), which is used to measure toxicity caused by an extended (48-hr) exposure to the test material, and the neutral red release assay (NRR), which is used to measure toxicity caused by a short-term (1-min) exposure to the test material. Both methodologies used the normal human epidermal keratinocyte (NHEK)-based NeutralRed Bioassay supplied by Clonetics Corporation (San Diego, CA, USA). 10 materials (paracetamol, acetylsalicylic acid, ferrous sulphate, diazepam, amitriptyline, digoxin, ethylene glycol, methanol, ethanol and isopropanol), which are part of the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) panel, were tested. NRU(50) values for the 10 compounds covered more than an eight-log range from 0.004 mum (digoxin) to 1.0 x 10(6) mum (methanol). Because of solubility limits, NRR(50) values for diazepam, digoxin, ferrous sulphate and paracetamol could not be determined. NRR(50) values for the remaining six compounds covered approximately a three-log range from 3.2 x 10(3) to 7.1 x 10(6) mum. When compared with documented values for either the human acute oral lethal dose or the human acute lethal blood concentration, the NRU assay was found to be much more useful in predicting human acute toxicity than was the NRR assay.

Gene mutation assay of acrolein in the CHO/HGPRT test system.

The mutagenic potential of acrolein has been studied with a wide range of in vitro and in vivo genetic toxicity assays. The data often have been conflicting, especially with the Ames assay. This study was undertaken to assess the mutagenic potential of acrolein using the CHO/HGPRT assay, both with and without metabolic activation. This assay system was chosen because it provides eukaryotic DNA as the target and is capable of detecting a range of mutational events. Because of its considerable toxicity, acrolein was tested over a very narrow dose range of 0.2-2 nl ml-1 without exogenous activation and 0.5-8 nl ml-1 with rat S-9 activation. Multiple assays were performed under both conditions. The results indicated that while acrolein was clearly very cytotoxic, it did not induce a significant mutagenic response in the presence or absence of metabolic activation.

Genotoxicity of multifunctional acrylates in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/-assay.

Multifunctional acrylates are being used increasingly as replacements for solvents, and occupational and general population exposure to this structural class is expanding. Four multifunctional acrylates and acrylic acid were tested for mutagenicity in the Salmonella typhimurium and mouse lymphoma L5178Y TK+/-assays. In the Salmonella assay, two of the compounds (trimethylolpropane triacrylate and trimethylolpropane trimethacrylate) showed weakly positive results with a single tester strain (TA1535) in the presence of hamster liver S9; the other three compounds were negative. All five compounds were negative in the Salmonella assay without S9 activation. In the mouse lymphoma assay, two of the compounds (acrylic acid and ethylene glycol diacrylate) were positive in both the presence and the absence of S9, one compound was positive only in the presence of S9 (ethylene glycol dimethacrylate), and one compound was positive only in the absence of S9 (trimethylolpropane triacrylate).