Increase in thyroid follicular cell tumors in nelfinavir-treated rats observed in a 2-year carcinogenicity study is consistent with a rat-specific mechanism of thyroid neoplasia.

The carcinogenic potential of nelfinavir mesylate (nelfinavir) was evaluated in a 2-year oral (gavage) study on Sprague-Dawley rats at dose levels of 0 (control), 0 (vehicle control), 100, 300 and 1000 mg/kg per day. At the end of the treatment, increased incidences of thyroid follicular cell hyperplasia and neoplasms were observed at 300 (males) and 1000 mg/kg per day (both sexes). There were no other treatment-related effects and no tumors at other sites. Results from previous studies indicated a number of effects in the liver and thyroid, as well as metabolic profiles that suggested nelfinavir might cause thyroid hyperplasia/neoplasia secondary to hormone imbalance by altering thyroid hormone disposition. To investigate this hypothesis, the effects of nelfinavir on gene expression in rat hepatocytes and liver slices (in vitro), thyroxine plasma clearance, and thyroid gland function were evaluated. Compared to controls, gene expression analyses demonstrated an increased expression of glucuronyltransferase (UDPGT) and CYP450 3A1 in nelfinavir-treated rat hepatocytes and liver slices. In rats treated with nelfinavir (1000 mg/kg per day) for 4 weeks, liver weights and centrilobular hepatocellular hypertrophy were increased and minimal to mild diffuse thyroid follicular cell hypertrophy and follicular cell hyperplasia were evident in the thyroid gland. Thyroid-stimulating hormone (TSH) levels were significantly increased (three-fold), while tri-iodothyronine (T3)/tetra-iodothyronine (T4) and reverse T3(rT3) levels were unchanged, indicating that a compensated state to maintain homeostasis of T3/T4 had been achieved. Plasma 125I-thyroxine clearance was increased and the plasma thyroxine AUC0-48 was decreased (24%) compared to control. In conclusion, these data indicate that thyroid neoplasms observed in the nelfinavir-treated rats were secondary to thyroid hormone imbalance. Increased thyroxine clearance contributes to the effects of nelfinavir on thyroid gland function and is probably a result of UDPGT induction that leads to elevated TSH levels in the rat and eventual thyroid neoplasia. These results are consistent with a well-recognized rat-specific mechanism for thyroid neoplasms.

In vivo micronucleus assays on 2,4-dichlorophenoxyacetic acid and its derivatives.

The potential for 2,4-D and seven of its salts and esters to induce cytogenetic abnormalities in mammalian cells in vivo was investigated in the mouse bone marrow micronucleus test. All the test materials were administered to male and female mice by oral gavage and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow were determined at intervals of 24, 48 and 72 h following dosing. There were no significant increases in the incidence of MN-PCE in the treated mice at any of the bone marrow sampling times. These results are consistent with the reported lack of in vitro genetic toxicity for these materials in various in vitro genotoxicity assays as well as the absence of carcinogenic potential for 2,4-D in both mice and rats.

Lack of genotoxic effects of sucrose acetate isobutyrate (SAIB).

Sucrose acetate isobutyrate (SAIB) was tested for potential genotoxic activity in four different in vitro assay systems. Two independent trials of a Salmonella reverse mutation assay (using strains TA98, TA100, TA1535, TA1537 and TA1538) showed no increases in revertant frequencies at doses up to 10,000 microg/plate which was non-toxic but exceeded the solubility limit. Similarly, no mutagenic response was observed at doses up to 1000 microg/ml at the HGPRT locus in cultured CHO cells; SAIB was toxic and its solubility limit was exceeded at 50 microg/ml. No clastogenic activity was detected in cultured CHO cells at concentrations up to 2000 microg/ml. All three preceding in vitro tests were conducted both in the presence and absence of Aroclor 1254-induced rat liver S-9 metabolic activation systems. An unscheduled DNA synthesis assay also was performed using rat primary hepatocyte cultures with doses up to 1000 microg/ml, and no DNA repair was detectable. Thus, SAIB was stringently tested at doses exceeding the solubility limit in culture medium and causing toxicity to CHO cells without obtaining any evidence for genotoxic activity as a mutagen, clastogen, or DNA-damaging agent.

Report from working group on in vitro tests for chromosomal aberrations.

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro and in vivo assays of isopropanol for mutagenicity.

To assess the mutagenic potential of isopropanol, an in vitro Chinese hamster ovary (CHO) cell/HGPRT gene mutation assay and a bone marrow micronucleus study in mice were conducted. In the CHO/HGPRT assay, concentration levels ranged from 0.5 to 5.0 mg/ml. No elevated mutant frequencies attributable to treatment were observed in the test under either activated or non-activated conditions. In the micronucleus assay, mice were injected intraperitoneally (IP) with either 350, 1,173, or 2,500 mg/kg of isopropanol at constant volumes of 10 ml/kg. No increased incidence of micronuclei was observed in bone marrow polychromatic erythrocytes (PCEs) harvested at 24, 48, or 72 hr post-dosing. In both assays, negative and positive control mutant frequencies were within historical control ranges. These results, in conjunction with previously published data, clearly demonstrate that isopropanol is not a mutagen.

Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells.

Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.

Genetic toxicology studies comparing the activity of sidestream smoke from cigarettes which burn or only heat tobacco.

The results of in vitro genetic toxicology studies of sidestream cigarette smoke (SSCS) from cigarettes which heat but do not burn tobacco were compared to those of sidestream smoke from cigarettes which burn tobacco. SSCSs from 5 cigarettes were compared. Three of the cigarettes, the Kentucky reference research cigarette (1R4F), a commercially available ultra-low-tar brand (ULT) and a commercially available ultra-low-tar menthol brand (ULT-menthol) burn tobacco while two of the cigarettes, a regular (TEST) and a menthol (TEST-menthol) heat tobacco. SSCSs from all cigarettes were prepared by identical techniques, which involved collecting sidestream smoke particulate matter on Cambridge filter pads and combining the particulate matter with the vapor-phase materials collected by bubbling the smoke exiting the Cambridge pad through DMSO. The SSCSs obtained (equivalent to 0.4 cigarettes/ml DMSO) were evaluated at identical concentrations in an in vitro genetic toxicology test battery. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in Ames bacterial strains TA98, TA100, TA1537 and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative results in strain TA1535. In the absence of metabolic activation, 1R4F, ULT and ULT-menthol SSCSs were not significantly mutagenic. TEST and TEST-menthol SSCSs produced negative results in all 5 bacterial strains, both with and without metabolic activation. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in the CHO chromosomal aberration assay and in the CHO sister-chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol SSCSs produced negative results in both assays, either with or without metabolic activation. The SSCSs from 1R4F, ULT and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol SSCSs were negative in this assay. All 5 SSCSs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. SSCSs from the 1R4F, ULT and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol SSCSs were not cytotoxic under either condition. These results demonstrate that sidestream smoke from cigarettes which heat but do not burn tobacco (TEST and TEST-menthol) was neither genotoxic nor cytotoxic under conditions where sidestream smoke from cigarettes which burn tobacco (1R4F, ULT and ULT-menthol) was genotoxic and/or cytotoxic in a concentration-dependent manner.

Comparative studies on the genotoxic activity of mainstream smoke condensate from cigarettes which burn or only heat tobacco.

The in vitro genotoxic activity of mainstream cigarette smoke condensate (CSC) from cigarettes which heat but do not burn tobacco was compared to that of CSC from cigarettes which burn tobacco. CSCs from five cigarettes were compared. Three of the cigarettes [the Kentucky reference research cigarette (1R4F), a commercially available ultra-low tar brand (ULT) and a commercially available ultra-low tar menthol brand (ULT-menthol]) burn tobacco while two of the cigarettes [a regular (TEST) and a menthol (TEST-menthol]) heat tobacco. CSC from all cigarettes were collected by identical standard techniques, which involved collecting mainstream smoke particulate matter on Cambridge filter pads under FTC smoking conditions. The pads were extracted with DMSO, and the CSCs obtained [10 mg total particulate matter (TPM)/ml DMSO] were evaluated at identical concentrations in an in vitro genetic toxicology test battery. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were mutagenic in Ames bacterial strains TA98, TA100, TA1537, and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative in strain TA1535. In the absence of metabolic activation, 1R4F, ULT, and ULT-menthol CSCs were not mutagenic except for a weak response in strain TA1537 for the 1R4F and ULT CSCs. TEST and TEST-menthol CSCs were nonmutagenic in all five bacterial strains, both with and without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were positive in the CHO-chromosomal aberration assay and in the CHO--sister chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol CSCs were negative in both assays, either with or without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol CSCs were negative in this assay. All five CSCs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. CSCs from the 1R4F, ULT, and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol CSCs were not cytotoxic under either condition. These results demonstrate that mainstream CSCs from the TEST and TEST-menthol cigarettes are neither genotoxic nor cytotoxic under conditions where CSCs from 1R4F, ULT, and ULT-menthol cigarettes are genotoxic and/or cytotoxic in a concentration-dependent manner.

Chromosome aberration and sister chromatid exchange test results with 42 chemicals.

Forty-two chemicals were tested for their ability to induce cytogenetic change in Chinese hamster ovary cells using assays for chromosome aberrations (ABS) and sister chromatid exchanges (SCE). These chemicals were included in the National Toxicology Program's evaluation of the ability of four in vitro short-term genetic toxicity assays to distinguish between rodent carcinogens and noncarcinogens. The conclusions of this comparison are presented in Zeiger et al. [Zeiger E, Haseman JK, Shelby MD, Margolin BH, Tennant RW (1990): [Environ Molec Mutagen 16(Suppl 18): 1-14]. The in vitro cytogenetic testing was conducted at four laboratories, each using a standard protocol to evaluate coded chemicals with and without exogenous metabolic activation. Most chemicals were tested in a single laboratory; however, two chemicals, tribromomethane and p-chloroaniline, were tested at two laboratories as part of an interlaboratory comparison. Four chemicals (C.I. basic red 9 HCl, 2-mercaptobenzothiazole, oxytetracycline HCl, and rotenone) were tested for SCE in one laboratory and in a different laboratory for ABS. Tetrakis(hydroxymethyl)phosphonium sulfate was tested at one laboratory and the chloride form was tested at a different laboratory. Twenty-five of the 42 chemicals tested induced SCE. Sixteen of these also induced ABS; all chemicals that induced ABS also induced SCE. There was approximately 79% reproducibility of results in repeat tests, thus, we conclude that this protocol is effective and reproducible in detecting ABS and SCE.

Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals.

The National Toxicology Program has undertaken a study to assess the ability of four genetic toxicology assays to predict the carcinogenicity of chemicals in 2-year rodent studies [Tennant et al.: Science 236:933-941, 1987]. Two of the assays, used for evaluating in vitro cytogenetic damage, were the SCE and chromosome aberration assays in Chinese hamster ovary cells. The results and data for 15 of the chemicals tested in these two assays are presented here. Each chemical was tested with and without exogenous metabolic activation. The chemicals tested were bisphenol A, 2-chloroethanol, C.I. acid orange 10, C.I. disperse yellow 3, C.I. solvent yellow 14, cytembena, D&C red 9, 1,2-dibromoethane, FD&C yellow 6, malaoxon, D,L-menthol, phenol, sulfisoxazole, titanium dioxide, and tris(2-ethylhexyl)phosphate. In vitro cytogenetic results from the other chemicals presented by Tennant et al. (Science 236:933-941, 1987) have been published by Galloway et al. (Environmental and Molecular Mutagenesis 10(Suppl 10): 1-175, 1987), Gulati et al. (Environmental and Molecular Mutagenesis 13:133-193, 1989), and Love-day et al. (Environmental Mutagenesis 13:60-94).

Repair of sister-chromatid exchange-inducing lesions in mutagen-treated cultures of human whole blood and purified fresh or frozen lymphocytes.

Repair of mutagen-induced lesions that result in sister-chromatid exchanges was evaluated in 10 normal individuals. The mutagens used were mitomycin C (MMC), 4-nitroquinoline 1-oxide (4NQO), and N-methyl-N'nitro-N-nitrosoguanidine (MNNG). Cultures of whole blood, freshly purified lymphocytes, or purified lymphocytes cryopreserved for 6 months were analyzed after the mutagen treatments. All 3 mutagens induced reparable damage as evaluated by comparison of sister-chromatid exchanges between cultures that were given time to repair induced damage before 5-bromo-2'-deoxyuridine (BrdUrd) was added to the culture medium with those where BrdUrd was added immediately after the administration of the mutagens (MMC or 4NQO) or at culture initiation (MNNG). Repair of mutagen-induced DNA damage was detected in all 3 culture types; thus cryopreservation did not appear to alter the capacity of lymphocytes to repair mutagen-induced lesions. Quantitative differences in apparent repair capabilities were observed among individuals. Variability also existed among the different culture types within an individual, suggesting that caution should be exercised in interpreting these apparent differences.

Baseline and mutagen-induced sister-chromatid exchanges in cultures of human whole blood and purified fresh or frozen lymphocytes.

Baseline and mutagen-induced levels of sister-chromatid exchanges were evaluated in 10 normal individuals. Cultures with whole blood or purified lymphocytes, either freshly isolated or after 1 or 6 months of cryopreservation, were analyzed to determine whether frozen lymphocytes are suitable for SCE studies. Whole blood and freshly isolated lymphocytes were cultured from samples taken at the beginning of the study (Time 0) and 6 months later (Time 6). Cryopreserved lymphocytes were recovered after 1 month (Time 1) and 6 months (Time 6) of cryopreservation and then challenged with mutagens in culture. The mutagens used were mitomycin C, 4-nitroquinoline-1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine. Purified lymphocytes had consistently and significantly higher baseline SCE frequencies than cells from whole blood cultures and were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline-1-oxide. The response to mitomycin C was similar in all culture types. There was, overall, no consistent effect of freezing on baseline or induced sister-chromatid exchange frequencies in the purified lymphocytes. This suggests that purification and cryopreservation of human lymphocytes does not alter the baseline or mutagen-induced sister-chromatid exchange response and in certain epidemiological, occupational and monitoring situations may have logistical and technical advantages over the use of fresh whole blood.

The effect of agent treatment time on the induction of sister-chromatid exchanges in mouse bone marrow cells in vivo.

The effect of time of agent administration, via intraperitoneal injection, on the yield of SCEs in bone marrow cells of male B6C3F1 mice was determined for cyclophosphamide (CP), 7,12-dimethylbenz[a]anthracene (DMBA) and mitomycin C (MMC). Animals were treated with several doses of each carcinogen/mutagen at 3 different treatment times: -1, +1 and +8 h in relation to the onset of BrdUrd administration. The results of these studies indicate that the optimal treatment time for inducing a maximal SCE response is agent-specific. For CP, the slope of the SCE response was greatest at the +8 h treatment time while the maximal response for DMBA occurred at the -1 h treatment time. For MMC, the slope of the SCE response was independent of treatment time and of the method of bromodeoxyuridine administration (intravenous infusion vs. tablet implantation) but dependent on the laboratory conducting the study (Brookhaven National Laboratory vs. Oak Ridge Associated Universities). Based on the results of these studies, the +1 h acute treatment time is considered optimal for the in vivo cytogenetic evaluation of suspect chemicals for genotoxic activity when bone marrow is used as the target cell population.

Chemically induced aneuploidy in mammalian cells in culture.

Our objectives were to assess whether there exist useful aneuploidy tests in vitro, to identify chemicals that showed potential for mitotic aneuploidy induction, and to recommend some features of suitable protocols for such testing. From over 100 papers we selected 24 for review. The acceptable studies examined hyperdiploidy at metaphase, had concurrent negative controls with low background rates of hyperdiploidy, used a fixation time sufficient for cells to complete more than one cell cycle after treatment and had multiple dose levels with at least 100 cells scored per point. We judged that 12 compounds were positive, 7 inconclusive, and 4 negative with the reservation that 2 of the 4 compounds had not been tested up to toxic doses. Many of the positive compounds are also known to cause structural chromosome aberrations. We separately reviewed qualitative reports of 'C-mitotic' effects, anaphase lagging, multipolar mitoses, or altered DNA content, since these effects may sometimes by associated with aneuploidy induction. No well-validated in vitro aneuploidy assay exists, and much research is required to develop tests, perhaps using chromosome counts, DNA content, or effects on cell organelles necessary for mitosis. In test protocol development we should carefully consider choice of cell sample size, use of in vitro metabolic activation systems, and selection of doses, especially with regard to the problem of whether cytotoxic concentrations should be used.

Diethylstilbestrol-diphosphate induces chromosomal aberrations but not sister chromatid exchanges in murine bone marrow cells in vivo.

Diethylstilbestrol diphosphate (DES-dp) clastogenesis was examined in the bone marrow of C57Bl/6 male and female mice. Significant and sex-related dose effects were observed for the induction of chromatid-type chromosomal aberrations and for the inhibition of cellular proliferation. Females were more sensitive to the effects of DES-dp than males when assessed for either induced chromosomal aberrations or proliferative inhibition. Contrary to other published results, we did not observe either an increase in sister chromatid exchanges or an increased incidence of aneuploidy. Ovariectomy reduced the ability of DES-dp to inhibit cellular proliferation and decreased the high degree of variability between animals at high doses of DES-dp. The results of our studies show that DES is a clastogenic agent in vivo which may relate to its carcinogenicity.

Cytogenetic effects of inhaled ozone.

We have repeated as closely as possible the experiments of Zelac et al., who observed significantly elevated levels of chromosome aberrations in short-term cultures of peripheral lymphocytes from Chinese hamsters that had inhaled ozone. Unlike Zelac et al., we observed no increase in chromosome-type aberration levels, though a small increase in chromatid-aberration levels similar to that reported for exposed human subjects by Merz et al. was seen. No increase in the levels of any chromosomal aberration type was seen in parallel direct bone-marrow preparations. Sister-chromatid exchange (SCE) levels and cell-replication rates, which were determined in the Chinese hamster peripheral lymphocyte cultures and also in bone-marrow samples from similarly treated mice, failed to show any ozone-induced changes.