Rudolph Valentine

Testicular Degeneration and Spermatid Retention in Young Male Rats

The incidence of spontaneous testicular atrophy and its morphological changes in relation to stage-specific spermatogenesis were investigated in young Crl:CDr/BR male rats at 10–12 wk of age used as controls for toxicity screening during 1983–1990. The incidence of testicular degeneration was 2.5% (5/197) in control rats used for oral toxicity studies and 9.4% (31/327) in rats used for inhalation studies. The epididymal tubules of rats with testicular degeneration had exfoliated germ cells and low sperm density. The high incidence of testicular degeneration observed in the control rats used in inhalation studies may be related to the stress associated with immobilization in the restrainer during nose-only exposure conditions. The severity of testicular degeneration in the inhalation studies was mostly minimal. In these minimally affected testes, mature spermatids (step 19) were retained within normal-appearing germinal epithelium at spermatogenic stages IX-XIV. Also, eosinophilic globular bodies (EGBs) were formed with elongated or mature spermatids throughout all spermatogenic stages, but the general architecture of germinal epithelium was normal in appearance. By electron microscopy, EGBs were sequestered necrotic spermatids, and the germ cell degeneration was associated with cytoplasmic vacuolation of Sertoli cells. In moderate testicular degeneration, markedly decreased maturing spermatids (steps 15–19) and a slight depletion of round spermatids were observed in stages I-VIII. In severe testicular degeneration, seminiferous tubules were lined with 1–2 layers of round spermatids and spermatocytes with giant cell formation. The round spermatids served as a marker to identify spermatogenic stages (I-VIII) of the atrophic tubules. Also, in severe testicular degeneration, tubules in spermatogenic stages X-XIV had no elongated spermatids, and spermatocytes were exfoliated with occasional giant cell formation. Many seminiferous tubules were lined with only 1–2 layers of spermatocytes, and specific germ cell markers were not present.

Differentiating between local cytotoxicity, mitogenesis, and genotoxicity in carcinogen risk assessments: the case of vinyl acetate

Understanding the mode of action of carcinogens is critical to scientifically assessing exposure-related risk. Regulatory hazard classification schemes and dose-response assessment paradigms generally require basic knowledge of genotoxic potential to guide decisions on which scheme or paradigm is most appropriate. Although convention suggests that classification and dose-response assessment of genotoxic chemicals should be assessed using conservative assumptions of no threshold, several examples, such as vinyl acetate, exist that challenge this assumption. Vinyl acetate is carcinogenic at portals of entry (nasal cavity and upper gastrointestinal tract). Local metabolism of vinyl acetate produces DNA-reactive acetaldehyde but also produces acetic acid and protons, which contribute to intracellular acidification, cytotoxicity and cell proliferation. This paper reviews their relative contributions to the overall mode of action. Elevated cellular proliferation, well understood to be a risk factor for carcinogenesis, is observed at concentrations associated with tumor formation. Cytotoxicity and compensatory tissue regeneration is one pathway for stimulating cellular proliferation while intracellular acidification is a mitogenic stimulus. Both of these pathways may be operative in nasal tissues while mitogenic proliferation alone appears to be induced in the upper gastrointestinal tract. Using a physiologically-based pharmacokinetic model, quantitative relationships between critical tissue dosimeters and tissue responses are developed to assess the relative importance of genotoxicity and cell proliferation in the overall mode of action of vinyl acetate. This approach supports the concept that intracellular acidification is the sentinel response that precedes cytotoxicity and cellular proliferation. Secondarily, the carcinogenic potential of vinyl acetate is expressed only when tissue exposure to acetaldehyde is high and when cellular proliferation is simultaneously elevated. This mode of action suggests that exposure levels that do not increase intracellular acidification beyond homeostatic bounds will be adequately protective of adverse downstream responses including cancer. These mechanistic insights provide the scientific basis for a cancer classification that incorporates thresholds for cytotoxic and/or mitogenic cell proliferation secondary to intracellular acidification.

Nasal Lesion Development and Reversibility in Rats Exposed to Aerosols of Dibasic Esters

This study was conducted to investigate the initial tissue damage, morphogenesis, and reversibility of nasal lesions induced by the inhalation of dibasic esters (DBE). Young male rats were exposed, nose-only, to an aerosol/vapor mixture of DBE at a concentration of 5,900 mg/m3 for 4 hr and subsequently killed at 1, 4, 7, 14, 21, and 42 days after exposure. Nasal lesions were distributed along major inspiratory airflow routes. Widespread epithelial denudation occurred in the anterior nasal cavity, but the lesions were confined to the dorsal meatus, adjacent the nasal septum, and the lateral middle meatus in the mid-anterior nasal cavity. The lesions were markedly less severe in the posterior nasal cavity and sharply confined to the tips of dorsal ethmoturbinates and adjacent nasal septum. The damaged cuboidal/nonciliated and respiratory epithelium in the anterior nasal cavity regained a normal structure by 4 and 7 days postexposure, respectively. The regeneration of damaged olfactory epithelium was related to the severity of initial tissue damage. Slightly damaged epithelium regained a normal appearance within 1-2 weeks, but the extensively denuded epithelium of the dorsal meatus in the anterior nasal cavity failed to regain a normal structure by 6 weeks. The sustentacular cells of the olfactory epithelium appeared to be the initial site of DBE nasal injury. In the early stages of regeneration, the epithelium was repaired by proliferating stem cells derived from basal cells. Numerous mitotic figures and bromodeoxyuridine labeling were found in the regenerating basal cells, stem cells, and sustentacular cells at 4 and 7 days. As repair processes advanced, the numbers of olfactory neurons and vesicles were increased with a proportional decrease in stem cells.

The metabolism of β-chloroprene: preliminary in-vitro studies using liver microsomes

Based on analogy with butadiene and isoprene, the metabolism of beta-chloroprene (2-chloro-1,3-butadiene, CD) to reactive intermediates is likely to be a key determinant of tumor development in laboratory rodents exposed to CD by inhalation. The purpose of this study is to identify species differences in toxic metabolite (epoxide) formation and detoxification in rodents and humans. The in-vitro metabolism of CD was studied in liver microsomes of B6C3F1 mice, Fischer/344 and Wistar rats, Syrian hamsters, and humans. Microsomal oxidation of CD in the presence of NADP(+), extraction with diethyl ether, and analysis by GC-mass selective detection (MSD) indicated that (1-chloroethenyl)oxirane (CEO) was an important metabolite of CD in the liver microsomal suspensions of all species studied. Other potential water-soluble oxidative metabolites may have been present. The oxidation of CD was inhibited by 4-methyl pyrazole, an inhibitor of CYP 2E1. CEO was sufficiently volatile at 37 degrees C for vial headspace analysis using GC-MSD single ion monitoring (m/z=39). CEO was synthesized and used to conduct partition measurements along with CD and further explore CEO metabolism in liver microsomes and cytosol. The liquid-to-air partition coefficients for CD and CEO in the microsomal suspensions were 0.7 and 58, respectively. Apparent species differences in the uptake of CEO by microsomal hydrolysis were hamster approximately human>rats>mice. Hydrolysis was inhibited by 1,1,1-trichloropropene oxide, a competitive inhibitor of epoxide hydrolase. A preliminary experiment indicated that the uptake of CEO in liver cytosol by GSH conjugation was hamster>rats approximately mice (human cytosol not yet tested). In general, the results suggest that metabolism may help explain species differences showing a greater sensitivity for CD-induced tumorigenicity in mice, for example, compared with hamsters. Additional experiments are in progress to quantify the kinetic parameters of CD oxidation and CEO metabolism by enzymatic hydrolysis and conjugation by glutathione S-transferase for in cytosol. A future goal is to use the kinetic rates to parameterize a physiologically based toxicokinetic model and relate the burden of toxic metabolite to the cancer dose-response observed in experimental animals.

In vitro genotoxicity testing of (1-chloroethenyl)oxirane, a metabolite of β-chloroprene

(1-Chloroethenyl)oxirane (CEO) is a metabolite of beta-chloroprene (2-chloro-1,3-butadiene, CD). The purpose of this study was to evaluate the in vitro mutagenic and clastogenic (chromosome breaking) potential of CEO. For comparative purposes, the study also included an evaluation of the racemic compounds, 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Mutagenicity was evaluated in a bacterial reverse mutation test (Ames), using the pre-incubation method in the presence and absence of an exogenous metabolism system (Aroclor)-induced rat liver S9). Four Salmonella typhimurium tester strains, TA97a, TA98, TA100 and TA1535 were used. The exposure concentrations in the sealed incubation vials ranged from 0 to 69 mM for CEO, 0 to 102 mM for EB, and 0 to 83 mM for DEB. All three compounds showed signs of toxicity, with DEB being substantially more toxic than either CEO or EB. Mutagenic activity was observed with all three chemicals in primarily the base pair substitution strains (S. typhimurium TA100 and TA1535), but some activity was also seen in the frameshift elimination strains (S. typhimurium TA97a and TA98). The observed mutagenic responses after exposure with CEO or EB were greater than the observed response for DEB, most likely because of the higher toxicity of DEB. Generally, the mutagenic responses were unchanged in the frameshift strains and base pair substitution strains in the presence of S9 metabolism. In vitro clastogenicity was evaluated using the cytochalasin-B blocked micronucleus test in cultured Chinese hamster V79 cells. The test was conducted without S9 metabolism because of the absence of substantial changes in the Ames test. Exposure concentrations ranged from 0 to 0.943 mM for CEO, 0 to 3.0 mM for EB, and 0 to 0.035 mM for DEB, with the upper exposure concentrations dictated by cytotoxicity. Cytotoxicity, measured as a reduction in the proportion of binucleated cells and altered cell morphology, was observed for CEO at concentrations > or =0.175 mM. Exposure to EB led to a reduced proportion of binucleated cells at concentrations > or =2.0 mM, and cell death was observed after DEB exposure at concentrations > or =0.025 mM. No clastogenicity was observed in the V79 cells when tested up to cytotoxic concentrations of CEO, whereas an elevated frequency of micronuclei was observed after exposure to either EB (> or =1.0 mM) or DEB (> or =0.0125 mM). These results suggest that CEO-induced mutagenicity, but not clastogenicity, may contribute to the observed beta-chloroprene-induced carcinogenicity in the rodent bioassay studies.

Overview of the acute, subchronic, reproductive, developmental and genetic toxicology of β-chloroprene

beta-Chloroprene (CD), the 2-chloro derivative of 1,3-butadiene, is used for the manufacture of the synthetic rubber, polychloroprene. Acute inhalation studies show that CD is lethal to Crl:CD rats at >2300 p.p.m. (4 h); the primary target organ effects were pulmonary hemorrhage and edema, and hepatic necrosis. In 2- and 4-week inhalation studies in Fischer 344 (F344) and Wistar rats, early deaths occurred at 500 and > or =161 p.p.m., respectively. Organ system injury was found in the nose (degeneration/metaplasia of olfactory epithelium), liver (centrilobular necrosis), and blood (decreased red blood cell count in F344 rats only). In a 90-day inhalation study with F344 rats, degeneration/metaplasia of the olfactory epithelium and reduced nonprotein sulfhydryl content of lungs and liver were found in animals exposed to 80 p.p.m., and anemia, hepatocellular necrosis, and forestomach inflammation were observed at 200 p.p.m. In a 90-day study with B6C3F1 mice, CD caused deaths at 200 p.p.m., the highest concentration tested, and epithelial hyperplasia of the forestomach at 80 p.p.m. Other than a slight (<10%) reduction in sperm motility in male rats at 200 p.p.m., all other reproductive parameters (sperm count or morphology in males, and estrous cyclicity or cycle length in females) were unaffected in these 90-day rat/mouse studies. There were no significant indications of neurological toxicity. The study No-Observable Adverse Effect Level was 32 p.p.m. based on nasal injury in rats. Despite some early reports of reproductive system abnormalities at levels <1 p.p.m., recent studies show no embryotoxic or developmental toxicity in female Wistar or Crl:CD rats, or in New Zealand White rabbits at CD exposure concentrations up to 25 or 175 p.p.m., respectively. In a one-generation reproduction study with Wistar rats, CD produced growth retardation in the F(0) generation exposed to 100 p.p.m., and in the F(1) offspring at 33 and 100 p.p.m.; no effects on reproductive parameters or histopathology were found. CD is nonmutagenic in standard plate incorporation bacterial reverse mutation assays (Ames assays) but positive using direct gas-phase incubation methods. Bacterial mutagenicity (primarily base pair substitution) was either negative or weakly positive when freshly prepared CD was tested. Mutagenicity increased markedly with time, presumably from CD dimer formation, and also by addition of liver S9 metabolic activation system. In vivo micronucleus, chromosome aberration and sister chromatid exchange studies in mice showed no structural chromosomal damage. Overall, the pathological effects in the liver and nose dominate the subchronic toxicity of CD. The genotoxicity of CD is inconsistent and requires further study.

Dimethylacetamide pharmacokinetics following inhalation exposures to rats and mice

Whole-body inhalation exposures to N,N-dimethylacetamide (DMAC) were conducted with male rats (Crl:CD BR) and mice (Crl:CD-1 (ICR)BR). Exposure concentrations were 50, 150, 300 and 500 ppm. The exposure routines consisted of single 1-, 3-, or 6-h exposures and ten 6-h exposures (10 exposure days in 2 weeks). Area under the plasma concentration curve (AUC) values were determined for DMAC and its metabolite N-methylacetamide (NMAC), following 6-h exposures (single exposure or last in a series of 10 exposures). The range of exposures was chosen to assess the exposure-dependent nature of DMAC pharmacokinetics in rats and mice. Plasma profiles indicated mice metabolized DMAC rapidly with plasma half-lives from 0.3 to 0.5 h for DMAC. The DMAC AUC values from mice were underestimated due to the required time (< 30 min) between termination of exposure and the initial blood sample. DMAC plasma half-life in rats ranged from 0.6 to 1.5 h. The AUC values for DMAC in rats increased approximately 5-fold and 3-fold as exposure concentrations increased from 150 to 300 ppm and 300 to 500 ppm, respectively. NMAC persisted in plasma for at least 24 h after the 150, 300 and 500 ppm exposures to rats. NMAC was not detected in plasma from mice beyond the 12-h post-exposure timepoint for the 300 and 500 ppm exposures. Regardless of exposure level, repeated DMAC exposures to both rats and mice resulted in plasma profiles of DMAC and NMAC similar to those from a single exposure. The dose-dependent nature of the DMAC AUC data and the absence of effects of repeated 300 and 500 ppm DMAC exposures supported a toxicity-driven upper limit of 350 ppm for a chronic inhalation study.

Inhalation Toxicity and Genotoxicity of Hydrofluorocarbon (HFC)-236fa and HFC-236ea

The acute, subchronic, and developmental and genetic toxicity of hydrofluorocarbon (HFC)-236fa and HFC-236ea were evaluated to assist in establishing proper handling guidance. In acute inhalation studies, rats were exposed whole body for 4 hours to various concentrations of each isomer. Based on the lack of mortality, the approximate lethal concentration for HFC-236ea for male rats was > 85,000 ppm. For HFC-236fa, the LC50 for males and females (combined) was > 457,000 ppm. Narcotic-like effects, e.g., prostration, incoordination, and reduced motor activity, were observed only during exposure to either isomer, but were not evident after termination of exposure. In cardiac sensitization studies, HFC-236ea induced cardiac sensitization at ≥ 35,000 ppm, with fatal responses occurring at 50,000 ppm and greater. For HFC-236fa, a cardiac sensitization response was observed at 150,000 ppm and greater but not at 100,000 ppm. A fatal cardiac sensitization response was observed in one dog exposed to 150,000 ppm HFC-236fa. In 90-day subchronic inhalation studies, male and female rats were exposed whole body to HFC-236ea at concentrations of 0, 5000, 20,000, or 50,000 ppm for 6 hours/day, 5 days/week. Similarly, male and female rats were exposed whole body to HFC-236fa at concentrations of 0, 5000, 20,000, or 50,000 ppm for 6 hours/day, 5 days/week. During exposure, narcotic-like effect (reduced acoustic startle response) was observed at 50,000 ppm with both isomers, although there appeared to be an adaptive response to this effect as the study progressed. With HFC-236ea, dilatation of the seminiferous tubules, without effects on germ or Sertoli cells, was observed only in rats at 50,000 ppm. No other effects on in-life measures or on clinical or anatomic pathology, including histopathology, were observed for either isomer. In rat developmental toxicity studies, no evidence of embryotoxicity or teratogenicity was observed with either isomer exposed up to 50,000 ppm during gestational days 7 to 16. Also, no developmental toxicity was observed in rabbits exposed to HFC-236fa at concentrations of up to 50,000 ppm during gestational days 7 to 19. Neither of the HFC-236 isomers was mutagenic in the Ames reverse mutation assay or clastogenic in the chromosomal aberration assay with human lymphocytes. No increase in chromosomal aberrations was observed in in vivo micronucleus studies with either isomer.

Nonlinear responses for chromosome and gene level effects induced by vinyl acetate monomer and its metabolite, acetaldehyde in TK6 cells

Vinyl acetate monomer (VAM) produced rat nasal tumors at concentrations in the hundreds of parts per million. However, VAM is weakly genotoxic in vitro and shows no genotoxicity in vivo. A European Union Risk Assessment concluded that VAMs hydrolysis to acetaldehyde (AA), via carboxylesterase, is a critical key event in VAMs carcinogenic potential. In the following study, we observed increases in micronuclei (MN) and thymidine kinase (Tk) mutants that were dependent on the ability of TK6 cell culture conditions to rapidly hydrolyze VAM to AA. Heat‐inactivated horse serum demonstrated a high capacity to hydrolyze VAM to AA; this activity was highly correlated with a concomitant increase in MN. In contrast, heat‐inactivated fetal bovine serum (FBS) did not hydrolyze VAM and no increase in MN was observed. AAs ability to induce MN was not impacted by either serum since it directly forms Schiff bases with DNA and proteins. Increased mutant frequency at the Tk locus was similarly mitigated when AA formation was not sufficiently rapid, such as incubating VAM in the presence of FBS for 4 hr. Interestingly, neither VAM nor AA induced mutations at the HPRT locus. Finally, cytotoxicity paralleled genotoxicity demonstrating that a small degree of cytotoxicity occurred prior to increases in MN. These results established 0.25 mM as a consistent concentration where genotoxicity first occurred for both VAM and AA provided VAM is hydrolyzed to AA. This information further informs significant key events related to the mode of action of VAM‐induced nasal mucosal tumors in rats. Environ. Mol. Mutagen. 54:755–768, 2013.

Developmental toxicity of inhaled trans-1,2-dichloroethylene in the rat

The developmental toxicity of trans-1,2-dichloroethylene (t-DCE), a component of certain Freon cleaning agents, was examined in pregnant rats. t-DCE was administered by inhalation 6 hr daily on Days 7-16 of gestation (the day copulation was confirmed was termed Day 1 of gestation) at exposure levels of 0, 2000, 6000, or 12,000 ppm. The offspring were then examined on Day 22 of gestation. Overt maternal toxicity was expressed as a significant reduction in weight gain at 12,000 ppm and in feed consumption at 6000 and 12,000 ppm. During the exposure period, lacrimation and stained periocular hair, and signs of ocular irritation, were observed in all groups. In addition, increased incidences of alopecia, lethargy, and salivation were observed in the high-dose dams. Significant increases in the mean number of resorptions per litter were seen in the litters of dams exposed to 6000 and 12,000 ppm of t-DCE; however, these values are within the range of historical controls and not considered to be treatment related. The mean combined and female fetal weights were significantly reduced in the litters of dams exposed to the highest concentration (12,000 ppm) of t-DCE. Marginal effects on feed consumption, unaccompanied by other changes and reflective of the pattern seen at higher doses, were seen at 2000 ppm. Thus, marginal maternal toxicity was seen at 2000 ppm and exposures to 6000 ppm t-DCE or higher caused frank maternal toxicity while the fetus was affected only at 12,000 ppm. Therefore, t-DCE is not considered to be uniquely toxic to the rat conceptus.