A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Respiratory hypersensitivity to diphenylmethane-4,4'-diisocyanate in guinea pigs: comparison with trimellitic anhydride.

Published evidence demonstrates successful induction and elicitation of respiratory hypersensitivity in guinea pigs by the known human respiratory allergens trimellitic anhydride (TMA) and diphenylmethane-4,4'-diisocyanate (MDI). From these data it is apparent that TMA-related respiratory hyperresponsiveness can be elicited readily in guinea pigs upon inhalation challenge with the free chemical. Despite the interlaboratory variability in methodological procedures used for the sensitization as well as elicitation of response and the wide range of concentrations of TMA employed for challenge exposures (6-57 mg/m(3) air), TMA had been unequivocally identified as a benchmark respiratory sensitizer by measurements of the respiratory rate during challenge. The protocols were duplicated to examine the respiratory sensitizer MDI. In intradermally sensitized guinea pigs, changes in immediate-onset-like respiratory response were observed when MDI challenge concentrations exceeded approximately 30 mg MDI/m(3) air. Collective experimental evidence suggests that the respiratory responses observed upon challenge with TMA were markedly more pronounced and easier to identify than those recorded following challenge with MDI or MDI conjugate. In contrast to TMA, irritant concentrations of MDI had to be used to elicit any respiratory response and the differentiation of irritant and allergic responsiveness became increasingly difficult. Despite the absence of unequivocal changes in breathing patterns upon MDI challenge, MDI-sensitized animals displayed elevated anti-MDI immunoglobulin G1 (IgG1) antibodies, and a significant influx of eosinophilic granulocytes in the bronchial wall and lung-associated lymph nodes. Therefore, it is believed that the robustness of this animal model to identify low-molecular-weight agents as respiratory sensitizer is increased when several endpoints are considered. These are (1) positive respiratory response upon challenge with the hapten, and if negative, also challenge with the conjugate of the hapten; (2) an influx of eosinophilic granulocytes; and (3) increased specific IgG1 response. Furthermore, it appears that particles in the range of approximately 2-6 microm evoke more consistent respiratory response upon challenge exposure than particles in the 1-2 microm range.

Developmental toxicity evaluation of inhaled toluene diisocyanate vapor in CD rats.

Mated female CD (Sprague-Dawley) rats, 25/group, were exposed to toluene diisocyanate (TDI) vapor, for six h/day on gestational days (gd) 6 through 15, at 0.00, 0.02, 0.10, or 0.50 p.p.m.. Maternal clinical signs, body weights, and feed and water consumption were recorded throughout gestation. At termination (gd 21), maternal body, gravid uterine, and liver weights were recorded. Corpora lutea were counted, and implantation sites were identified: resorptions and dead and live fetuses. All live fetuses were examined for external alterations. One-half of the live fetuses/litter were examined for visceral (including craniofacial) alterations. The remaining intact fetuses/litter were stained with alizarin red S and examined for ossified skeletal alterations. Maternal toxicity at 0.50 ppm consisted of reduced body weights, body weight gains, feed consumption, and clinical signs of toxicity. Water consumption was unaffected. Gestational parameters exhibited no significant treatment-related changes, including pre- and postimplantation loss, sex ratio/litter, or fetal body weights/litter. Incidences of individual malformations, malformations by category (external, visceral, and skeletal), total malformations, individual external and visceral variations, variations by category, and total variations were unaffected. Of 111 skeletal variants observed, only 1, incidence of poorly ossified cervical centrum 5, was increased at 0.50 ppm, indicating possible minimal fetotoxicity, although it occurred in the absence of any other indications of developmental toxicity. Therefore, exposure to TDI vapor by inhalation, during major organogenesis in CD rats, resulted in maternal toxicity and minimal fetotoxicity at 0.50 ppm no observed adverse effect level (NOAEL) for maternal and developmental toxicity was 0.10 ppm. No treatment-related embryotoxicity or teratogenicity was observed.

Two-generation reproductive toxicity study of inhaled toluene diisocyanate vapor in CD rats.

Twenty-eight 42-day-old pups/sex/group (F0) were exposed to toluene diisocyanate vapor (TDI; 80% 2,4-TDI, 20% 2,6-TDI) by inhalation at 0.0, 0.02, 0.08, or 0.3 ppm, 6 h/day, 5 days/week, for 10 weeks, then mated within groups for 3 weeks, with exposure 7 days/week during mating, gestation, and lactation. F0 maternal animals were not exposed from gestational day (gd) 20 through postnatal day (pnd) 4; maternal exposures resumed on pnd 5. Twenty-eight weanlings/sex/group continued exposure for 12 weeks (starting on pnd 28) and were bred as described above. F0 and F1 parents and ten F1 and F2 weanlings/sex/group were necropsied, and adult reproductive organs, pituitary, liver, kidneys, and upper respiratory tract (target organs) were evaluated histologically in ten/sex/group. Adult toxicity was observed in both sexes and generations at 0.08 and 0.3 ppm, including occasional reductions in body weights and weight gain, clinical signs of toxicity at 0.08 and 0.3 ppm, and histologic changes in the nasal cavities at 0.02, 0.08, and 0.3 ppm (including rhinitis, a nonspecific response to an irritating vapor, at all concentrations). There was no reproductive toxicity, reproductive organ pathology, or effect on gestation or lactation at any exposure concentration. Postnatal toxicity and reduced body weights and weight gains during lactation occurred only in F2 litters at 0.08 and 0.3 ppm. Therefore, under the conditions of this study, a no observed adverse effect level (NOAEL) was not determined for adult toxicity; the NOAEL for reproductive toxicity was at least 0.3 ppm, and the NOAEL for postnatal toxicity was 0.02 ppm.

Chronic toxicity and oncogenicity of picloram in Fischer 344 rats.

The chronic toxicity and oncogenicity of the herbicide picloram was studied in male and female Fischer 344 rats administered 0, 20, 60, or 200 mg/kg.d technical-grade picloram via their feed for 2 yr. A comprehensive set of in-life and clinical pathology parameters was measured and an extensive list of tissues was examined grossly and by light microscopy from control and treatment groups of animals. The primary treatment-related effect observed in the study was hepatocellular swelling and altered tinctorial properties in the central regions of the liver lobules of both sexes of rats ingesting 60 or 200 mg/kg.d picloram. Males were more affected than females. Increases in liver weights accompanied these changes in both sexes of rats ingesting the high dose level of picloram. All other histopathologic lesions observed were typical of those that normally occur in aged Fischer 344 rats. There were no treatment-related increases in the incidence of any particular tumor type or in total tumors. No treatment-related effects were observed in rats ingesting 20 mg/kg.d of the test material.

Chlorpyrifos: a 13-week nose-only vapor inhalation study in Fischer 344 rats.

Fischer 344 rats were exposed by the nose-only inhalation route to chlorpyrifos vapors at concentrations of 0, 5.2, 10.3, or 20.6 ppb, 6 hr/day, 5 days/week for 13 weeks. The exposure concentrations were limited by the low vapor pressure of chlorpyrifos (theoretical maximum vapor concentration of 25 ppb at 25 degrees C). No treatment-related signs of toxicity or changes in body weights were detected during the course of the study. Urinalysis, hematology, clinical chemistry, organ weights, gross pathologic, and histopathologic evaluations were performed at the end of the study with no treatment-related effects observed. In addition, no differences from controls were noted in plasma, red blood cell, or brain cholinesterase activities. The results of this study indicate that the no-observed-effect level for chlorpyrifos vapor was the highest attainable concentration, 20.6 ppb, in male and female Fischer 344 rats.

Ethyl chloride: 11-day continuous exposure inhalation toxicity study in B6C3F1 mice.

Groups of seven B6C3F1 mice per sex were exposed for 23 hr/day to 0, 250, 1250, or 5000 ppm ethyl chloride (EtCl) for 11 consecutive days to evaluate the potential toxicity of EtCl under near-continuous exposure conditions. On the day following the last exposure, a neurobehavioral observation battery was performed, samples were obtained for clinical chemistry and hematology, and necropsies were conducted. Histopathologic examination was subsequently performed. The only observed effects were increased relative liver weights and a slight increase in hepatocellular vacuolation (glycogen or fat) in 5000 ppm-exposed mice. Exposures to EtCl were well tolerated despite the unusually long exposure periods.

Dietary toxicity of picloram herbicide in rats.

The toxicity of orally administered technical-grade picloram was evaluated in male and female Fischer 344 rats. Dietary dose levels were up to 2000 mg/kg body weight (bw) X d for 2 wk, 500 mg/kg bw X d for 13 wk, or 200 mg/kg bw X d for 12 mo. Routine indices of toxicity were evaluated at all of the respective time periods. Body weight, food consumption, clinical chemistries, urinalyses, and hematological determinations were considered unaffected by treatment. The only treatment-related effect, regardless of the duration of exposure, was in the liver of both male and female rats. This was generally manifested as an increase in the liver-to-body weight ratio and slight hypertrophy and pallor of the centrilobular hepatocytes. These effects were consistently present in rats receiving 1000 mg/kg bw X d for 2 wk, 300 mg/kg bw X d for 13 wk, or 200 mg/kg bw X d for 6 or 12 mo. Similar effects were marginally evident for rats receiving 500 mg/kg bw X d for 2 wk, 150 mg/kg bw X d for 13 wk, or 60 mg/kg bw X d for 6 or 12 mo. At 60 mg/kg bw X d, the effects were not progressive from 6 to 12 mo. The no-observable-effect level (NOEL) was 20 mg/kg bw X d for male and female rats fed picloram for 12 mo.

Pharmacokinetics of inhaled methyl chloride (CH3Cl) in male volunteers.

Six volunteers, 25-41 years of age, were exposed for 6 hr on separate days to 50 and 10 ppm of CH3Cl. Blood and expired air CH3Cl concentrations reached an apparent plateau during the first hour of the exposure and were proportional to the exposure concentration. Consistent with previous reports, the volunteers could be separated into two discrete groups based on the differences observed in their blood and expired air CH3Cl concentrations. Both groups eliminated CH3Cl rapidly once the exposure was terminated, but CH3Cl was eliminated more rapidly by those volunteers with the lower blood and expired air CH3Cl concentrations. The existence of these two groups can be explained by a twofold difference in the rate at which they metabolized CH3Cl; however, this difference is of questionable toxicological significance. Urinary excretion of the putative metabolite S-methyl cysteine was not related to the exposure; thus, it is not a valid means of monitoring occupational exposure to CH3Cl.

Neurotoxicity of methyl chloride in continuously versus intermittently exposed female C57BL/6 mice.

This study evaluated the relationship between methyl chloride (MeCl) exposure duration and neurotoxicity. Female C57BL/6 mice were exposed to MeCl for 11 days, either continuously (22 hr/day) to 15, 50, 100, 150, or 200 ppm, or intermittently (5.5 hr/day) to 150, 400, 800, 1600, or 2400 ppm. This strain and sex of mouse was chosen because it is sensitive to MeCl neurotoxicity and was a good candidate to allow the evaluation of morphological effects and the quantitation of functional effects. A simple quantitative relationship between neurotoxicity and continuous vs intermittent exposure was not observed. Although the no-observable-effect levels for continuous and intermittent MeCl exposures were very nearly proportionate to exposure concentration multiplied by duration, the dose-response curve was much steeper for continuously exposed mice. Cerebellar granular cell layer degeneration was observed in mice exposed continuously to 100 ppm MeCl and in mice exposed intermittently to 400 ppm. This histopathologic effect was observed at lower concentrations than a decrement in rotating rod running performance. No effects were observed in mice exposed to 50 ppm continuously or to 150 ppm intermittently. Continuous exposure to MeCl produced the cerebellar lesion with less effect on other tissues than did intermittent exposure. In mice exposed to 2400 ppm intermittently, there were renal and hematopoietic effects in addition to relatively slight cerebellar granular cell layer degeneration. These 2400-ppm exposed mice developed hemoglobinuria, apparently as a result of intravascular hemolysis. Although the effect of exposure duration on MeCl toxicity was complex, this study indicated that careful judgment is necessary when extrapolating intermittent exposure data to a continuous exposure situation.

Ethylene glycol monomethyl ether and propylene glycol monomethyl ether: metabolism, disposition, and subchronic inhalation toxicity studies.

Short-term and subchronic vapor inhalation studies have shown that there are pronounced differences in the toxicological properties of ethylene glycol monomethyl ether (EGME) and propylene glycol monomethyl ether (PGME). Overexposure to EGME has resulted in adverse effects on testes, bone marrow and lymphoid tissues in laboratory animals. PGME does not affect these tissues, and instead, overexposure to PGME has been associated with increases in liver weight and central nervous system depression. EGME is primarily oxidized to methoxyacetic acid in male rats, while PGME apparently undergoes O-demethylation to form propylene glycol. Since methoxyacetic acid has been shown to have the same spectrum of toxicity as EGME in male rats, the observed differences in the toxicological properties of EGME and PGME are thought to be due to the fact that the two materials are biotransformed via different routes to different types of metabolites.

Dipropylene glycol monomethyl ether: a 13-week inhalation toxicity study in rats and rabbits.

Fischer 344 rats (10/sex/exposure concentration) and New Zealand White rabbits (7/sex/exposure concentration) were exposed to 0, 15, 50, or 200 ppm (0, 91, 303, or 1212 mg/m3) of dipropylene glycol monomethyl ether (DPGME) for 6 hr/day, 5 days/week for 13 weeks. Criteria of response included general observations, body weights, clinical chemistry, hematology, urinalyses (rats only), necropsy, organ weights, and histopathology. There were no effects attributed to exposure to DPGME at any exposure concentration in either male or female rats or rabbits. The highest concentration tested (200 ppm) was approximately 40% of a saturated DPGME atmosphere. Based on the low vapor pressure of DPGME, and results in this 13-week study, DPGME appears to have a low subchronic vapor inhalation toxicity hazard.

Propylene glycol monomethyl ether: a 13-week inhalation toxicity study in rats and rabbits.

Fischer 344 rats (10/sex/exposure concentration) and New Zealand White rabbits (7/sex/exposure concentration) were exposed to 0, 300, 1000, or 3000 ppm (0, 1.09, 3.62, or 10.9 mg/L) of propylene glycol monomethyl ether (PGME) for 6 hr/da, 5 da/wk, for 13 weeks. Minimal effects were observed in animals exposed to 3000 ppm. Indications of a transient central nervous system depression were observed in rats and rabbits exposed to 3000 ppm. There were also small increases (6 to 8%) in mean relative liver weights of 3000 ppm exposed male and female rats relative to controls. Minimal histologic effects were observed in the livers of 3000 ppm exposed female rats. These were suggestive of hepatocellular hypertrophy but were without evidence of degenerative changes. There was an increase in the urinary pH of male rats exposed to 3000 ppm PGME for 4 weeks, but this was not evident after 12 weeks of exposure. There was no indication of histopathological effects in the kidneys of either species, and there were no hematological effects. No treatment-related effects were found in either rats or rabbits exposed to 300 or 1000 ppm.

Pulmonary physiology and inhalation dosimetry in rats: development of a method and two examples.

Methods were developed to measure simultaneously respiratory frequency, tidal volume, minute volume, and net uptake of an inhaled vapor in rats. During steady state, if metabolism is the only significant route of elimination, net uptake rate of the inhaled vapor is equal to its rate of metabolism. The rates of metabolism of methyl chloride in 50- and 1000-ppm-exposed rats were 0.20 and 3.3 nmol/min/g, respectively; the rates of metabolism of methylene chloride in 50- and 1500-ppm-exposed rats were 0.57 and 2.8 nmol/min/g, respectively. The uptake values obtained for both solvents were consistent with pharmacokinetic and metabolism data that were previously obtained in our laboratory. A pharmacokinetic model incorporating the metabolic rate at steady state, blood concentration versus time, and respiratory minute volume was used to describe the fate of inhaled methyl chloride in F344 rats, and to estimate the inhaled "effective" dose in 50- and 1000-ppm 6-hr-exposed rats (3.8 and 67 mg/kg, respectively). The approach used in these studies appears to be a useful method for the evaluation of metabolic rates and for inhalation dosimetry.

Pharmacokinetics and metabolism of inhaled methyl chloride in the rat and dog.

Methyl chloride (MeCl) metabolism and pharmacokinetics were studied in male Fischer 344 rats and male beagle dogs. Apparent steady-state blood MeCl concentrations were proportionate to exposure concentration in rats and dogs exposed to 50 and 1000 ppm. Furthermore, blood MeCl concentrations were similar in both species when they were exposed to the same concentration. A linear two-compartment open model described the blood MeCl data: alpha and beta phase elimination half-times corresponded to approximately 4 and 15 min, respectively, in rats, and 8 and 40 min in dogs. Rats exposed for 6 hr to 0, 50, 225, 600, or 1000 [14C]MeCl were evaluated for tissue nonprotein sulfhydryl (NPSH), total 14C activity, nonextractable tissue 14C activity, and urinary metabolites. MeCl-induced NPSH depletion was dose-related and was greatest in liver. Total 14C in liver and kidney was approximately proportionate to exposure concentrations. Relative concentrations of nonextractable 14C decreased at 600 to 1000 ppm MeCl suggesting a dose-dependent metabolic pathway for MeCl in the rat. Metabolites in urine included N-acetyl-S-methylcysteine, methylthioacetic acid sulfoxide, and N-(methylthioacetyl)glycine. These metabolites are likely to be products of a reaction between MeCl and glutathione. A nonradiometric analysis of a putative MeCl metabolite (S-methylcysteine) was performed in dogs exposed to MeCl; this method was not a sensitive indicator of MeCl exposure.

Ethyl chloride: a two-week inhalation toxicity study and effects on liver non-protein sulfhydryl concentrations.

Male and female Fischer 344 rats (6/sex/exposure concentration) and male beagle dogs (2/exposure concentration) exposed to 0, 1600, 4000 or 10 000 ppm ethyl chloride (EtCl) for 6 hr/da, 5 da/wk for 2 weeks showed no toxicologically significant treatment-related effects on body weights; clinical chemistry, hematology, or urinalysis parameters; neurology (dogs only were examined); gross pathology or histopathology. The only treatment-related differences in organ or relative organ weights (in rats or dogs) were slight, but statistically significant increases in liver to body weight ratios of male rats exposed to 4000 or 10,000 ppm EtCl (4.9 and 7.5% respectively). Liver non-protein sulfhydryl (NPSH) concentration was measured in male Fischer rats and male B6C3F1 mice that were exposed for 6 hours to 0, 1600, 4000 or 10,000 ppm EtCl (mice were exposed to 0 or 4000 ppm EtCl only). Liver NPSH, measured 1/2 hr post exposure, was less than control values in 4000 ppm exposed rats (88% of control value), 4000 ppm exposed mice (64%), and 10,000 ppm exposed rats (89%). The slight decreases in rat liver NPSH seem consistent with the increased liver to body weight ratios. The toxicity data indicate that 2-week repeated exposures to EtCl concentrations that were up to 10 times the current A.C.G.I.H. T.L.V. (1000 ppm) caused minimal treatment-related effects in dogs and rats.