* BioReliance Corporation, 14920 Broschardt Road, Rockville, Maryland 20850; and
MPI Research, Mattawan, Michigan 49071
Received October 15, 1999; accepted February 18, 2000
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ABSTRACT |
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Key Words: 1,6-hexamethylene diisocyanate (HDI); aliphatic diisocyanate; mutagenicity; gene mutation; micronucleus.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Bacterial reverse mutation assay.
HDI was tested for its ability to induce reverse mutations in Salmonella typhimurium both in the presence and in the absence of a rat liver microsome S9 mix. The test was performed using the following tester strains: TA98, TA100, TA1535, and TA1537. Although following standard methodology (Maron and Ames, 1983; Vogel and Bonner, 1956
), the assay was modified for vapor-phase exposure. The first phase, the preliminary toxicity assay, was used to establish the dose range for the second phase, the mutagenicity assay. The exposures were accomplished by delivering undiluted HDI to the bottom of 9-liter desiccators and exposing the cultures for a fixed period of time. Initial exposures were carried out for 24 h but were found to be highly toxic. In addition to adjusting the volumes of HDI placed into the desiccators, the exposure time was decreased to 8 h. With the exception of the exposure, the assay itself was conducted using standard procedures.
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, 5 days prior to sacrifice. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2, and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4.
One-half (0.5) milliliter of S9 or Sham mix and 100 µl of tester strain were added to 2.0 ml of molten selective top agar at 45 ± 2°C. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. The overlaid plates were inverted and placed uncovered in the appropriate number of 9-liter desiccators, segregated by test substance dose level, positive control (all contained together), and untreated control (all contained together). Measured volumes of HDI were placed in an uncovered petri dish on the bottom of each desiccator. Untreated controls were plated following the above method, without the addition of test substance. The desiccators were incubated for 8 to 24 h at 37 ± 2°C. Following incubation with HDI, the plates were removed from the desiccators and incubated with the lids replaced at 37 ± 2°C for an additional 8 to 64 h. The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Toxicity was scored relative to the untreated control plates. 2-Aminoanthracene at 1 µg/plate was used as the positive control for all strains in the presence of S9 activation. The following positive controls were used in the absence of S9 activation: 2-nitrofluorene at 1 µg/plate for TA98, sodium azide at 1 µg/plate for TA100 and TA1535, and 9-aminoacridine at 75 µg/plate for TA1537. Exposure to the positive controls was by plate incorporation.
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. For a test substance to be evaluated as positive, it must cause a dose-related increase in the mean revertants per plate in at least one tester strain, with a minimum of two increasing concentrations of test substance. Data sets for strains TA1535 and TA1537 are judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean untreated control value. Data sets for strains TA98 and TA100 are judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean untreated control value.
CHO/HGPRT gene mutation assay.
CHO-K1-BH4 cells were obtained from Dr. Abraham Hsie, Oak Ridge National Laboratories, Oak Ridge, TN. Cell cultures were exposed to HDI using the desiccator methodology in the presence and absence of metabolic activation. The desiccator methodology has been shown to be an effective method for detecting the genotoxic activity of volatile and gaseous test articles (Wagner et al., 1992). Aroclor 1254-induced rat liver S9 was used as the metabolic activation system and prepared as described above.
Preliminary toxicity assays were used to establish the optimal dose levels for the mutagenesis assay and consisted of evaluation of test article effect on colony-forming efficiency. CHO cells were exposed for 5 or 7.5 h at 37 ± 1°C to seven concentrations of HDI ranging from 0.05 to 5.0 ml and a negative control in both the absence and presence of S9-activation. The initial and independent repeat mutagenesis assays were used to evaluate the mutagenic potential of HDI. CHO cells were exposed for 7.5 h at 37 ± 1°C to a negative control, appropriate positive controls, and five concentrations of HDI, in duplicate, in both the absence and presence of S9. An untreated control, incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 7.5 h instead of in a desiccator, was included with the independent repeat assay.
The mutagenesis assay was performed according to a protocol developed from published methodologies (Hsie et al., 1981; O'Neill et al., 1977
; Wagner et al., 1992
). The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2 x 106 cells: 10 plates at 2 x 105 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 106 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony.
Mouse micronucleus test.
All in vivo portions of the study, including inhalation exposures, were performed by MPI Research, L.L.C. Animal euthanasia, bone marrow collection, slide preparation, and evaluation were performed by BioReliance (Heddle, 1973; Heddle et al., 1983
).
The assay was performed in two phases (Matter and Grauwiler, 1974). The first phase, designed to set exposure levels for the definitive study, consisted of a preliminary toxicity study (range-finding study). The second phase, the micronucleus study, evaluated the potential of the test substance to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control articles were administered by a single 6-h whole-body inhalation.
The inhalation exposures were conducted using a 1000-liter stainless steel and glass whole-body chamber. A minimum chamber airflow rate of 200 l/min supplied by the generation and HVAC systems resulted in at least 12 chamber air changes per hour, and a chamber equilibration time (T99) of 23 min. The chamber environment was maintained to the maximum extent possible at a temperature between 20 to 24°C and a relative humidity between 40 to 60%. Chamber temperature, percent relative humidity, and airflow rate were monitored continuously and recorded at 30-min intervals during the exposure period.
Chamber supply air was supplied by the HVAC system, with flow being controlled by chamber exhaust and monitored with a magnehelic gauge. The chamber inlet was open to room air during the exposure. Chamber airflow measurements were based on a pressure differential of the exhaust air through a 1.6-cm orifice plate.
For the exposure, the animals were removed from their home cages and placed in the inhalation caging prior to the generation of test substance atmosphere. Food and water were not available to the animals during the exposure period. Following the required exposure duration, the animals were returned to their individual home cages, where food and water were made available. The chamber size and flow rate were considered adequate to maintain the oxygen level above 19%. Prior to initiation of animal exposures, samples were obtained to demonstrate that the test substance was evenly distributed throughout the breathing zone of the animals.
Exposure atmospheres were generated in the following fashion: a known weight of HDI was added to the bubbler and compressed air (filtered and dried, 1% humidity) was metered by a flowmeter through the test substance. The resulting vapor entered the exposure chamber through 3/8 inch Teflon tubing placed into the chamber inlet, where it was mixed and diluted with chamber supply air. To enhance vapor generation, the test article bubblers for groups 4 and 5 (0.75- and 1.5-ppm groups) were placed in heated water baths (37°C). Following the exposure, the bubbler was weighed to determine the amount of test substance used for nominal calculations. The animals were exposed to vapor atmospheres of the test substance for 6 h (measured from the end of the T99 chamber equilibration time).
A nominal concentration was determined. The amount of HDI vaporized and delivered to the exposure chamber during the generation of test substance atmosphere was divided by the total volume of air passing through the chamber to give the nominal concentration. Actual concentrations were determined by high-performance liquid chromatography.
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed and cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were prepared for analysis by standard methods. An individual not involved with the scoring process coded slides using a random number table. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables, which are based on the binomial distribution (Kastenbaum and Bowman, 1970; Mackey and MacGregor, 1979
). In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
A test substance is considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes is observed and one or more doses are statistically elevated relative to the air control (p 0.05, Kastenbaum-Bowman tables) at any sampling time. A test substance is considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent air control is observed at any sampling time.
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RESULTS |
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The results of the mutagenicity assay are presented in Table 1. No precipitate was observed, but toxicity was generally observed at
6.0 µl per desiccator, with non-uniform toxicity over at least 25% of the surface of each affected plate. The non-uniform toxicity profile appears to be unique to 1,6-hexamethylene diisocyanate; it was not observed by this testing facility using this methodology with several volatile liquids tested under a contract with the NTP (Wagner et al., 1992
). No mutagenic activity was observed with any of the tester strains in the presence and absence of S9 activation (Table 1
). Although it can be argued that a completely nontoxic concentration was not tested, the complete lack of mutagenic activity at minimally and highly toxic doses suggests that the negative conclusion is valid. The positive control substances gave the expected increases, depending on strain, from 3.5 to 80 times the solvent control revertant frequencies.
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In the second trial, CHO cells in open 60-mm dishes were exposed for 7.5 h at seven dose levels of test article ranging from 0.05 to 5.0 ml in each desiccator in the absence and presence of S9 reaction mixture. A negative control was also included. The culture vessels were changed from flasks to dishes to improve cellular exposure to HDI, and because the quantities of HDI added to the dessicators did not appear to be limiting, the exposure time was increased to achieve a higher level of cytotoxicity. Cloning efficiency relative to the negative controls (RCE) was 27% at 5.0 ml without activation and 67% at 5.0 ml with S9 activation. Based on the results of the toxicity test, the doses chosen for the mutagenesis assay ranged from 1.0 to 5.0 ml for the nonactivated cultures and from 1.0 to 10 ml for the S9-activated cultures.
The cytotoxic effects of the test article (concurrent cytotoxicity) in both the initial and confirmatory assay are presented in Table 2. Mutagenicity data are presented in Tables 3
(initial study) and 4 (confirmatory study). In the initial nonactivated system, cultures treated with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 ml were cloned. In the S9-activated system, cultures treated with concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, and 10 ml were cloned. Relative cloning efficiency was 121% and 78% at the highest dose tested in the nonactivated and S9-activated systems, respectively. None of the treated cultures exhibited significantly elevated mutant frequencies (Tables 3 and 4
). Similar results were observed in the confirmatory study. In both the nonactivated system and the S9-activated system, cultures treated with concentrations of 1.0, 3.0, 5.0, 7.5, and 10 ml were cloned. Relative cloning efficiency was 74% and 114% at the highest dose tested in the nonactivated and S9-activated systems, respectively. Again, none of the treated cultures exhibited elevated mutant frequencies. Although it can be argued that little, or in some instances, no concurrent cytotoxicity was seen in the mutagenesis assays, it is believed that the highest possible practical dose was achieved. Because all the HDI added to the dessicators did not vaporize, the atmosphere was considered to be saturated and addition of more HDI would not have produced additional toxicity.
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DISCUSSION |
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NOTES |
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REFERENCES |
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