Lack of Mutagenic Activity of 1,6-Hexamethylene Diisocyanate

Valentine O. Wagner*, Richard H. C. San*, Ramadevi Gudi*, Roger J. Hilaski{dagger} and David Jacobson-Kram*,1

* BioReliance Corporation, 14920 Broschardt Road, Rockville, Maryland 20850; and {dagger} MPI Research, Mattawan, Michigan 49071

Received October 15, 1999; accepted February 18, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1,6-Hexamethylene diisocyanate (HDI) is an aliphatic diisocyanate used in the manufacture of higher molecular weight biuret and trimer polyisocyanate resins. These resins are commonly used in polyurethane paints, resulting in potential occupational, and to a lesser extent consumer exposures. Because some isocyanates have been reported to be mutagenic, HDI was tested in the bacterial reverse mutation assay (Ames test), CHO/HGPRT gene mutation assay, and in the mouse micronucleus test, using vapor-phase exposures. Although indicators of toxicity were observed in each test, HDI did not induce mutagenic or clastogenic effects in any of the three assays.

Key Words: 1,6-hexamethylene diisocyanate (HDI); aliphatic diisocyanate; mutagenicity; gene mutation; micronucleus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1,6-Hexamethylene diisocyanate (HDI) is an aliphatic diisocyanate used in the manufacture of higher molecular weight biuret and trimer polyisocyanate resins (Sangha et al., 1981Go). These resins are commonly used in polyurethane paints. The use of HDI resins in paints can result in occupational exposures; limited consumer exposures may also occur. Previous studies on a series of isocyanates suggested that at least some are mutagenic in a bacterial reverse mutation assay (Anderson et al., 1980Go). HDI was the object of an enforceable consent agreement between the Environmental Protection Agency and the Chemical Manufacturers Association (Federal Register, 1997Go). As part of the consent agreement, HDI was tested for potential mutagenicity in the bacterial reverse mutation assay (Ames test), CHO/HGPRT (Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase) gene mutation assay, and in the mouse micronucleus test. Because of its volatile properties, HDI was tested using vapor-phase exposures. All studies were performed in strict compliance with Good Laboratory Practices (GLPs). The two in vitro studies are designed to determine whether a test material induces mutations in a specific gene. In the case of the bacterial test, mutations revert cells from auxotrophy to prototrophy. Positive results, depending on the tester strain, indicate that the test material induced base substitutions or frameshift mutations. The mammalian gene mutation assay is a forward mutation test. The results indicate whether a test material is capable of mutating a gene such that the gene product is no longer functional. The in vivo mouse micronucleus test indicates whether a test material has the ability to induce chromosome breakage in bone marrow cells. This assay also indicates if test materials are disrupting the mitotic process and inducing aneuploidy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test chemical and positive controls.
HDI (CAS #822-06-0), a product of Bayer Corporation, was received on February 9, 1998, with a reported purity of at least 99.5%. It was stored in the dark at room temperature with a blanket of nitrogen. The following positive controls were used in the bacterial mutation assay: 2-aminoanthracene (Sigma), 2-nitrofluorene (Aldrich), sodium azide (Sigma), and 9-aminoacridine (Sigma). Ethyl methanesulfonate (Aldrich) and benzo(a)pyrene (Sigma) were used as positive controls in CHO/HGPRT assay, while cyclophosphamide (Sigma) was used in the mouse micronucleus test.

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, 1983Go; Vogel and Bonner, 1956Go), 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., 1992Go). 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., 1981Go; O'Neill et al., 1977Go; Wagner et al., 1992Go). 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, 1973Go; Heddle et al., 1983Go).

The assay was performed in two phases (Matter and Grauwiler, 1974Go). 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, 1970Go; Mackey and MacGregor, 1979Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Mutation Assay
In the initial toxicity assay, the maximum dose tested was 5 ml per desiccator; this dose was achieved by delivering neat test substance to the desiccator and exposing the plates for 24 h. Lower doses were achieved by varying the aliquot of neat test substance delivered to each desiccator. No precipitate was observed but nearly complete toxicity was observed at all dose levels. The preliminary toxicity assessment was repeated using sequentially lower doses. Toxicity was observed beginning at either 10 or 33 µl per desiccator, regardless of strain or activation condition. In addition, many plates exhibited non-uniform toxicity. The non-uniform toxicity did not appear to be a function of the location or orientation of the plates in the desiccators. Additional toxicity studies were performed and the exposures were decreased to 8 h. Toxicity was generally observed at >= 100 µl per desiccator, with non-uniform toxicity across many plates. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 150 µl per desiccator, with an exposure time of 8 h. The lowest dose level was decreased from 10 µl to 6.0 µl per desiccator in an attempt to achieve an appropriate number of nontoxic dose levels. An aliquot of 6.0 µl per desiccator was the smallest volume of test substance that could be accurately delivered to the test system by the vapor-phase exposure method.

The results of the mutagenicity assay are presented in Table 1Go. 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., 1992Go). No mutagenic activity was observed with any of the tester strains in the presence and absence of S9 activation (Table 1Go). 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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TABLE 1 Salmonella Mutagenicity Assay
 
CHO/HGPRT Gene Mutation Assay
In the first preliminary cytotoxicity trial, CHO cells in 25 cm2 flasks were exposed for 5 h at seven dose levels of HDI 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. Cloning efficiency relative to the negative controls (RCE) was 78% at 5.0 ml without activation and 129% at 5.0 ml with S9 activation.

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 2Go. Mutagenicity data are presented in Tables 3Go (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 4GoGo). 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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TABLE 2 CHO/HGPRT Mutation Assay: Concurrent Cytotoxicity Test
 

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TABLE 3 CHO/HGPRT Mutation Assay
 

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TABLE 4 CHO/HGPRT Mutation Assay: Confirmatory Assay
 
Mouse Micronucleus Test
In the range-finding study, male and female mice were exposed with the target exposure levels of 0.2 (actual 0.195 ± 0.090 ppm), 0.4 (actual 0.361 ± 0.105 ppm), 1.0 ppm (actual 1.34 ± 0.383 ppm), and 1.5 ppm (actual 1.08 ± 0.154 ppm), or with just air for 6 h. Based on the analytical results, animals exposed with the target exposure level of 1.0 ppm were considered as high-exposure group. Four groups of three mice per sex per group were exposed to HDI vapors and a negative control group consisting of six mice per sex was exposed to air only on each exposure day. Mice were observed for clinical signs prior to, during, and immediately after the inhalation exposure and each day thereafter for 2 days. No mortality was observed in male or female mice regardless of the exposure levels tested. Clinical signs observed during the 48-h period following inhalation included decreased activity and slow respiration in male and female mice at 0.4 ppm; decreased activity, labored breathing, slow respiration, abnormal vocalization, and tremors in male and female mice at 1.08 ppm and 1.5 ppm; and wheezing in male and female mice at 1.5 ppm. A significant loss of body weight on day 3 postdosing was observed (> 10%) in both male and female mice at the two higher exposure levels (Table 5Go). Two days after the inhalation exposure, bone marrow smears were prepared from all animals. Reductions of 4 to 15% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the HDI-exposure groups relative to their respective air controls. Reduction of 12% and 15% in the ratio of polychromatic erythrocytes was observed in male mice at 1.08 ppm and 1.34 ppm, respectively. The exposure levels for the main study were selected to be 0.15 ppm, 0.75 ppm, and 1.5 ppm. The top exposure was considered to be the maximum tolerated exposure based on extensive clinical signs and loss of body weight.


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TABLE 5 Toxicity Study in CD-1 Mice: Body Weight and Mortality Data
 
In the micronucleus assay, male and female mice were exposed with the target exposure levels of 0.15 ppm (actual 0.14 ± 0.043 ppm), 0.75 ppm (actual 0.80 ± 0.126 ppm), and 1.5 ppm (actual 1.47 ± 0.309 ppm), or with just air for 6 h. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs on the day of exposure included increased activity during the exposure of 0.15 ppm, 0.75 ppm, and 1.5 ppm in male and female mice; slow respiration in male and female mice at 0.75 ppm and only in female mice at 1.5 ppm; labored breathing in one male mouse at 0.75 ppm and female mice at 1.5 ppm; and abnormal vocalization in one female mouse at 1.5 ppm. The increased activity was considered to be a transient phenomenon occurring during the early stages of exposure. Significant weight loss was seen in both males and females at the two higher doses on post-treatment day 2 (Table 6Go). Bone marrow cells, collected 24 and 48 h after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions of 2 to 17% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in HDI-treated males relative to the air control group at 48-h harvest.


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TABLE 6 Body Weight Changes in CD-1 Mice Examined in Micronucleus Study
 
No significant increase in micronucleated polychromatic erythrocytes in HDI-treated groups relative to the respective air control group was observed in male or female mice at 24 or 48 h after exposure (p > 0.05, Kastenbaum-Bowman, Table 7Go). The results of the assay indicate that under the test conditions, hexamethylene diisocyanate did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. Hexamethylene diisocyanate was concluded to be negative in the mouse micronucleus assay.


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TABLE 7 Summary of Bone Marrow Micronucleus Study Using Hexamethylene Diisocyanate
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies with other isocyanates used in the production of polyurethanes yielded positive results in the Salmonella reverse mutation assay (Anderson, 1980). All positive responses were seen only in the presence of S9 metabolic activation. Toluene diisocyanate was positive in TA1538, TA100, and TA98; 4-4`methylene diphenyl isocyanate was positive in TA100 and TA98, whereas 1-naphthyl isocyanate was positive only in TA100. The authors suggested that isocyanates react with hydrogen and through hydrolysis may produce mutagenic aromatic amines. The current studies show that the widely used isocyanate HDI is not mutagenic or clastogenic in three commonly used genetic toxicology assays. Although the pattern of toxicity seen in the bacterial mutation is unusual, HDI clearly induced a cytotoxic response, as seen by reduced numbers of revertant colonies compared to the vehicle. In the presence of a significant level of toxicity, the results were clearly negative. A comparable situation was seen in the CHO/HGPRT assay; concurrent cytotoxicity data were somewhat variable. Nevertheless, under conditions where cytotoxicities were in the range of 50%, there were no significant increases in mutant frequencies. In cases where there was not a significant level of concurrent cytotoxicity, HDI was tested to the highest dose that could be practically achieved. In the micronucleus assay, the two highest HDI doses resulted in clinical signs and significant weight loss, especially on day 2. The types of clinical signs noted and the clear reduction in absolute body weights argue strongly that an MTD had been reached. Again, there was no indication of increased frequencies of micronucleated erythrocytes. The results suggest that HDI was tested at sufficiently high concentration and that no mutagenic responses were noted.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (301) 738-2362. E-mail: djacobson-kram{at}bioreliance.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ames, B. N., McCann J., and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347-364.[ISI][Medline]

Anderson, M., Binderup, M.-L., Kiel, P., Larsen, H., and Maxild, J. (1980). Mutagenic action of isocyanates used in the production of polyurethane. Scand. J. Work Environ. Health 6, 222-226.

Federal Register (1997). Testing consent order for 1,6-hexamethylene diisocyanate. 64, 51107-51108.

Heddle, J. A. (1973). A rapid in vivo test for chromosomal damage. Mutat. Res. 18, 187-190.[ISI][Medline]

Heddle, J. A., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J. T. Newell, G. W., and Salamone, M. (1983). The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 123, 61-118.[ISI][Medline]

Hsie, A. W., Casciano, D. A., Couch, D. B., Krahn, B.F., O'Neill, J.P., and Whitfield, B.L. (1981). The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. A report of the Gene-Tox Program. Mutat. Res. 86, 193-214.[ISI][Medline]

Kastenbaum, M. A., and Bowman, K. O. (1970). Tables for determining the statistical significance of mutation frequencies. Mutat. Res. 9, 527-549.[ISI][Medline]

Mackey, B. E., and MacGregor, J. T. (1979). The micronucleus test: statistical design and analysis. Mutat. Res. 64, 195-204.[ISI][Medline]

Maron, D. M., and Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173-215.[ISI][Medline]

Matter, B. E., and Grauwiler, J. (1974). Micronuclei in mouse bone marrow cells. A simple in vivo model for the evaluation of drug-induced chromosomal aberrations. Mutat. Res. 23, 239-249.[ISI][Medline]

O'Neill, J. P., Brimer, P. A., Machanoff, R., Hirsch, G. P., and Hsie, A. W. (1977). A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): development and definition of the system. Mutat. Res. 45, 91-101.[ISI][Medline]

Sangha, G. K., Matijak, M., and Alarie, Y. (1981). Comparison of some mono- and diisocyanates as sensory irritants. Toxicol. Appl. Pharmacol. 57, 241-246.[ISI][Medline]

Vogel, H. J., and Bonner, (1956). Acetylornithinase of E. coli: partial purification and some properties. J. Biol. Chem. 218, 97-106.[Free Full Text]

Wagner, V. O., III, San, R. H., and Zeiger, E. (1992). Desiccator methodology for Salmonella mutagenicity assay of vapor-phase and gas-phase test materials. Environ. Mol. Mutagen. 19(Suppl 20), 68.





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