Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
13‐week inhalation toxicity study and fertility assessment in rats exposed to atmospheres containing adiponitrile R. D. Short , W. V. Roloff , L. D. Kier & W. E. Ribelin To cite this article: R. D. Short , W. V. Roloff , L. D. Kier & W. E. Ribelin (1990) 13‐week inhalation toxicity study and fertility assessment in rats exposed to atmospheres containing adiponitrile, Journal of Toxicology and Environmental Health, 30:3, 199-207, DOI: 10.1080/15287399009531423 To link to this article: http://dx.doi.org/10.1080/15287399009531423
Published online: 20 Oct 2009.
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13-WEEK INHALATION TOXICITY STUDY AND FERTILITY ASSESSMENT IN RATS EXPOSED TO ATMOSPHERES CONTAINING ADIPONITRILE R. D. Short
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Department of Medicine and Health Sciences, Monsanto Company, St. Louis, Missouri W. V. Roloff, L. D. Kier, W. E. Ribelin Environmental Health Laboratory, Monsanto Company, St. Louis, Missouri
Adiponitrile is an aliphatic dinitrile that is used as an intermediate in the synthesis of hexamethylenediamine. In order to asses potential health effects associated with industrial exposure, rats were exposed 6 h/d, 5 d/wk for 4 or 13 wk to atmospheres containing a range of adiponitrile concentrations and observed for signs of toxicity. A fertility assessment was also included as a component of the 13-wk study. Mortality and reduced weight gain were observed within 1 wk only in rats exposed to 493 mg/m3. Evidence of slight anemia was present in rats exposed to 99 mg/m3 and above. There was no histopathological evidence of organ toxicity in about 30 tissues from both sexes exposed up to 99 mg/m3, the highest concentration tested, for 13 wk. In addition, fertility, as monitored by reproductive performance and litter parameters, was normal in both males and females similarly exposed.
INTRODUCTION Adiponitrile (hexanedinitrile, CAS number 111-69-3), an aliphatic dinitrile, is a chemical intermediate in the synthesis of hexamethylenediamine, which is used in the production of nylon. The National Institute for Occupational Safety and Health (NIOSH) reviewed the available information on select nitriles and concluded that insufficient quantitative inhalation toxicity data were available for recommending workplace exposure limits for these materials (NIOSH, 1978). NIOSH adopted, as an alternative, a comparative toxicity approach using subcutaneous LD50 values in female rats and the available information for acetonitrile as a basis for recommending workplace exposure limits for the various nitriles they considered. Using this approach NIOSH recommended in 1978 that employee exposure to adiponitrile not exceed 18 mg/m3 (4 This manuscript was reviewed by the Monsanto Quality Assurance unit to assure consistency with the original reports and data developed during the conduct of these studies. Correspondence should be sent to Robert D. Short, Ph.D., Monsanto Company, Mail Zone A3ND, 800 N. Lindbergh Blvd, St. Louis, MO 63167.
199 Journal of Toxicology and Environmental Health, 30:199-207, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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ppm) as a time-weighted average limit for up to a 10-h workshift in a 40-h work week. Currently, neither the Occupational Safety and Health Administration nor the American Conference of Governmental Industrial Hygienists has adopted a workplace standard for adiponitrile. The literature contains the following information about adiponitrile. It is moderately toxic following a single exposure by the oral (rat), dermal (rabbit), and inhalation (rat) routes of exposure (Johannsen and Levinskas, 1986). After repeated exposure to atmospheres containing 268 mg/m3 6 h/d, 5 d/wk for 2 wk, rats showed a weight loss during the first week, and some clinical pathological changes at the end of the treatment period (Smith and Kennedy, 1982). These changes included decreased erythrocyte count, hemoglobin concentration, leukocyte count, relative number of eosinophils, and urine osmolality, and increased blood glucose, blood urea nitrogen, and urine glucose. All values were normal 2 wk later and there was no histopathologic evidence of target organ toxicity at the end of the treatment period. In a longer-time study, by a different route of exposure, rats were given drinking water containing 0.5, 5.0, and 50 ppm of adiponitrile for 2 yr (Svirbely and Floyd, 1964). No adverse effects on body weights, organ weight ratios (liver, kidney, and spleen), or hematological parameters were reported. Advanced adrenal degeneration, however, was reported in females at all exposure levels and in males exposed to the high concentration. The present studies were undertaken to provide additional information to assess possible health effects associated with adiponitrile in the industrial setting. Accordingly, rats were exposed to atmospheres containing adiponitrile for up to 13 wk and evaluated for treatment-related effects, and a fertility component was also included. A preliminary 4-wk pilot study was conducted to select concentrations for the 13-wk study and is also reported. METHODS Experimental Design CD rats (Charles River Breeding Laboratories, Portage, Mich.) were used in this study, which was conducted in two phases. In the first phase rats (18/sex/ group) were exposed to adiponitrile at target concentrations of 0, 50,100, and 500 mg/m for 4 wk and the results were used to select exposure levels for the next phase. In the second phase rats (15/sex/ group) were exposed to adiponitrile at target concentrations of 0,10, 30, and 100 mg/m3 for 13 wk. Additional rats (12 males and 24 females/group) were included to provide an assessment of male and female fertility by evaluating the outcome of pregnancies that resulted from the mating of treated with untreated rats. In this portion of the study males were exposed for at least 10 wk while females were exposed for at least 3 wk
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prior to mating. Exposures were discontinued for females after mating and for males the day before sacrifice. All exposures, with the exception of the female fertility study, where 6 h/d, 5 d/wk. Females in the fertility study were exposed 6 h/d, 7 d/wk.
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Exposure Test atmospheres were generated using adiponitrile with a purity of greater than 99%. All rats were exposed in 10-m3 Rochester-style inhalation chambers to air or air containing various concentrations of adiponitrile. Airflow was maintained between 9.8 and 12 chamber volumes per hour, temperature at 24 ± 2°C, and relative humidity at 50 ± 2%. Test atmospheres were generated using a Laskin-style nebulizer (Drew et al., 1978). The concentration of adiponitrile in the chambers was controlled by regulating its flow rate into the nebulizer. A vertical aerosol particle size separator prevented most of the large, nonrespirable aerosol particles from entering the chamber, and the large particles were collected in the bottom of the separator. Nominal concentrations of adiponitrile represent the net amount of material entering the chamber per unit time divided by the total air flow through the chamber per unit time. The analytical concentrations were determined by drawing atmosphere samples through an impinger containing 15 ml of methanol and measuring the concentration of adiponitrile in solution using a gas chromatographic method with a nitrogen phosphorus detector. Particle size of the aerosol was measured using a nonviable, nine-stage Andersen impactor (Anderson Samples Inc., Atlanta, Ga.).
Observations Rats were observed twice daily during each exposure for signs of toxicity and weighed at weekly intervals during the treatment period. In the 4-wk and 13-wk studies, blood was obtained from the periorbital sinus of the eye (4-wk study) or posterior vena cava (13-wk study) prior to sacrifice and analyzed for hematologic and blood chemistry parameters. A gross necropsy was performed on all survivors, organs were weighed (adrenals, testes with epididymides, heart, kidneys, liver, pituitary, and spleen), and the following tissues were preserved and processed for subsequent histopathological evaluation: abdominal aorta, adrenals, bone and bone marrow (femur), brain, esophagus, eyes with optic nerve, gonads (ovaries, and testes with epididymides), heart, intestine (colon and ileum), kidneys, liver, lung, lymph node (mesenteric), mammary gland, nasal turbinates, pancreas, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin, spinal cord (thoraco-lumbar), spleen, stomach, thymus, trachea, thyroid/parathyroid, urinary bladder, uterus (corpus and cervix), and gross lesions. In the male fertility study, each treated male remained on treatment and was given the opportunity to mate with 3 untreated females by hous-
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ing 1 male and 1 female for 5 consecutive nights or until evidence of mating (sperm positive vaginal smear or copulatory plug) was observed. Treatments were discontinued the day before sacrifice. Untreated females were sacrificed at midgestation and examined for the number of implantation sites, resorptions, live implantations, and corpora lutea. Males were subsequently sacrificed and a gross necropsy was performed. In the female fertility study, each treated female remained on treatment and was given the opportunity to mate with 1 or 2 untreated males by housing 1 female with 1 male for 7 consecutive nights or until evidence of mating was observed. All exposures were discontinued after mating. Females were sacrificed at midgestation and examined for the number of implantation sites, resorptions, live implantations, and corpora lutea. Statistical Analysis Continuous data (body weight, hematology, blood chemistry, and litter data) were analyzed using an analysis of variance and Dunnett's test (Dunnett, 1955). Discontinuous data (fertility) were evaluated using the Mann-Whitney U-test (Siegel, 1956) or Fisher's exact test (Fisher, 1950). The level of significance was selected as p < .05. RESULTS Chamber Concentrations During the 4-wk study, the mean analytical concentrations of adiponitrile ranged from 64 to 493 mg/m3 (Table 1). The nominal to analytical ratios were 2.0,1.2, and 1.2 for the low, mid, and high exposure groups, respectively. The agreement between the analytical and gravametric concentrations suggest that the low and mid exposures were mostly vapor whereas the high exposure level was mostly aerosol. Greater than 90% of the adiponitrile aerosol particles were less than 10 /*m at all three concentrations, and the mass medium aerodynamic diameter (MMAD) of these particles was 3-4 jtim. During the 13-wk study, the mean analytical concentrations ranged from 12.9 to 99 mg/m3 (Table 1). Because rats for the female fertility studies were exposed for only a portion of this period, their mean actual exposures were slightly different from the above and ranged from 13 to 104 mg/m3. The nominal to analytical ratios were 2.3, 2.1, and 1.5 for the low, mid, and high exposure groups, respectively. A comparison between the analytical and gravametric concentrations suggests that a significant fraction of the material at the low and mid dose groups was
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TABLE 1. Adiponitrile Concentrations in Chambers Used to Treat Rats for 4- and 13-week periods
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Adiponitrile concentration (mg/m3) Low
Mid
High
4-wk Treatment Analytical Nominal
64 ± 7a 130 ± 10
114 ± 10 140 ± 20
493 ± 48 580 ± 170
13-wk Treatment Analytical Nominal
12.9 ± 2.2 29.9 + 6.0
30.6 ± 3.2 64.8 ± 8.4
99.0 ± 13.0 146.0 ± 18.0
a Mean ± SD for 20 or 66 daily values for chamber connections of adiponitrile expressed in terms of mg/m3.
present as vapors. The MMAD of aerosol particles less than 10 /im at the high concentration was between 3.5 and 4.2 4-Week Study Mortality and reduced weight gain were observed only after exposure to 493 mg/m3 of adiponitrile Gable 2). All of the males and half of the females died during the first week at this level. In addition, the body weights of surviving females were reduced to about 90% of control values after the first week of exposure, and this reduction persisted throughout the study. Females surviving at this level exhibited changes in various red blood cells parameters (Table 3). These changes included a reduction in the red blood cell count, hematocrit, hemoglobin concentration, and mean corpuscular hemoglobin concentration, and an inTABLE 2. Survival and Body Weights of Rats Exposed for 4 Weeks to Adiponitrile Adiponitrile (mg/rr!3) 0 Male Mortality (%)a Body weight (g) WkO Wk1 Wk2 Wk3 Wk4
0 169 b 237 293 341 372
64 Female
Male
114
Female
Female
Male
Female
0
0
0
0
0
100
50
152 179 200 219 235
169 236 291 337 369
151 179 200 219 233
169 231 288 338 371
151 178 199 217 232
169 NSC NS NS NS
152 156°* 186 d 198 d 213 d
Percentage of rats (18/sex/group) dying during exposure. b Mean. c
Male
493
No survivors.
"'Significantly different from control.
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TABLE 3 . Hematology Parameters for Rats Exposed to Adiponitrile for 4 or 13 Weeks Adiponitrile Concentration Low
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Control
White blood cells/mm 3 (x10 3 ) Red blood cells/mm 3 (X106) Hematocrit (%) Hemoglobin (g/100 ml) Mean corpuscular Volume Otm3) Hemoglobin (pg) Hemoglobin cone. (%) White blood cells/mm 3 (x10 3 ) Red blood cells/mm 3 (X106) Hematocrit (%) Hemaglobin (g/100 ml) Mean corpuscular Volume (urn3) Hemoglobin (pg) Hemoglobin cone. (%)
Male
Female
13.4 6 8.8 46.0 19.1
High
Male
Female
Male
Female
4-Week treatment 3 7.5 7.6 13.5 8.4 8.6 8.7 47.7 47.6 48.8 18.1 18.4 18.1
12.2 8.5 48.0 17.8 d
7.8 8.5 47.6 18.1
NSC NS NS NS
8.1 7.8 d 45.1'' 16.5 d
52.6 21.6 42.1
55.9 21.0 37.8
56.9 20.9 d 37.2
56.6 21.3 38.0
NS NS NS
58.6 d 21.2 36.5 d
12.8 7.8 41.6 16.5
13-Week 10.6 7.5 42.1 16.8
treatment6 11.2 9.1 7.3 7.8 40.9 41.0 16.4 16.5
9.9 7.8 40.5 16.2
8.9 7.4 41.3 16.6
11.3 7.7 40.3 16.3
9.6 7.0 d 39.6 d
53.0 21.1 39.5
56.0 22.5 39.9
56.0 22.5 40.0
52.0 20.8 39.8
56.0 22.4 40.1
53.0 21.3 40.3 d
57.0 22.5 39.6
Male
56.8 21.1 37.8
53.0 21.3 40.0
Female
Mid
57.0 21.4 37.9
a
Rats exposed to 0, 64,114, and 493 mg/m3 of adiponitrile. Mean of at least 10 rats or all survivors. c No survivors. ''Significantly different from control. e Rats exposed to 0, 12.9, 30.6, or 99 mg/m3 of adiponitrile. b
crease in mean corpuscular volume. Males exposed to 114 mg/mg3 of adiponitrile had a statistically significant reduced hemoglobin concentration and mean corpuscular hemoglobin. Histopathological evaluation of tissues from surviving mid and high dose rats revealed an excessive presence of a hemosiderin-like pigment and hemopoiesis in the spleen. These effects ranged in severity from slight to moderate at the high concentration and slight at the midconcentration. No other microscopic changes were considered treatment-related in these rats or rats from the low dose. Although elevated alkaline phosphatase and serum glutamic pyruvic transaminase levels were observed in females from the high exposure level, these values remained within the normal range from the laboratory, and there were no microscopic lesions in the liver to suggest a treatment-related effect. 13-Week and Fertility Studies Target concentrations of 13, 30, and 100 mg/m3 were selected for these studies based on the significant mortality present at 493 mg/m3 in
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the previous study. The actual concentrations achieved are indicated in Table 1. No rats died during a 13-wk exposure to adiponitrile at concentrations up to 99 mg/m3, and the weight gain of treated animals were comparable to control values (Table 4). A statistically significantly reduced red blood cell count, hematocrit, and hemoglobin concentration were present in females exposed to 99 mg/m3 of adiponitrile (Table 3). Males had an increased mean corpuscular hemoglobin concentration at this level. Hematological values for rats exposed up to 30.6 mg/m3 of adiponitrile were comparable to controls. No treatment-related histopathological changes were observed in the 13-wk study. The slight to moderate splenic changes reported above in the 4-wk study were not detected in rats exposed to up to 99 mg/m3 of adiponitrile for 13 wk. No adverse effects on fertility were observed in males exposed to up to 99 mg/m3 of adiponitrile and mated with unexposed females or females exposed to up to 104 mg/m3 and mated with untreated males (Table 5). Although two males from the low dose group were judged to be nonfertile and there was an increased preimplantation loss in this group, these are probably not treatment-related effects for the following reasons. First, since all males exposed to higher concentrations of adiponitrile were fertile, and there was no increase in preimplantation loss of these levels, there was no evidence of a dose-response relationship. Second, there was no histopathological evidence that adiponitrile was toxic to male reproductive organs. DISCUSSION Rats were exposed for 4 or 13 wk to concentrations of adiponitrile that ranged from approximately 10 to 500 mg/m3. Although atmospheres contained both aerosols and vapors, a particle size analysis indicated that the MMAD of aerosols was less than 4 /im. Thus, while the physical form of the test material varied with the concentration, all atmospheres contained adiponitrile that was primarily in a respirable form. Various effects were observed in rats during the present study. Mortality and reduced weight gain were evident only after exposure to 493 mg/m3, and males appeared to be more sensitive than females to the lethal effects of adiponitrile. Hematological changes in various red blood cell parameters, indicative of anemia, were present. They occurred following a 4-wk exposure of males to 114 mg/m3 and females to 493 mg/m3 and a 13-wk exposure to 99 mg/m3 in females but not males. Similar changes have also been reported following a 2-wk inhalation exposure (Smith and Kennedy, 1982). No treatment-related histopathological changes were observed in rats exposed to 99 mg/m3 of adiponitrile for 13 wk in the present study or 268 mg/m3 for 2 wk (Smith and Kennedy, 1982).
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TABLE 4. Survival and Body Weights of Rats Exposed for 13 Weeks to Adiponitrile Adiponitrile (mg/m3)
Male
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Mortality (%)a Body weight (g) WkO Wk3 Wk6 Wk9
Wk13
Female
342 424 465 511
Male
Female
Male
99
Female
Male
Female
0
0
0
0
0
0
0
159 228 263 282 301
200 337 418 466 509
159 223 258 277 300
200 337 419 462 511
159 222 254 273 294
200 334 416 459 512
158 222 255 279 302
0
200b
30.6
12.9
0
Percentage of rats (15/sex/group) dying during exposure. 6 Mean.
Thus, with the exception of some slight hematological changes, adiponitrile does not appear to have a specific target organ toxicity in rats following inhalation exposure. Furthermore, atmospheres containing approximately 100 mg/m of adiponitrile did not adversely affect the fertility of male or female rats. In addition, no teratogenic or embryotoxic effects TABLE 5. Fertility of Male and Female Rats Exposed to Adiponitrile and Mated with Unexposed Rats Adiponitrile concentration Control
Low
Mid
High
Males exposeda Fertile males6 Fertility (unexposed females)c Live implants/dam Preimplantation loss (%) Postimplantation loss (%) Fertility (exposed females)c Live implants/dam Preimplantation loss (%) Postimplantation loss (%)
12
10
27/32d 13.5 + 0.4 6 +3d 2± 1 8 + 1 6 ± 1 Females exposed' 23/24 23/24 13.0 ± 0.7 12.7 + 0.9 9+ 4 15 + 6 6 ± 2 3 + 1 34/34 13.8 ± 0.3e
12
12
31/34 13.5 ± 0.4 4+ 2 7 + 1
30/31 13.1 ± 0.3 3 + 1 8 +1
20/24 13.4 + 0.7 6 ± 3 9 + 2
19/23 13.5 ± 0.4
4 ±2 6+2
a Males (12/group) exposed to 0, 12.9, 30.6, and 99 mg/m3 of adiponitrile for at least 10 wk and mated with unexposed females. ''Number of males producing at least one pregnant female. c Number of pregnant female/number mated females. ^Significantly different from control. e Mean ± SE. 'Females (24/group) exposed to 0,13.0, 31.8, and 104 mg/m3 of adiponitrile for at least 3 wk and mated with unexposed males.
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were observed in the offspring of rats treated orally with up to 80 mg/kg/d of adiponitrile on d 6-15 of gestation (Johannsen et al., 1986). NIOSH recommended that employee exposures not exceed 18 mg/m3 as a time-weighted average for a 10-h work shift based on a comparison of acute toxicity data (NIOSH, 1978). The Monsanto Company has adopted an exposure limit of 9 mg/m3 as an 8-h time-weighted average. This level was selected to provide a 10-fold safety factor below the level of the most subtle effect seen in the present 13-wk study, which was a slight anemia present only in female rats exposed to 99 mg/m3. The results of animal studies, nevertheless, indicate that adiponitrile does not possess a potent target organ toxicity, it does not affect fertility, and it does not alter fetal development. REFERENCES Drew, R. T., Bernstein, D. M., and Laskin, S. 1978. The Laskin generator. J. Toxicol. Environ. Health 4:661-670. Dunnett, C. W. 1955. Multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50:1096-1121. Fisher, R. A. 1950. Statistical Methods for Research Workers. New York: Hafner. Johannsen, F. R., and Levinskas, G. J. 1986. Relationships between toxicity and structure of aliphatic nitriles. Fundam. Appl. Toxicol. 7:690-697. Johannsen, F. R., Levinskas, C. L., Berteau, P. E., and Rodwell, D. E. 1986. Evaluation of the teratogenic potential of three aliphatic nitriles in the rat. Fundam. Appl. Toxicol. 7:33-40. National Institute for Occupational Safety and Health. 1978. Criteria for a Recommended Standard—Occupational Exposure to Nitriles. NIOSH Publication No. 78-212. Siegel, S. 1956. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill. Smith, L. W., and Kennedy, G. L. 1982. Inhalation toxicity of adiponitrile in rats. Toxicol. Appl. Pharmacol 65:257-263. Svirbely, J. L., and Floyd, E. P. 1964. Toxicological Studies of Acrylonitrile, Adiponitrile, and (ββoxydipropionitrile-III. Chronic Studies. U.S. Department of Health, Education, and Welfare, Public Health Service. A. Taft Engineering Center, J-4614. Received December 6, 1989 Accepted February 28, 1990
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
13‐week inhalation toxicity study and fertility assessment in rats exposed to atmospheres containing adiponitrile R. D. Short , W. V. Roloff , L. D. Kier & W. E. Ribelin To cite this article: R. D. Short , W. V. Roloff , L. D. Kier & W. E. Ribelin (1990) 13‐week inhalation toxicity study and fertility assessment in rats exposed to atmospheres containing adiponitrile, Journal of Toxicology and Environmental Health, 30:3, 199-207, DOI: 10.1080/15287399009531423 To link to this article: http://dx.doi.org/10.1080/15287399009531423
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 10 November 2015, At: 16:44
13-WEEK INHALATION TOXICITY STUDY AND FERTILITY ASSESSMENT IN RATS EXPOSED TO ATMOSPHERES CONTAINING ADIPONITRILE R. D. Short
Downloaded by [University of Florida] at 16:44 10 November 2015
Department of Medicine and Health Sciences, Monsanto Company, St. Louis, Missouri W. V. Roloff, L. D. Kier, W. E. Ribelin Environmental Health Laboratory, Monsanto Company, St. Louis, Missouri
Adiponitrile is an aliphatic dinitrile that is used as an intermediate in the synthesis of hexamethylenediamine. In order to asses potential health effects associated with industrial exposure, rats were exposed 6 h/d, 5 d/wk for 4 or 13 wk to atmospheres containing a range of adiponitrile concentrations and observed for signs of toxicity. A fertility assessment was also included as a component of the 13-wk study. Mortality and reduced weight gain were observed within 1 wk only in rats exposed to 493 mg/m3. Evidence of slight anemia was present in rats exposed to 99 mg/m3 and above. There was no histopathological evidence of organ toxicity in about 30 tissues from both sexes exposed up to 99 mg/m3, the highest concentration tested, for 13 wk. In addition, fertility, as monitored by reproductive performance and litter parameters, was normal in both males and females similarly exposed.
INTRODUCTION Adiponitrile (hexanedinitrile, CAS number 111-69-3), an aliphatic dinitrile, is a chemical intermediate in the synthesis of hexamethylenediamine, which is used in the production of nylon. The National Institute for Occupational Safety and Health (NIOSH) reviewed the available information on select nitriles and concluded that insufficient quantitative inhalation toxicity data were available for recommending workplace exposure limits for these materials (NIOSH, 1978). NIOSH adopted, as an alternative, a comparative toxicity approach using subcutaneous LD50 values in female rats and the available information for acetonitrile as a basis for recommending workplace exposure limits for the various nitriles they considered. Using this approach NIOSH recommended in 1978 that employee exposure to adiponitrile not exceed 18 mg/m3 (4 This manuscript was reviewed by the Monsanto Quality Assurance unit to assure consistency with the original reports and data developed during the conduct of these studies. Correspondence should be sent to Robert D. Short, Ph.D., Monsanto Company, Mail Zone A3ND, 800 N. Lindbergh Blvd, St. Louis, MO 63167.
199 Journal of Toxicology and Environmental Health, 30:199-207, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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ppm) as a time-weighted average limit for up to a 10-h workshift in a 40-h work week. Currently, neither the Occupational Safety and Health Administration nor the American Conference of Governmental Industrial Hygienists has adopted a workplace standard for adiponitrile. The literature contains the following information about adiponitrile. It is moderately toxic following a single exposure by the oral (rat), dermal (rabbit), and inhalation (rat) routes of exposure (Johannsen and Levinskas, 1986). After repeated exposure to atmospheres containing 268 mg/m3 6 h/d, 5 d/wk for 2 wk, rats showed a weight loss during the first week, and some clinical pathological changes at the end of the treatment period (Smith and Kennedy, 1982). These changes included decreased erythrocyte count, hemoglobin concentration, leukocyte count, relative number of eosinophils, and urine osmolality, and increased blood glucose, blood urea nitrogen, and urine glucose. All values were normal 2 wk later and there was no histopathologic evidence of target organ toxicity at the end of the treatment period. In a longer-time study, by a different route of exposure, rats were given drinking water containing 0.5, 5.0, and 50 ppm of adiponitrile for 2 yr (Svirbely and Floyd, 1964). No adverse effects on body weights, organ weight ratios (liver, kidney, and spleen), or hematological parameters were reported. Advanced adrenal degeneration, however, was reported in females at all exposure levels and in males exposed to the high concentration. The present studies were undertaken to provide additional information to assess possible health effects associated with adiponitrile in the industrial setting. Accordingly, rats were exposed to atmospheres containing adiponitrile for up to 13 wk and evaluated for treatment-related effects, and a fertility component was also included. A preliminary 4-wk pilot study was conducted to select concentrations for the 13-wk study and is also reported. METHODS Experimental Design CD rats (Charles River Breeding Laboratories, Portage, Mich.) were used in this study, which was conducted in two phases. In the first phase rats (18/sex/ group) were exposed to adiponitrile at target concentrations of 0, 50,100, and 500 mg/m for 4 wk and the results were used to select exposure levels for the next phase. In the second phase rats (15/sex/ group) were exposed to adiponitrile at target concentrations of 0,10, 30, and 100 mg/m3 for 13 wk. Additional rats (12 males and 24 females/group) were included to provide an assessment of male and female fertility by evaluating the outcome of pregnancies that resulted from the mating of treated with untreated rats. In this portion of the study males were exposed for at least 10 wk while females were exposed for at least 3 wk
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prior to mating. Exposures were discontinued for females after mating and for males the day before sacrifice. All exposures, with the exception of the female fertility study, where 6 h/d, 5 d/wk. Females in the fertility study were exposed 6 h/d, 7 d/wk.
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Exposure Test atmospheres were generated using adiponitrile with a purity of greater than 99%. All rats were exposed in 10-m3 Rochester-style inhalation chambers to air or air containing various concentrations of adiponitrile. Airflow was maintained between 9.8 and 12 chamber volumes per hour, temperature at 24 ± 2°C, and relative humidity at 50 ± 2%. Test atmospheres were generated using a Laskin-style nebulizer (Drew et al., 1978). The concentration of adiponitrile in the chambers was controlled by regulating its flow rate into the nebulizer. A vertical aerosol particle size separator prevented most of the large, nonrespirable aerosol particles from entering the chamber, and the large particles were collected in the bottom of the separator. Nominal concentrations of adiponitrile represent the net amount of material entering the chamber per unit time divided by the total air flow through the chamber per unit time. The analytical concentrations were determined by drawing atmosphere samples through an impinger containing 15 ml of methanol and measuring the concentration of adiponitrile in solution using a gas chromatographic method with a nitrogen phosphorus detector. Particle size of the aerosol was measured using a nonviable, nine-stage Andersen impactor (Anderson Samples Inc., Atlanta, Ga.).
Observations Rats were observed twice daily during each exposure for signs of toxicity and weighed at weekly intervals during the treatment period. In the 4-wk and 13-wk studies, blood was obtained from the periorbital sinus of the eye (4-wk study) or posterior vena cava (13-wk study) prior to sacrifice and analyzed for hematologic and blood chemistry parameters. A gross necropsy was performed on all survivors, organs were weighed (adrenals, testes with epididymides, heart, kidneys, liver, pituitary, and spleen), and the following tissues were preserved and processed for subsequent histopathological evaluation: abdominal aorta, adrenals, bone and bone marrow (femur), brain, esophagus, eyes with optic nerve, gonads (ovaries, and testes with epididymides), heart, intestine (colon and ileum), kidneys, liver, lung, lymph node (mesenteric), mammary gland, nasal turbinates, pancreas, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin, spinal cord (thoraco-lumbar), spleen, stomach, thymus, trachea, thyroid/parathyroid, urinary bladder, uterus (corpus and cervix), and gross lesions. In the male fertility study, each treated male remained on treatment and was given the opportunity to mate with 3 untreated females by hous-
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ing 1 male and 1 female for 5 consecutive nights or until evidence of mating (sperm positive vaginal smear or copulatory plug) was observed. Treatments were discontinued the day before sacrifice. Untreated females were sacrificed at midgestation and examined for the number of implantation sites, resorptions, live implantations, and corpora lutea. Males were subsequently sacrificed and a gross necropsy was performed. In the female fertility study, each treated female remained on treatment and was given the opportunity to mate with 1 or 2 untreated males by housing 1 female with 1 male for 7 consecutive nights or until evidence of mating was observed. All exposures were discontinued after mating. Females were sacrificed at midgestation and examined for the number of implantation sites, resorptions, live implantations, and corpora lutea. Statistical Analysis Continuous data (body weight, hematology, blood chemistry, and litter data) were analyzed using an analysis of variance and Dunnett's test (Dunnett, 1955). Discontinuous data (fertility) were evaluated using the Mann-Whitney U-test (Siegel, 1956) or Fisher's exact test (Fisher, 1950). The level of significance was selected as p < .05. RESULTS Chamber Concentrations During the 4-wk study, the mean analytical concentrations of adiponitrile ranged from 64 to 493 mg/m3 (Table 1). The nominal to analytical ratios were 2.0,1.2, and 1.2 for the low, mid, and high exposure groups, respectively. The agreement between the analytical and gravametric concentrations suggest that the low and mid exposures were mostly vapor whereas the high exposure level was mostly aerosol. Greater than 90% of the adiponitrile aerosol particles were less than 10 /*m at all three concentrations, and the mass medium aerodynamic diameter (MMAD) of these particles was 3-4 jtim. During the 13-wk study, the mean analytical concentrations ranged from 12.9 to 99 mg/m3 (Table 1). Because rats for the female fertility studies were exposed for only a portion of this period, their mean actual exposures were slightly different from the above and ranged from 13 to 104 mg/m3. The nominal to analytical ratios were 2.3, 2.1, and 1.5 for the low, mid, and high exposure groups, respectively. A comparison between the analytical and gravametric concentrations suggests that a significant fraction of the material at the low and mid dose groups was
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TABLE 1. Adiponitrile Concentrations in Chambers Used to Treat Rats for 4- and 13-week periods
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Adiponitrile concentration (mg/m3) Low
Mid
High
4-wk Treatment Analytical Nominal
64 ± 7a 130 ± 10
114 ± 10 140 ± 20
493 ± 48 580 ± 170
13-wk Treatment Analytical Nominal
12.9 ± 2.2 29.9 + 6.0
30.6 ± 3.2 64.8 ± 8.4
99.0 ± 13.0 146.0 ± 18.0
a Mean ± SD for 20 or 66 daily values for chamber connections of adiponitrile expressed in terms of mg/m3.
present as vapors. The MMAD of aerosol particles less than 10 /im at the high concentration was between 3.5 and 4.2 4-Week Study Mortality and reduced weight gain were observed only after exposure to 493 mg/m3 of adiponitrile Gable 2). All of the males and half of the females died during the first week at this level. In addition, the body weights of surviving females were reduced to about 90% of control values after the first week of exposure, and this reduction persisted throughout the study. Females surviving at this level exhibited changes in various red blood cells parameters (Table 3). These changes included a reduction in the red blood cell count, hematocrit, hemoglobin concentration, and mean corpuscular hemoglobin concentration, and an inTABLE 2. Survival and Body Weights of Rats Exposed for 4 Weeks to Adiponitrile Adiponitrile (mg/rr!3) 0 Male Mortality (%)a Body weight (g) WkO Wk1 Wk2 Wk3 Wk4
0 169 b 237 293 341 372
64 Female
Male
114
Female
Female
Male
Female
0
0
0
0
0
100
50
152 179 200 219 235
169 236 291 337 369
151 179 200 219 233
169 231 288 338 371
151 178 199 217 232
169 NSC NS NS NS
152 156°* 186 d 198 d 213 d
Percentage of rats (18/sex/group) dying during exposure. b Mean. c
Male
493
No survivors.
"'Significantly different from control.
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TABLE 3 . Hematology Parameters for Rats Exposed to Adiponitrile for 4 or 13 Weeks Adiponitrile Concentration Low
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Control
White blood cells/mm 3 (x10 3 ) Red blood cells/mm 3 (X106) Hematocrit (%) Hemoglobin (g/100 ml) Mean corpuscular Volume Otm3) Hemoglobin (pg) Hemoglobin cone. (%) White blood cells/mm 3 (x10 3 ) Red blood cells/mm 3 (X106) Hematocrit (%) Hemaglobin (g/100 ml) Mean corpuscular Volume (urn3) Hemoglobin (pg) Hemoglobin cone. (%)
Male
Female
13.4 6 8.8 46.0 19.1
High
Male
Female
Male
Female
4-Week treatment 3 7.5 7.6 13.5 8.4 8.6 8.7 47.7 47.6 48.8 18.1 18.4 18.1
12.2 8.5 48.0 17.8 d
7.8 8.5 47.6 18.1
NSC NS NS NS
8.1 7.8 d 45.1'' 16.5 d
52.6 21.6 42.1
55.9 21.0 37.8
56.9 20.9 d 37.2
56.6 21.3 38.0
NS NS NS
58.6 d 21.2 36.5 d
12.8 7.8 41.6 16.5
13-Week 10.6 7.5 42.1 16.8
treatment6 11.2 9.1 7.3 7.8 40.9 41.0 16.4 16.5
9.9 7.8 40.5 16.2
8.9 7.4 41.3 16.6
11.3 7.7 40.3 16.3
9.6 7.0 d 39.6 d
53.0 21.1 39.5
56.0 22.5 39.9
56.0 22.5 40.0
52.0 20.8 39.8
56.0 22.4 40.1
53.0 21.3 40.3 d
57.0 22.5 39.6
Male
56.8 21.1 37.8
53.0 21.3 40.0
Female
Mid
57.0 21.4 37.9
a
Rats exposed to 0, 64,114, and 493 mg/m3 of adiponitrile. Mean of at least 10 rats or all survivors. c No survivors. ''Significantly different from control. e Rats exposed to 0, 12.9, 30.6, or 99 mg/m3 of adiponitrile. b
crease in mean corpuscular volume. Males exposed to 114 mg/mg3 of adiponitrile had a statistically significant reduced hemoglobin concentration and mean corpuscular hemoglobin. Histopathological evaluation of tissues from surviving mid and high dose rats revealed an excessive presence of a hemosiderin-like pigment and hemopoiesis in the spleen. These effects ranged in severity from slight to moderate at the high concentration and slight at the midconcentration. No other microscopic changes were considered treatment-related in these rats or rats from the low dose. Although elevated alkaline phosphatase and serum glutamic pyruvic transaminase levels were observed in females from the high exposure level, these values remained within the normal range from the laboratory, and there were no microscopic lesions in the liver to suggest a treatment-related effect. 13-Week and Fertility Studies Target concentrations of 13, 30, and 100 mg/m3 were selected for these studies based on the significant mortality present at 493 mg/m3 in
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the previous study. The actual concentrations achieved are indicated in Table 1. No rats died during a 13-wk exposure to adiponitrile at concentrations up to 99 mg/m3, and the weight gain of treated animals were comparable to control values (Table 4). A statistically significantly reduced red blood cell count, hematocrit, and hemoglobin concentration were present in females exposed to 99 mg/m3 of adiponitrile (Table 3). Males had an increased mean corpuscular hemoglobin concentration at this level. Hematological values for rats exposed up to 30.6 mg/m3 of adiponitrile were comparable to controls. No treatment-related histopathological changes were observed in the 13-wk study. The slight to moderate splenic changes reported above in the 4-wk study were not detected in rats exposed to up to 99 mg/m3 of adiponitrile for 13 wk. No adverse effects on fertility were observed in males exposed to up to 99 mg/m3 of adiponitrile and mated with unexposed females or females exposed to up to 104 mg/m3 and mated with untreated males (Table 5). Although two males from the low dose group were judged to be nonfertile and there was an increased preimplantation loss in this group, these are probably not treatment-related effects for the following reasons. First, since all males exposed to higher concentrations of adiponitrile were fertile, and there was no increase in preimplantation loss of these levels, there was no evidence of a dose-response relationship. Second, there was no histopathological evidence that adiponitrile was toxic to male reproductive organs. DISCUSSION Rats were exposed for 4 or 13 wk to concentrations of adiponitrile that ranged from approximately 10 to 500 mg/m3. Although atmospheres contained both aerosols and vapors, a particle size analysis indicated that the MMAD of aerosols was less than 4 /im. Thus, while the physical form of the test material varied with the concentration, all atmospheres contained adiponitrile that was primarily in a respirable form. Various effects were observed in rats during the present study. Mortality and reduced weight gain were evident only after exposure to 493 mg/m3, and males appeared to be more sensitive than females to the lethal effects of adiponitrile. Hematological changes in various red blood cell parameters, indicative of anemia, were present. They occurred following a 4-wk exposure of males to 114 mg/m3 and females to 493 mg/m3 and a 13-wk exposure to 99 mg/m3 in females but not males. Similar changes have also been reported following a 2-wk inhalation exposure (Smith and Kennedy, 1982). No treatment-related histopathological changes were observed in rats exposed to 99 mg/m3 of adiponitrile for 13 wk in the present study or 268 mg/m3 for 2 wk (Smith and Kennedy, 1982).
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TABLE 4. Survival and Body Weights of Rats Exposed for 13 Weeks to Adiponitrile Adiponitrile (mg/m3)
Male
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Mortality (%)a Body weight (g) WkO Wk3 Wk6 Wk9
Wk13
Female
342 424 465 511
Male
Female
Male
99
Female
Male
Female
0
0
0
0
0
0
0
159 228 263 282 301
200 337 418 466 509
159 223 258 277 300
200 337 419 462 511
159 222 254 273 294
200 334 416 459 512
158 222 255 279 302
0
200b
30.6
12.9
0
Percentage of rats (15/sex/group) dying during exposure. 6 Mean.
Thus, with the exception of some slight hematological changes, adiponitrile does not appear to have a specific target organ toxicity in rats following inhalation exposure. Furthermore, atmospheres containing approximately 100 mg/m of adiponitrile did not adversely affect the fertility of male or female rats. In addition, no teratogenic or embryotoxic effects TABLE 5. Fertility of Male and Female Rats Exposed to Adiponitrile and Mated with Unexposed Rats Adiponitrile concentration Control
Low
Mid
High
Males exposeda Fertile males6 Fertility (unexposed females)c Live implants/dam Preimplantation loss (%) Postimplantation loss (%) Fertility (exposed females)c Live implants/dam Preimplantation loss (%) Postimplantation loss (%)
12
10
27/32d 13.5 + 0.4 6 +3d 2± 1 8 + 1 6 ± 1 Females exposed' 23/24 23/24 13.0 ± 0.7 12.7 + 0.9 9+ 4 15 + 6 6 ± 2 3 + 1 34/34 13.8 ± 0.3e
12
12
31/34 13.5 ± 0.4 4+ 2 7 + 1
30/31 13.1 ± 0.3 3 + 1 8 +1
20/24 13.4 + 0.7 6 ± 3 9 + 2
19/23 13.5 ± 0.4
4 ±2 6+2
a Males (12/group) exposed to 0, 12.9, 30.6, and 99 mg/m3 of adiponitrile for at least 10 wk and mated with unexposed females. ''Number of males producing at least one pregnant female. c Number of pregnant female/number mated females. ^Significantly different from control. e Mean ± SE. 'Females (24/group) exposed to 0,13.0, 31.8, and 104 mg/m3 of adiponitrile for at least 3 wk and mated with unexposed males.
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were observed in the offspring of rats treated orally with up to 80 mg/kg/d of adiponitrile on d 6-15 of gestation (Johannsen et al., 1986). NIOSH recommended that employee exposures not exceed 18 mg/m3 as a time-weighted average for a 10-h work shift based on a comparison of acute toxicity data (NIOSH, 1978). The Monsanto Company has adopted an exposure limit of 9 mg/m3 as an 8-h time-weighted average. This level was selected to provide a 10-fold safety factor below the level of the most subtle effect seen in the present 13-wk study, which was a slight anemia present only in female rats exposed to 99 mg/m3. The results of animal studies, nevertheless, indicate that adiponitrile does not possess a potent target organ toxicity, it does not affect fertility, and it does not alter fetal development. REFERENCES Drew, R. T., Bernstein, D. M., and Laskin, S. 1978. The Laskin generator. J. Toxicol. Environ. Health 4:661-670. Dunnett, C. W. 1955. Multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50:1096-1121. Fisher, R. A. 1950. Statistical Methods for Research Workers. New York: Hafner. Johannsen, F. R., and Levinskas, G. J. 1986. Relationships between toxicity and structure of aliphatic nitriles. Fundam. Appl. Toxicol. 7:690-697. Johannsen, F. R., Levinskas, C. L., Berteau, P. E., and Rodwell, D. E. 1986. Evaluation of the teratogenic potential of three aliphatic nitriles in the rat. Fundam. Appl. Toxicol. 7:33-40. National Institute for Occupational Safety and Health. 1978. Criteria for a Recommended Standard—Occupational Exposure to Nitriles. NIOSH Publication No. 78-212. Siegel, S. 1956. Nonparametric Statistics for the Behavioral Sciences. New York: McGraw-Hill. Smith, L. W., and Kennedy, G. L. 1982. Inhalation toxicity of adiponitrile in rats. Toxicol. Appl. Pharmacol 65:257-263. Svirbely, J. L., and Floyd, E. P. 1964. Toxicological Studies of Acrylonitrile, Adiponitrile, and (ββoxydipropionitrile-III. Chronic Studies. U.S. Department of Health, Education, and Welfare, Public Health Service. A. Taft Engineering Center, J-4614. Received December 6, 1989 Accepted February 28, 1990
