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Dimethylethanolamine: Acute, 2-week, and 13-week inhalation toxicity studies in rats
FUNDAMENTAL
AND
9,5 12-52 1 (1987)
APPLIEDTOXICOLOGY
Dimethylethanolamine: Acute, 2-Week, and 13-Week Inhalation Toxicity Studies in Rats DENNIS
R. KLONNE,’
DAROL E. DODD, IRVIN M. PRITTS, DONALD J. NACHREINER, EDWARD H. FOWLER, CATHERINE M. TROUP, ELTON R. HOMAN,
ANDBRYANBALLANTYNE Bushy
Run Research
Dimethylethanolamine: KLONNE, D. R., DODD, M., HOMAN, E. R., AND
Center,
Union
Carbide
Corporation,
Export,
Pennsylvania
15632
Acute, 2-Week, and 13-Week Inhalation Toxicity Studies in Rats. D. E., PRITTS, BALLANTYNE,
I. M., NACHREINER, B. (1987). Fundam.
D. J., FOWLER,
E. H., TROUP,
C.
9,5 12-52 1. Dimethylethanolamine (DMEA) is a volatile, water-soluble amine that has applications in the chemical and pharmaceutical industries. These studies evaluated the acute and subchronic inhalation toxicity of DMEA. Acute (4-hr) exposures of Wistar rats to DMEA vapor resulted in an LC50 value (95% confidence limits) of 1641 (862-3 125) ppm. Clinical signs of nasal and ocular initation, respiratory distress, and body weight loss were observed in rats exposed to 1668 ppm DMEA and higher. In the 2-week study, F-344 rats exposed to 98,288, or 586 ppm DMEA for 9 days (6 hr/day) during an 11-day period also exhibited signs of respiratory and ocular irritation (except the 98 ppm group). All animals of the 586 ppm group and 4 of 15 male rats of the 288 ppm group died. Body weight values for the 288 ppm group were reduced to about 75% of preexposure values, while the 98 ppm group gained 35% less weight than controls. Statistically significant differences in clinical pathology parameters (288 ppm group) and in organ weight values (288 and 98 ppm groups) probably resulted from the decreased food consumption and not from specific target organ toxicity. In the groups evaluated histologically (the 98 and 288 ppm groups) the eye and nasal mucosa were the primary target organs. In the 13-week subchronic study, F-344 rats were exposed to 0,8,24, or 76 ppm DMEA for 6 hr/day, 5 days/week for 13 weeks. The principal exposure-related changes were transient comeal opacity in the 24 and 76 ppm groups; decreased body weight gain for the 76 ppm group; and histopathologic lesions of the respiratory and olfactory epithelium of the anterior nasal cavity of the 76 ppm group and of the eye of several 76 ppm group females. Rats maintained for a 5-week recovery period only exhibited histological lesions of the nasal tissue, with the lesions being decreased in incidence and severity. DMEA acts primarily as an ocular and upper respiratory tract irritant and toxicant at vapor concentrations of 76 ppm, while 24 ppm or less produced no biologically significant toxicity in rats. Thus, 24 ppm was considered to be the no-observable-effect level. 0 I987 Society of Toxicology.
The aliphatic amines are a class of volatile, water-soluble chemicals with a wide variety of industrial applications. They are generally well absorbed from the gastrointestinal and respiratory tracts and may even produce lethality by a dermal route of exposure (Beard and Noe, 1981). The most apparent effect of ’ To whom correspondence should be addressed at Bushy Run Research Center, R.D. 4, Mellon Road, Export, PA 15632. 0272-0590187 $3.00 Copyright 0 I987 by the Society of Toxicology. All rights of reproduction in any form reserved.
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Appl. Toxicol.
inhalation of amines is the irritation of the mucous membranes of the eyes and respiratory tract. In animals, single exposures to high vapor concentrations can produce inflammation throughout the respiratory tract and pulmonary edema. In man, the vapors also produce irritation of the eyes, mouth, throat, and skin. Systemic effects following inhalation include headache, nausea, fainting, and anxiety (Beard and Noe, 198 1). Dimethylethanolamine (DMEA; 2-(di-
DMEA INHALATION
methylamino)ethanol) has applications as a desulfurizing agent in the treatment of natural gas; as an intermediate in the synthesis of corrosion inhibitors, pharmaceuticals, and dyestuffs; and as a curing agent for epoxy, amine, and polyamide resins. In previous studies, the acute peroral LD50 (95% confidence limits) value in rats for DMEA, administered as a 20% aqueous solution, was found to be 2.34 (2.26-2.42) g/kg (Smyth et al., 195 1). The 24-hr occluded percutaneous LD50 value for rabbits was 1.37 (1.21-1.55)ml/kg(Smythetal., 1951).Thus, DMEA is moderately toxic by acute oral and dermal routes of administration. A 90-day dietary feeding study in rats of DMEA at dosages up to 0.89 g/kg/day produced increases in the kidney to body weight value at the highest dosage (BRRC, 1949; Smyth et al., 195 1). No microscopic renal lesions accompanied this change. No effects were found in the groups fed 0.18 or 0.045 g/kg/day. Consistent with the properties of other aliphatic amines, DMEA was previously found to be severely irritating in a 4-hr occluded primary dermal irritation test in rabbits (BRRC, 1976), with 6 of 6 animals exhibiting dermal necrosis, and severely irritating in the rabbit ocular irritation test (Smyth et al., 195 1). DMEA is a normal constituent of mammalian metabolism, although it is present only in minute quantities (Hochschild, 1973). It is a precursor of choline synthesis and can cross the blood-brain barrier. Since choline is involved in the biosynthesis of acetylcholine and cellular membranes, DMEA may be indirectly involved with nerve impulse propagation and membrane biosynthesis. Thus, nervous system activity could be affected by exogenous administration of DMEA. The purpose of these studies was to obtain information on the acute and subchronic inhalation toxicity of DMEA that would be useful in determining potential human health risks from inhalation of DMEA vapor.
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METHODS Test material. DMEA (CAS 108-01-O) was obtained from Union Carbide Corp. (South Charleston, WV). Compositional analyses before and after each study indicated that the material was at least 99% pure. Animals and exposure regimen. Male and female Wistar albino rats (Hilltop Lab Animals, Inc., Scottdale, PA) with a body weight range of 200-300 g were used for the acute exposure. (Wistar rats were used for the acute studies because their faster growth rate (in comparison to F-344 rats) makes it easier to detect exposure-related effects on body weight.) Fischer-344 rats (Charles River Breeding Laboratories, Kingston, NY) were used for the 2-week and 13-week studies. Food (Purina Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO) and water were provided ad libitum except during exposures. All animals were identified by toe clipping and were maintained on a 12-hr light/dark cycle throughout the quarantine, exposure, and recovery periods. For the acute study, 5 rats/sex were exposed for 4 hr at each exposure concentration, then maintained for an additional 14-day observation period before their sacrifice. For the 2-week study, 10 rats/sex, approximately 8 weeks old, with mean body weights of 167 g for males and 12 1 g for females, were randomly assigned to all study groups to be used for clinical and anatomic pathology evaluations. Additionally, 5 male rats were assigned to the control, middle, and high concentration groups for possible ultrastructural (electron microscopic) evaiuation of nerve tissue (not performed since no behavioral abnormalities or light microscopic lesions of nerve tissue were observed). Rats were exposed for 6 hrjday, 5 days/ week, for 9 exposures during an 1 l-day period (2 days without exposure between Exposure Days 5 and 6). All animals were sacrificed on the morning after the ninth exposure. For the 13-week study, 20 rats/sex, approximately 9 weeks old, with mean body weights of 180 g for males and 130 g for females, were randomly assigned to each of four study groups. Additionally. 10 male rats were assigned to the control, middle, and high concentration groups for possible ultrastructural evaluation of nerve tissue (not performed since no behavioral abnormalities or light microscopic lesions of nerve tissue were observed). Rats were exposed for 6 hr/day, 5 days/week, for 13 weeks. One-half of all rats per sex per group were sacrificed after at least 2 days ofexposure during the 14th week of the study; the remaining rats were sacrificed after 5 complete weeks of recovery. Generation and analysis of exposure atmospheres. The acute study was conducted in a 900-liter stainless-steel and glass chamber (airflow of 200 liters/min) at mean analytical vapor concentrations of 1668,2408, or 33 11 ppm DMEA. For the 2-week study, rats were exposed in 1330-liter stainless-steel and glass chambers at an airflow of 300 liters/min. The target concentrations of DMEA
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KLONNE ET AL.
were 0, 100, 300, and 600 ppm. The 13-week study was conducted in 4320~liter stainless-steel and glass chambers at airflows of 800-1000 liters/min. The target concentrations of DMEA vapor were 0, 8, 25, and 75 ppm. Chamber temperature and relative humidity for all studies were generally maintained at 25°C and 46%, respectively. DMEA vapor was generated by metering the liquid into a heated, spiral-grooved evaporator with a countercurrent airstream, similar in design to the one used by Carpenter et al. (1975). Chamber concentrations of DMEA were analyzed at approximately 40-min intervals with a Perkin-Elmer 3920B gas chromatograph (CC) equipped with a flame ionization detector. The CC column was a 5 ft X i in stainless-steel column packed with 20% YIP-2100 on 80/ 100 mesh Supelcoport (Supelco, BeIlefonte, PA), maintained at 200°C. Injections of gas standards were used to calibrate the GC. Chamber atmosphere samples were automatically injected into the GC with the use of a Perkin-Elmer environmental sampler. Biological procedures. In the acute inhalation study, rats were observed for signs of toxicity during exposure and for 14 days postexposure. Body weights were determined prior to exposure and at 7 and 14 days after exposure. Necropsies were performed on all rats. For the 2and 13-week studies, biological assessments included daily animal observation, neurobehavioral evaluations (tremors, convulsions, tail elevation, impaired gait, paresis, salivation, lacrimation, diarrhea, piloerection, hypothermia, stereotyphy, surface and midair righting reflex, wire grasping, body tone, limb rotation, pupil size, skin color, respiration, locomotor activity, cornea1 response. tail and toe pinch response, and auditory startle response; Irwin, 1968), body weight and organ (brain, kidney, liver, lungs, testes, and adrenals) weight determinations, ophthalmology (evaluation of the eye with a light source and magnifying lens), clinical pathology, and gross and light microscopic examination of tissues. Organ weight determinations, gross pathologic examination, and blood and urine sample collections were made the morning following the last DMEA exposure day (except for recovery animals). Blood samples for hematologic and serum chemistry measurements were obtained by retroorbital bleeding of nonfasted rats anesthetized with methoxyflurane. Hematologic determinations (Coulter Counter S-Plus IV, Coulter Electronics, Inc., Hialeah, FL) included white blood cell (WBC) count, red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH concentration, and platelet count; WBC differentials were also performed. Serum chemistries (performed on a CentrifiChem, Baker Instruments, Pleasantville, NY, or an Astra, Beckman Instruments, Inc., Brea, CA) included creatinine, aspartate aminotransferase, alanine aminotransferase, total protein, albumin, globulin, total bilirubin, y-glutamyl transpeptidase, sorbitol dehydrogenase, sodium, potassium, and chloride. Individual urine samples were col-
lected during a 16-hr period prior to sacrifice from rats held in polycarbonate metabolism cages. Semiquantitative measurements on urine (Ames Multistix, read on the Clinitek, Ames Division, Miles Laboratories, Elkhart, IN) included pH, protein, bilirubin, urobilinogen, glucose, ketones, and blood. In addition, osmolality determinations (Cryomatic osmometer, Advanced Instruments, Inc., Needham Heights, MA) and microscopic evaluations of the urine sediment were performed. Food and water consumption were also measured during the 16-hr urine collection period. Selected tissues examined histologically included brain (five sections: frontal lobe and basal ganglia, parietal and temporal lobes with thalamus and hypothalamus, occipital lobes with midbrain, cerebellum and pons, and the medulla oblongata), sciatic nerve, kidneys, liver. larynx, lungs, nasal turbinates, spleen, trachea, gonads, eyes,and all gross lesions. Histopathologic examinations for the 2-week study were performed on tissues from rats of the control and middle exposure groups (due to complete mortality in the high exposure group), with nasal turbinates also being evaluated for the low exposure group rats. For the 13-week study, additional tissues for histopathologic examination included: sternal bone and bone marrow, epididymides, thymus, urinary bladder, thyroids and parathyroids, adrenals, heart, cervical lymph nodes. eyes, pituitary, gastrocnemius muscle, spinal cord, and uterus. These examinations were performed on rats from the control and high exposure groups, with nasal turbinates also being evaluated for the middle exposure group rats. Statisticalanalysis. Results of quantitative continuous variables were intercompared among the DMEA concentration levels and the control group by Bartlett’s homogeneity of variance (Sokal and Rohlf, 1969) analysis of variance (ANOVA), and Duncan’s multiple range test (Snedecor and Cochran, 1967). Duncan’s test was used when a significant F value from an ANOVA was observed. For heterogeneous group variances, the groups were compared by ANOVA for unequal variances (Brown and Forsythe, 1974) and either Student’s t test or Cochran’s t test (Snedecor and Cochran, 1967) was used. Corrected Bonferroni probabilities were used for t test comparisons (Miller, 1966). The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons. For the calculation of the LC50, the method of Finney (1964) was used.
RESULTS Acute Study The mean (*SD) chamber concentrations of DMEA were 1668 (+138), 2408 (+178), and 33 11 (+ 156) ppm. Mortality occurred at
DMEA
INHALATION
TOXICITY
IN RATS
515
all exposure concentrations with 5/10, 7/10, and 8/ 10 rats dying 1 to 12 days after exposure at 1668, 2408, and 33 11 ppm, respectively. No sex-related differences were observed in mortality, and the LC50 value (95% confidence limits) for the combined sexes was 1641 (862 to 3 125) ppm. Clinical signs, observed in all exposure groups, included lacrimation, excessive salivation, ocular, oral, and nasal discharge and encrustation, respiratory difficulties, decreased motor activity, coordination loss, and swelling and bleeding of extremities (feet and nose) from excessive preening (highest concentration only). Substantial body weight losses occurred for all but one survivor on postexposure Day 7, with only two males of the 1668 ppm group surpassing the preexposure body weight by Day 1 2 3 4 5 6 7 6 9 101112 study w 14. Discolored lungs, livers, kidneys, and FIG. 1. Mean body weight change for F-344 rats during spleens were seen at the necropsy of animals which died on study as well as in the two sur- the DMEA 2-week vapor inhalation study. vivors of the 33 11 ppm group. Survivors of the 2408 and 1668 ppm groups did not have exposure-related macroscopic lesions at the ppm group died 8- 12 days after the initiation end of the 14-day postexposure observation of exposures. There was a concentration-reperiod. lated effect on body weight in this study (Fig. 1). Weight loss occurred after one exposure in both sexes of the 288 and 586 ppm groups, 2-Week Study with the final mean body weight of the 288 ppm group being about 75% of preexposure The means (+-SD) of the daily mean cham- values. Although the 98 ppm group gained ber concentrations were 98 (+ 15) 288 (+ 14), weight, they gained 35% less than controls. and 586 (+36) ppm of DMEA vapor. The re- There was also a statistically significant conspective analytical to nominal concentration centration-related decrease in food consumpratios were 0.75, 0.86, and 0.87, indicating tion for both the 98 and 288 ppm groups. that DMEA was mildly reactive with the sur- Mean water consumption was equivalent for faces or contents of the chamber. all groups. The ophthalmic examination inSigns of respiratory distress (audible respi- dicated that animals of the 288 ppm group ration, mouth breathing), ocular and nasal ir- had conjunctivitis, including chemosis, and ritation (discharge and encrustation), and cornea1 opacity. comeal opacity were observed in both sexes Most of the serum chemistry, hematology, of the 288 and 586 ppm exposure groups. and urinalysis values for the 288 ppm group There were no exposure-related effects on the were statistically significantly different from neurobehavioral evaluations. All animals of control values, a further indication of the the 586 ppm group died or were sacrificed in stressed and debilitated condition of the ania moribund state 4-8 days after the initiation mals. No similar exposure-related effects of exposures. Four of fifteen males of the 288 were observed for the 98 ppm exposure
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ET AL.
TABLE 1 TERMINAL BODY WEIGHT AND SELECTED ORGAN WEIGHTS FOR F-344 RATS OFTHE DMEA ~-WEEK INHALATION S~JDY” Males Parameter
Bodywt (g) Brain wt (g) Brain/body wt (%) Liver wt (g) Liver/body wt (%) Kidney wt (g) Kidney/body wt (%) Lung w (9) Lung/body wt (%) Adrenal wt (g X IO’) Adrenal/body wt (%X 10’) Testes wt (g) Testes/body wt (%)
Females
Control
98 mm
288 ppm
Control
98 mm
288 ppm
198 (10.4) 1.70 (0.04) 0.86 (0.04) 7.91 ( 1.OO) 3.99 (0.35) 1.53 (0.14) 0.77 (0.04) 1.08 (0.19) 0.54 (0.09) 4.88 (1.97) 0.25 (0.09) 2.34 (0.26) 1.18 (0.11)
184** (12.2) 1.65* (0.05) 0.90 (0.04) 7.32 (0.69) 3.98 (0.19) 1.49 (0.08) 0.81* (0.02) 0.98 (0.12) 0.53 (0.05) 4.35 (1.07) 0.24 (0.06) 2.41 (0.18) 1.32** (0.10)
126** (10.9) 1.63** (0.03) 1.30** (0.12) 5.07** (0.50) 4.03 (0.30) 1.24** (0.06) 0.99** (0.08) 1.02 (0.19) 0.81** (0.11) 5.73 (0.89) 0.45** (0.05) 1.94** (0.17) 1.54** (0.06)
137 (3.8) 1.64 (0.05) 1.19 (0.04) 5.04 (0.23) 3.67 (0.18) 1.09 (0.05) 0.79 (0.05) 0.80 (0.10) 0.58 (0.07) 5.00 (1.21) 0.36 (0.09)
131** (3.1) 1.62 (0.05) 1.23 (0.04) 4.69** (0.34) 3.57 (0.21) 1.09 (0.05) 0.83 (0.04) 0.88 (0.14) 0.67 (0.1 I) 4.95 (1.34) 0.38 (0.11)
94** (3.6) 1.54** (0.04) 1.65** (0.08) 3.97*+ (0.34) 4.23** (0.29) 1.05 (0.09) 1.12** (0.09) 0.83 (0.18) 0.88** (0.18) 5.89 (0.53) 0.63** (0.W
a Values represent mean (&SD) for 7 to IO rats. * p < 0.05 from control. ** p < 0.0 I from control.
group, except for a statistically significant decrease in urine volume for both sexes. Similarly, many of the absolute and relative (as a percentage of body weight) organ weight values for the 288 ppm group were significantly different from those of control values (Table 1). Because of the substantial reduction in body weight gain, aswell as the lack of histological evidence for organ toxicity at the higher concentration (288 ppm), the few statistically significant differences in organ weight values for the 98 ppm group (Table
1) were not considered to result from specific organ toxicity. At necropsy, grosschangesincluded ocular opacity, thymic atrophy, and reduced body fat in most rats of the 288 ppm group. The principal histologic lesions were in the upper respiratory tract of the 98 and 288 ppm groups (Table 2) and in the eyes of the 288 ppm group. In the nasal tissuesof the 98 ppm group, rhinitis, squamous metaplasia, and ulceration of the nasal mucosa (Fig. 2A) were observed. Similar lesions occurred at a higher
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TABLE 2 INCIDENCE
OF SELECZTED NAVAL TISSUE HISTOPATHOLOGIC LESIONS IN F-344 RATS EXPOSED TO FOR 2 OR 13 WEEKS AND SACR~FKED THE DAY AVER THE FINAL EXPOSURE
I3-week studyb
2-week studya Males DMEA concentration (ppm): 0 Total No. examined Unremarkable Rhinitis Squamous metaplasia Epithelial erosion Mucosal ulceration Degeneration of olfactory mucosa/epithelium Degeneration of respiratory epithelium Atrophy of olfactory epithelium Microcysts in respiratory epithelium
DMEA VAPOR
Males
Females
Females
98
288
0
98
288
0
24
16
0
24
76
10 10 0 0 0 0
10 1 5 8 1 4
7 0 6 7 4 6
10 9’ 0 0 0 0
10 5 5 2 0 2
10 0 I 10 0 8
10 9d 0 0 0 0
10 8 2 0 0 0
10 0 0 9 0 0
10 9d 0 0 0 0
10 8 2 0 0 0
10 I I 4 0 0
0
0
1
0
0
9
0
0
0
0
0
0
0
0
0
0
0
4
0
0
8
0
0
7
0
0
0
0
0
0
0
0
10
0
0
3
0
0
0
0
0
0
0
0
10
0
0
3
a All animals of the 586 ppm group died on study and were not histologically evaluated. Incidence values in the table also do not include those four male rats of the 288 ppm group which died on study. ’ Rats of the 8 ppm group were not histologically evaluated as only minimal rhinitis was observed in 2 rats/sex of the 24 ppm group. ‘One control had a hemorrhage in the nasolacrimal duct, ‘One control had minimal mineralization of the olfactory epithelium.
incidence in the 288 ppm group (Table 2). atrophy in the 288 ppm exposure group was Additional nasal lesions in the 288 ppm verified by the microscopic observation of degroup included respiratory epithelial degen- creased numbers of cortical lymphocytes. eration (presence of necrotic cells scattered The microscopic lesions in the eyes of the 288 throughout the epithelium) and erosion ppm group primarily occurred in the cornea (much of the pseudostratified ciliated colum(with some involvement of the anterior nar epithelium was absent-primarily stem chamber) and ranged from cornea1 edema to cells remained) and degeneration of the olfac- ulcerative keratitis. Although the nervous tory mucosal epithelium (Fig. 2B). The nasal system was a suspected target organ, microlesions were primarily limited to the anterior scopic lesions of the central (brain) or periphportions of the nose. The incidence and se- eral (sciatic nerve) nervous systems were not verity of the nasal lesions indicated increased observed. toxicity with increasing exposure concentration. 13- Week Study Mild squamous metaplasia of the laryngeal epithelium was observed histologically in the 288 ppm group. However, metaplasia did not The mean (*SD) chamber concentrations extend into the upper trachea or into the for DMEA were 8 (*0.8), 24 (+ 1.2), and 76 lung. The macroscopic observation of thymic (k2.6) ppm. The respective analytical to
FIG. 2A. Squamous metaplasia and mucosal ulceration of the lateral wall of the nasal cavity with inflammatory exudate covering the nasal mucosa. Inflammation is also present in the submucosa. Male rat exposed to 98 ppm of DMEA and sacrificed on the day following the ninth exposure. H&E, X280. FIG. 2B. Moderate degeneration of olfactory epithelium in the anteriodotsal portion of the nasal cavity in a male rat exposed 9 days to 288 ppm of DMEA. H&E, X440. FIG. 2C. Epithelial hyperplasia and intraepithelial cystic glands (microcysts) within the respiratory mucosa and inflammation in the submucosa of the nasaI cavity of a male rat exposed 13 weeks to 76 ppm of DMEA. H&E, X440.
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TABLE 3 BODY WEIGHT
GAIN FOR F-344 AND MAINTAINED
RATS EXPOSED TO DMEA VAPOR FOR 13 WEEKS FOR A S-WEEK RECOVERY F’ERIOD~
Mean exposure concentration (ppm) Interval (weeks)
0
8
24
76
113+ 11.1 157 t 10.2 172 k 13.2
102 + 8.0* 147 + 10.6* 169 * 9.2
5 1 f 5.0 70 f 6.3 71 k5.1
49 * 6.8** 64 k 6.6** 71 k4.0
Males 8’ 14’ 19d
108 f 12.2 154& 11.5 167 f 16.7
112k8.6
157k9.9 166k7.1 Females
8 14 19
54 k 4.7 73 f 5.4 76 f 6.8
53 f 5.9 70 + 5.8 13 + 7.7
’ Values represent mean f SD. b Week 8 was the first week that a consistent statistically significant depression in body weight gain was observed for both sexesof the 76 ppm group. ’ Final weight gain value obtained after two or three exposures during Week 14. d End of 5-week postexposure recovery period. * p i 0.05 from controls. ** p < 0.0 1 from controls.
nominal concentration ratios were 0.89, 0.76, and 0.85. Corneal opacity occurred in the 24 and 76 ppm groups at the end of the daily exposure, beginning approximately 2-3 weeks after initiation of exposures. The opacity regressed during the night-time nonexposure hours. There was also a moderate incidence (approximately 25%) of audible respiration in rats of the 76 ppm group. No animals died during the study. Body weight gains at selected intervals are presented in Table 3. The body weight gains for both sexes of the 76 ppm group were statistically significantly lower than control values for most of the latter half of the 13-week
exposure regimen. The body weight gain values for the 76 ppm group returned to control values during the recovery period. There were no exposure-related alterations of body weight gain for rats exposed to 8 or 24 ppm of DMEA. There were no exposure-related effects on the neurobehavioral, food and water consumption, hematologic, serum chemistry, or urinalysis evaluations, on organ weights, or on the gross appearance of organs. Exposure-related nasal lesions were observed histologically at the termination of exposures in both sexes of the 76 ppm group, but were generally not observed in rats of the 24 ppm group (Table 2). The lesions were
FIG. 2D. Mucous cell hyperplasia involving the ventromedial respiratory mucosa in the anterior nasal cavity from a female rat exposed 13 weeks to 76 ppm of DMEA. H&E, X440. FIG. 2E. Intraepithelial cysts and epithelial atrophy in the junctional area between respiratory and olfactory mucosa in a female rat exposed 13 weeks to 76 ppm of DMEA. H&E, X280. FIG. 2F. Comeal epithelial vacuolization together with comeal dystrophy (mineralization) (arrows) in the eye of a female rat exposed 13 weeks to 76 ppm of DMEA. H&E, X440.
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limited to the anterior nasal cavity and included squamous metaplasia, microcysts (cystic intraepithelial glands) (Fig. 2C) and mucous cell hyperplasia (Fig. 2D) of the respiratory epithelium, mild rhinitis, and atrophy of the dorsal olfactory epithelium (Fig. 2E). The incidence and severity of these lesions were decreased at the end of the recovery period, indicating some degree of repair. Additionally, 4/ 10 males had laryngitis and two of these rats also had tracheitis. No similar lesions were found in female rats. Vacuolization of the corneal epithelium (Fig. 2F) was observed in 3/10 female rats of the 76 ppm group at the termination of exposures but not at the end of the recovery period. DISCUSSION Similar to other aliphatic amines, DMEA vapor acts primarily as an irritant to the eyes and respiratory tract. In these studies the potential for exogenously administered DMEA vapor to affect neurologic function or structure appears to be minimal, despite DMEA being an endogenous compound involved in the synthesis of acetylcholine. The decreased motor activity and loss of coordination observed in rats exposed acutely to 1668 ppm DMEA or greater were the only neurobehavioral changes associated with DMEA exposure. These concentrations killed 50% or greater of the exposed rats. In the 2-week study, a concentration of 586 ppm of DMEA, which resulted in complete mortality 4-8 days into the study, or a concentration of 288 ppm, which produced 27% mortality in the male rats, caused neither clinical signs nor microscopic pathology indicative of neurologic involvement. There was also no indication of neurologic toxicity from DMEA exposure in the 13-week study. It has been postulated that the water solubility of respiratory irritants affects their site of action in the respiratory tract (Casarett, 1975; Buckley et al., 1984), although other
ET AL.
factors such as reactivity of the chemical and flow rate through the nasal passages may also be important determinants in the absorption of chemicals by the nasal tissues (Stott et al., 1986). Irritants which are highly water soluble (e.g., formaldehyde, dimethylamine, ammonia) tend to produce upper respiratory tract lesions, while irritants which have limited water solubility (e.g., nitrogen dioxide, phosgene) tend to produce pulmonary damage. DMEA is a highly water-soluble compound (100% soluble at 20°C) and, thus, would be expected to produce lesions primarily in the upper nasal passages. If DMEA vapor concentrations exceed the capacity of the upper respiratory tract to “scrub” the chemical from the inhaled air, then pulmonary lesions would be expected. In the 2-week study, some rats of the 288 ppm group did have squamous metaplasia of the laryngeal epithelium, but there was no histologic evidence of pulmonary toxicity. Thus, at DMEA concentrations of 288 ppm and less, irritation and/ or toxicity was limited to the eyes and upper respiratory tract. In conclusion, the irritation of the eyes and nasal passages induced by DMEA exposure were consistent with effects observed with other alkyl amines in other animal studies and in cases of human exposure. At a concentration of 24 ppm of DMEA, biologically significant lesions were not observed in the eyes or nasal mucosa, and this concentration was considered to be the no-observable-effectlevel for the 13-week study. ACKNOWLEDGMENTS The authors thank Mssrs. F. L. Howard, D. W. Fait, and W. J. Kintigh for their technical assistance, and Ms. F. C. Wilt for her secretarial assistance.
REFERENCES BEARD, R. R., AND NOE, J. T. (1981). Aliphatic and alicyclic amines. In Patty’s Industrial Hygiene and
DMEA INHALATION Toxicology (G. D. Clayton and F. E. Clayton, Eds.), Chapter 44, Vol. IIB, pp. 3135-3176. Wiley, New York. BROWN, M. B., AND FORSYTHE, A. B. (1974). The small sample behavior of some statistics which test the equality of several means. Technometrics 16, 129-132. BUCKL.EY, L. A., JIANG, X. Z., JAMES, R. A., MORGAN, K. T., AND BARROW, C. S. (1984). Respiratory tract lesions induced by sensory irritants at the RDSO concentration. Toxicol. Appl. Pharmacol. 74, 4 17429. Bushy Run Research Center (BRRC) Report Numbers: 12-9, 1949: 39-99. 1976. Union Carbide Corp., Export, PA. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975). Petroleum hydrocarbon toxicity studies. I. Methodology. Toxicol. Appl. Pharmacol. 32,246-262. CASARETT, L. J. (1975). Toxicology of the respiratory system. In Toxicology: The Basic Science of Poisons (L. J. Casarett and J. Doull, Eds.), pp. 204-205. Macmillan. New York.
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D. J. (1964). Probit Analysis, 2nd ed. Cambridge Univ. Press, London. HOCHSCHILD, R. (1973). Effect of dimethylaminoethano1 on the lifespan of senile male A/J mice. Exp. Geronto/. 8, 185- 19 1. IRWIN, S. (1968). Comprehensive observational assessment: la. A systematic quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia 13,222-257. MILLER, R. G., JR. (1966). Simultaneous Statistical Inference, pp. 6-10. McGraw-Hill, New York. SMYTH, H. F., CARPENTER, C. P., AND WEIL, C. S. (1951). Range-finding toxicity data: List IV. AMA Arch. Ind. Hyg. Occup. Med. 4,119-122. SNEDECOR, G. W., AND COCHRAN, W. G. ( 1967). Statistical Methods, Iowa State Univ. Press, Ames. SOKAL, R. R., AND ROHLF, F. J. (1969). Biometry. Freeman, San Francisco. STOTT, W. T.. RAMSEY, J. C., AND MCKENNA, M. J. (1986). Absorption of chemical vapors by the upper respiratory tract of rats. In To,xico/ogy of the Nasal Passages (C. S. Barrow, Ed.), pp. 191-210. Hemisphere, New York. FINNEY,
AND
9,5 12-52 1 (1987)
APPLIEDTOXICOLOGY
Dimethylethanolamine: Acute, 2-Week, and 13-Week Inhalation Toxicity Studies in Rats DENNIS
R. KLONNE,’
DAROL E. DODD, IRVIN M. PRITTS, DONALD J. NACHREINER, EDWARD H. FOWLER, CATHERINE M. TROUP, ELTON R. HOMAN,
ANDBRYANBALLANTYNE Bushy
Run Research
Dimethylethanolamine: KLONNE, D. R., DODD, M., HOMAN, E. R., AND
Center,
Union
Carbide
Corporation,
Export,
Pennsylvania
15632
Acute, 2-Week, and 13-Week Inhalation Toxicity Studies in Rats. D. E., PRITTS, BALLANTYNE,
I. M., NACHREINER, B. (1987). Fundam.
D. J., FOWLER,
E. H., TROUP,
C.
9,5 12-52 1. Dimethylethanolamine (DMEA) is a volatile, water-soluble amine that has applications in the chemical and pharmaceutical industries. These studies evaluated the acute and subchronic inhalation toxicity of DMEA. Acute (4-hr) exposures of Wistar rats to DMEA vapor resulted in an LC50 value (95% confidence limits) of 1641 (862-3 125) ppm. Clinical signs of nasal and ocular initation, respiratory distress, and body weight loss were observed in rats exposed to 1668 ppm DMEA and higher. In the 2-week study, F-344 rats exposed to 98,288, or 586 ppm DMEA for 9 days (6 hr/day) during an 11-day period also exhibited signs of respiratory and ocular irritation (except the 98 ppm group). All animals of the 586 ppm group and 4 of 15 male rats of the 288 ppm group died. Body weight values for the 288 ppm group were reduced to about 75% of preexposure values, while the 98 ppm group gained 35% less weight than controls. Statistically significant differences in clinical pathology parameters (288 ppm group) and in organ weight values (288 and 98 ppm groups) probably resulted from the decreased food consumption and not from specific target organ toxicity. In the groups evaluated histologically (the 98 and 288 ppm groups) the eye and nasal mucosa were the primary target organs. In the 13-week subchronic study, F-344 rats were exposed to 0,8,24, or 76 ppm DMEA for 6 hr/day, 5 days/week for 13 weeks. The principal exposure-related changes were transient comeal opacity in the 24 and 76 ppm groups; decreased body weight gain for the 76 ppm group; and histopathologic lesions of the respiratory and olfactory epithelium of the anterior nasal cavity of the 76 ppm group and of the eye of several 76 ppm group females. Rats maintained for a 5-week recovery period only exhibited histological lesions of the nasal tissue, with the lesions being decreased in incidence and severity. DMEA acts primarily as an ocular and upper respiratory tract irritant and toxicant at vapor concentrations of 76 ppm, while 24 ppm or less produced no biologically significant toxicity in rats. Thus, 24 ppm was considered to be the no-observable-effect level. 0 I987 Society of Toxicology.
The aliphatic amines are a class of volatile, water-soluble chemicals with a wide variety of industrial applications. They are generally well absorbed from the gastrointestinal and respiratory tracts and may even produce lethality by a dermal route of exposure (Beard and Noe, 1981). The most apparent effect of ’ To whom correspondence should be addressed at Bushy Run Research Center, R.D. 4, Mellon Road, Export, PA 15632. 0272-0590187 $3.00 Copyright 0 I987 by the Society of Toxicology. All rights of reproduction in any form reserved.
512
Appl. Toxicol.
inhalation of amines is the irritation of the mucous membranes of the eyes and respiratory tract. In animals, single exposures to high vapor concentrations can produce inflammation throughout the respiratory tract and pulmonary edema. In man, the vapors also produce irritation of the eyes, mouth, throat, and skin. Systemic effects following inhalation include headache, nausea, fainting, and anxiety (Beard and Noe, 198 1). Dimethylethanolamine (DMEA; 2-(di-
DMEA INHALATION
methylamino)ethanol) has applications as a desulfurizing agent in the treatment of natural gas; as an intermediate in the synthesis of corrosion inhibitors, pharmaceuticals, and dyestuffs; and as a curing agent for epoxy, amine, and polyamide resins. In previous studies, the acute peroral LD50 (95% confidence limits) value in rats for DMEA, administered as a 20% aqueous solution, was found to be 2.34 (2.26-2.42) g/kg (Smyth et al., 195 1). The 24-hr occluded percutaneous LD50 value for rabbits was 1.37 (1.21-1.55)ml/kg(Smythetal., 1951).Thus, DMEA is moderately toxic by acute oral and dermal routes of administration. A 90-day dietary feeding study in rats of DMEA at dosages up to 0.89 g/kg/day produced increases in the kidney to body weight value at the highest dosage (BRRC, 1949; Smyth et al., 195 1). No microscopic renal lesions accompanied this change. No effects were found in the groups fed 0.18 or 0.045 g/kg/day. Consistent with the properties of other aliphatic amines, DMEA was previously found to be severely irritating in a 4-hr occluded primary dermal irritation test in rabbits (BRRC, 1976), with 6 of 6 animals exhibiting dermal necrosis, and severely irritating in the rabbit ocular irritation test (Smyth et al., 195 1). DMEA is a normal constituent of mammalian metabolism, although it is present only in minute quantities (Hochschild, 1973). It is a precursor of choline synthesis and can cross the blood-brain barrier. Since choline is involved in the biosynthesis of acetylcholine and cellular membranes, DMEA may be indirectly involved with nerve impulse propagation and membrane biosynthesis. Thus, nervous system activity could be affected by exogenous administration of DMEA. The purpose of these studies was to obtain information on the acute and subchronic inhalation toxicity of DMEA that would be useful in determining potential human health risks from inhalation of DMEA vapor.
TOXICITY
IN RATS
513
METHODS Test material. DMEA (CAS 108-01-O) was obtained from Union Carbide Corp. (South Charleston, WV). Compositional analyses before and after each study indicated that the material was at least 99% pure. Animals and exposure regimen. Male and female Wistar albino rats (Hilltop Lab Animals, Inc., Scottdale, PA) with a body weight range of 200-300 g were used for the acute exposure. (Wistar rats were used for the acute studies because their faster growth rate (in comparison to F-344 rats) makes it easier to detect exposure-related effects on body weight.) Fischer-344 rats (Charles River Breeding Laboratories, Kingston, NY) were used for the 2-week and 13-week studies. Food (Purina Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO) and water were provided ad libitum except during exposures. All animals were identified by toe clipping and were maintained on a 12-hr light/dark cycle throughout the quarantine, exposure, and recovery periods. For the acute study, 5 rats/sex were exposed for 4 hr at each exposure concentration, then maintained for an additional 14-day observation period before their sacrifice. For the 2-week study, 10 rats/sex, approximately 8 weeks old, with mean body weights of 167 g for males and 12 1 g for females, were randomly assigned to all study groups to be used for clinical and anatomic pathology evaluations. Additionally, 5 male rats were assigned to the control, middle, and high concentration groups for possible ultrastructural (electron microscopic) evaiuation of nerve tissue (not performed since no behavioral abnormalities or light microscopic lesions of nerve tissue were observed). Rats were exposed for 6 hrjday, 5 days/ week, for 9 exposures during an 1 l-day period (2 days without exposure between Exposure Days 5 and 6). All animals were sacrificed on the morning after the ninth exposure. For the 13-week study, 20 rats/sex, approximately 9 weeks old, with mean body weights of 180 g for males and 130 g for females, were randomly assigned to each of four study groups. Additionally. 10 male rats were assigned to the control, middle, and high concentration groups for possible ultrastructural evaluation of nerve tissue (not performed since no behavioral abnormalities or light microscopic lesions of nerve tissue were observed). Rats were exposed for 6 hr/day, 5 days/week, for 13 weeks. One-half of all rats per sex per group were sacrificed after at least 2 days ofexposure during the 14th week of the study; the remaining rats were sacrificed after 5 complete weeks of recovery. Generation and analysis of exposure atmospheres. The acute study was conducted in a 900-liter stainless-steel and glass chamber (airflow of 200 liters/min) at mean analytical vapor concentrations of 1668,2408, or 33 11 ppm DMEA. For the 2-week study, rats were exposed in 1330-liter stainless-steel and glass chambers at an airflow of 300 liters/min. The target concentrations of DMEA
514
KLONNE ET AL.
were 0, 100, 300, and 600 ppm. The 13-week study was conducted in 4320~liter stainless-steel and glass chambers at airflows of 800-1000 liters/min. The target concentrations of DMEA vapor were 0, 8, 25, and 75 ppm. Chamber temperature and relative humidity for all studies were generally maintained at 25°C and 46%, respectively. DMEA vapor was generated by metering the liquid into a heated, spiral-grooved evaporator with a countercurrent airstream, similar in design to the one used by Carpenter et al. (1975). Chamber concentrations of DMEA were analyzed at approximately 40-min intervals with a Perkin-Elmer 3920B gas chromatograph (CC) equipped with a flame ionization detector. The CC column was a 5 ft X i in stainless-steel column packed with 20% YIP-2100 on 80/ 100 mesh Supelcoport (Supelco, BeIlefonte, PA), maintained at 200°C. Injections of gas standards were used to calibrate the GC. Chamber atmosphere samples were automatically injected into the GC with the use of a Perkin-Elmer environmental sampler. Biological procedures. In the acute inhalation study, rats were observed for signs of toxicity during exposure and for 14 days postexposure. Body weights were determined prior to exposure and at 7 and 14 days after exposure. Necropsies were performed on all rats. For the 2and 13-week studies, biological assessments included daily animal observation, neurobehavioral evaluations (tremors, convulsions, tail elevation, impaired gait, paresis, salivation, lacrimation, diarrhea, piloerection, hypothermia, stereotyphy, surface and midair righting reflex, wire grasping, body tone, limb rotation, pupil size, skin color, respiration, locomotor activity, cornea1 response. tail and toe pinch response, and auditory startle response; Irwin, 1968), body weight and organ (brain, kidney, liver, lungs, testes, and adrenals) weight determinations, ophthalmology (evaluation of the eye with a light source and magnifying lens), clinical pathology, and gross and light microscopic examination of tissues. Organ weight determinations, gross pathologic examination, and blood and urine sample collections were made the morning following the last DMEA exposure day (except for recovery animals). Blood samples for hematologic and serum chemistry measurements were obtained by retroorbital bleeding of nonfasted rats anesthetized with methoxyflurane. Hematologic determinations (Coulter Counter S-Plus IV, Coulter Electronics, Inc., Hialeah, FL) included white blood cell (WBC) count, red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH concentration, and platelet count; WBC differentials were also performed. Serum chemistries (performed on a CentrifiChem, Baker Instruments, Pleasantville, NY, or an Astra, Beckman Instruments, Inc., Brea, CA) included creatinine, aspartate aminotransferase, alanine aminotransferase, total protein, albumin, globulin, total bilirubin, y-glutamyl transpeptidase, sorbitol dehydrogenase, sodium, potassium, and chloride. Individual urine samples were col-
lected during a 16-hr period prior to sacrifice from rats held in polycarbonate metabolism cages. Semiquantitative measurements on urine (Ames Multistix, read on the Clinitek, Ames Division, Miles Laboratories, Elkhart, IN) included pH, protein, bilirubin, urobilinogen, glucose, ketones, and blood. In addition, osmolality determinations (Cryomatic osmometer, Advanced Instruments, Inc., Needham Heights, MA) and microscopic evaluations of the urine sediment were performed. Food and water consumption were also measured during the 16-hr urine collection period. Selected tissues examined histologically included brain (five sections: frontal lobe and basal ganglia, parietal and temporal lobes with thalamus and hypothalamus, occipital lobes with midbrain, cerebellum and pons, and the medulla oblongata), sciatic nerve, kidneys, liver. larynx, lungs, nasal turbinates, spleen, trachea, gonads, eyes,and all gross lesions. Histopathologic examinations for the 2-week study were performed on tissues from rats of the control and middle exposure groups (due to complete mortality in the high exposure group), with nasal turbinates also being evaluated for the low exposure group rats. For the 13-week study, additional tissues for histopathologic examination included: sternal bone and bone marrow, epididymides, thymus, urinary bladder, thyroids and parathyroids, adrenals, heart, cervical lymph nodes. eyes, pituitary, gastrocnemius muscle, spinal cord, and uterus. These examinations were performed on rats from the control and high exposure groups, with nasal turbinates also being evaluated for the middle exposure group rats. Statisticalanalysis. Results of quantitative continuous variables were intercompared among the DMEA concentration levels and the control group by Bartlett’s homogeneity of variance (Sokal and Rohlf, 1969) analysis of variance (ANOVA), and Duncan’s multiple range test (Snedecor and Cochran, 1967). Duncan’s test was used when a significant F value from an ANOVA was observed. For heterogeneous group variances, the groups were compared by ANOVA for unequal variances (Brown and Forsythe, 1974) and either Student’s t test or Cochran’s t test (Snedecor and Cochran, 1967) was used. Corrected Bonferroni probabilities were used for t test comparisons (Miller, 1966). The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons. For the calculation of the LC50, the method of Finney (1964) was used.
RESULTS Acute Study The mean (*SD) chamber concentrations of DMEA were 1668 (+138), 2408 (+178), and 33 11 (+ 156) ppm. Mortality occurred at
DMEA
INHALATION
TOXICITY
IN RATS
515
all exposure concentrations with 5/10, 7/10, and 8/ 10 rats dying 1 to 12 days after exposure at 1668, 2408, and 33 11 ppm, respectively. No sex-related differences were observed in mortality, and the LC50 value (95% confidence limits) for the combined sexes was 1641 (862 to 3 125) ppm. Clinical signs, observed in all exposure groups, included lacrimation, excessive salivation, ocular, oral, and nasal discharge and encrustation, respiratory difficulties, decreased motor activity, coordination loss, and swelling and bleeding of extremities (feet and nose) from excessive preening (highest concentration only). Substantial body weight losses occurred for all but one survivor on postexposure Day 7, with only two males of the 1668 ppm group surpassing the preexposure body weight by Day 1 2 3 4 5 6 7 6 9 101112 study w 14. Discolored lungs, livers, kidneys, and FIG. 1. Mean body weight change for F-344 rats during spleens were seen at the necropsy of animals which died on study as well as in the two sur- the DMEA 2-week vapor inhalation study. vivors of the 33 11 ppm group. Survivors of the 2408 and 1668 ppm groups did not have exposure-related macroscopic lesions at the ppm group died 8- 12 days after the initiation end of the 14-day postexposure observation of exposures. There was a concentration-reperiod. lated effect on body weight in this study (Fig. 1). Weight loss occurred after one exposure in both sexes of the 288 and 586 ppm groups, 2-Week Study with the final mean body weight of the 288 ppm group being about 75% of preexposure The means (+-SD) of the daily mean cham- values. Although the 98 ppm group gained ber concentrations were 98 (+ 15) 288 (+ 14), weight, they gained 35% less than controls. and 586 (+36) ppm of DMEA vapor. The re- There was also a statistically significant conspective analytical to nominal concentration centration-related decrease in food consumpratios were 0.75, 0.86, and 0.87, indicating tion for both the 98 and 288 ppm groups. that DMEA was mildly reactive with the sur- Mean water consumption was equivalent for faces or contents of the chamber. all groups. The ophthalmic examination inSigns of respiratory distress (audible respi- dicated that animals of the 288 ppm group ration, mouth breathing), ocular and nasal ir- had conjunctivitis, including chemosis, and ritation (discharge and encrustation), and cornea1 opacity. comeal opacity were observed in both sexes Most of the serum chemistry, hematology, of the 288 and 586 ppm exposure groups. and urinalysis values for the 288 ppm group There were no exposure-related effects on the were statistically significantly different from neurobehavioral evaluations. All animals of control values, a further indication of the the 586 ppm group died or were sacrificed in stressed and debilitated condition of the ania moribund state 4-8 days after the initiation mals. No similar exposure-related effects of exposures. Four of fifteen males of the 288 were observed for the 98 ppm exposure
516
KLONNE
ET AL.
TABLE 1 TERMINAL BODY WEIGHT AND SELECTED ORGAN WEIGHTS FOR F-344 RATS OFTHE DMEA ~-WEEK INHALATION S~JDY” Males Parameter
Bodywt (g) Brain wt (g) Brain/body wt (%) Liver wt (g) Liver/body wt (%) Kidney wt (g) Kidney/body wt (%) Lung w (9) Lung/body wt (%) Adrenal wt (g X IO’) Adrenal/body wt (%X 10’) Testes wt (g) Testes/body wt (%)
Females
Control
98 mm
288 ppm
Control
98 mm
288 ppm
198 (10.4) 1.70 (0.04) 0.86 (0.04) 7.91 ( 1.OO) 3.99 (0.35) 1.53 (0.14) 0.77 (0.04) 1.08 (0.19) 0.54 (0.09) 4.88 (1.97) 0.25 (0.09) 2.34 (0.26) 1.18 (0.11)
184** (12.2) 1.65* (0.05) 0.90 (0.04) 7.32 (0.69) 3.98 (0.19) 1.49 (0.08) 0.81* (0.02) 0.98 (0.12) 0.53 (0.05) 4.35 (1.07) 0.24 (0.06) 2.41 (0.18) 1.32** (0.10)
126** (10.9) 1.63** (0.03) 1.30** (0.12) 5.07** (0.50) 4.03 (0.30) 1.24** (0.06) 0.99** (0.08) 1.02 (0.19) 0.81** (0.11) 5.73 (0.89) 0.45** (0.05) 1.94** (0.17) 1.54** (0.06)
137 (3.8) 1.64 (0.05) 1.19 (0.04) 5.04 (0.23) 3.67 (0.18) 1.09 (0.05) 0.79 (0.05) 0.80 (0.10) 0.58 (0.07) 5.00 (1.21) 0.36 (0.09)
131** (3.1) 1.62 (0.05) 1.23 (0.04) 4.69** (0.34) 3.57 (0.21) 1.09 (0.05) 0.83 (0.04) 0.88 (0.14) 0.67 (0.1 I) 4.95 (1.34) 0.38 (0.11)
94** (3.6) 1.54** (0.04) 1.65** (0.08) 3.97*+ (0.34) 4.23** (0.29) 1.05 (0.09) 1.12** (0.09) 0.83 (0.18) 0.88** (0.18) 5.89 (0.53) 0.63** (0.W
a Values represent mean (&SD) for 7 to IO rats. * p < 0.05 from control. ** p < 0.0 I from control.
group, except for a statistically significant decrease in urine volume for both sexes. Similarly, many of the absolute and relative (as a percentage of body weight) organ weight values for the 288 ppm group were significantly different from those of control values (Table 1). Because of the substantial reduction in body weight gain, aswell as the lack of histological evidence for organ toxicity at the higher concentration (288 ppm), the few statistically significant differences in organ weight values for the 98 ppm group (Table
1) were not considered to result from specific organ toxicity. At necropsy, grosschangesincluded ocular opacity, thymic atrophy, and reduced body fat in most rats of the 288 ppm group. The principal histologic lesions were in the upper respiratory tract of the 98 and 288 ppm groups (Table 2) and in the eyes of the 288 ppm group. In the nasal tissuesof the 98 ppm group, rhinitis, squamous metaplasia, and ulceration of the nasal mucosa (Fig. 2A) were observed. Similar lesions occurred at a higher
DMEA INHALATION
TOXICITY
517
IN RATS
TABLE 2 INCIDENCE
OF SELECZTED NAVAL TISSUE HISTOPATHOLOGIC LESIONS IN F-344 RATS EXPOSED TO FOR 2 OR 13 WEEKS AND SACR~FKED THE DAY AVER THE FINAL EXPOSURE
I3-week studyb
2-week studya Males DMEA concentration (ppm): 0 Total No. examined Unremarkable Rhinitis Squamous metaplasia Epithelial erosion Mucosal ulceration Degeneration of olfactory mucosa/epithelium Degeneration of respiratory epithelium Atrophy of olfactory epithelium Microcysts in respiratory epithelium
DMEA VAPOR
Males
Females
Females
98
288
0
98
288
0
24
16
0
24
76
10 10 0 0 0 0
10 1 5 8 1 4
7 0 6 7 4 6
10 9’ 0 0 0 0
10 5 5 2 0 2
10 0 I 10 0 8
10 9d 0 0 0 0
10 8 2 0 0 0
10 0 0 9 0 0
10 9d 0 0 0 0
10 8 2 0 0 0
10 I I 4 0 0
0
0
1
0
0
9
0
0
0
0
0
0
0
0
0
0
0
4
0
0
8
0
0
7
0
0
0
0
0
0
0
0
10
0
0
3
0
0
0
0
0
0
0
0
10
0
0
3
a All animals of the 586 ppm group died on study and were not histologically evaluated. Incidence values in the table also do not include those four male rats of the 288 ppm group which died on study. ’ Rats of the 8 ppm group were not histologically evaluated as only minimal rhinitis was observed in 2 rats/sex of the 24 ppm group. ‘One control had a hemorrhage in the nasolacrimal duct, ‘One control had minimal mineralization of the olfactory epithelium.
incidence in the 288 ppm group (Table 2). atrophy in the 288 ppm exposure group was Additional nasal lesions in the 288 ppm verified by the microscopic observation of degroup included respiratory epithelial degen- creased numbers of cortical lymphocytes. eration (presence of necrotic cells scattered The microscopic lesions in the eyes of the 288 throughout the epithelium) and erosion ppm group primarily occurred in the cornea (much of the pseudostratified ciliated colum(with some involvement of the anterior nar epithelium was absent-primarily stem chamber) and ranged from cornea1 edema to cells remained) and degeneration of the olfac- ulcerative keratitis. Although the nervous tory mucosal epithelium (Fig. 2B). The nasal system was a suspected target organ, microlesions were primarily limited to the anterior scopic lesions of the central (brain) or periphportions of the nose. The incidence and se- eral (sciatic nerve) nervous systems were not verity of the nasal lesions indicated increased observed. toxicity with increasing exposure concentration. 13- Week Study Mild squamous metaplasia of the laryngeal epithelium was observed histologically in the 288 ppm group. However, metaplasia did not The mean (*SD) chamber concentrations extend into the upper trachea or into the for DMEA were 8 (*0.8), 24 (+ 1.2), and 76 lung. The macroscopic observation of thymic (k2.6) ppm. The respective analytical to
FIG. 2A. Squamous metaplasia and mucosal ulceration of the lateral wall of the nasal cavity with inflammatory exudate covering the nasal mucosa. Inflammation is also present in the submucosa. Male rat exposed to 98 ppm of DMEA and sacrificed on the day following the ninth exposure. H&E, X280. FIG. 2B. Moderate degeneration of olfactory epithelium in the anteriodotsal portion of the nasal cavity in a male rat exposed 9 days to 288 ppm of DMEA. H&E, X440. FIG. 2C. Epithelial hyperplasia and intraepithelial cystic glands (microcysts) within the respiratory mucosa and inflammation in the submucosa of the nasaI cavity of a male rat exposed 13 weeks to 76 ppm of DMEA. H&E, X440.
518
DMEA INHALATION
TOXICITY
519
IN RATS
TABLE 3 BODY WEIGHT
GAIN FOR F-344 AND MAINTAINED
RATS EXPOSED TO DMEA VAPOR FOR 13 WEEKS FOR A S-WEEK RECOVERY F’ERIOD~
Mean exposure concentration (ppm) Interval (weeks)
0
8
24
76
113+ 11.1 157 t 10.2 172 k 13.2
102 + 8.0* 147 + 10.6* 169 * 9.2
5 1 f 5.0 70 f 6.3 71 k5.1
49 * 6.8** 64 k 6.6** 71 k4.0
Males 8’ 14’ 19d
108 f 12.2 154& 11.5 167 f 16.7
112k8.6
157k9.9 166k7.1 Females
8 14 19
54 k 4.7 73 f 5.4 76 f 6.8
53 f 5.9 70 + 5.8 13 + 7.7
’ Values represent mean f SD. b Week 8 was the first week that a consistent statistically significant depression in body weight gain was observed for both sexesof the 76 ppm group. ’ Final weight gain value obtained after two or three exposures during Week 14. d End of 5-week postexposure recovery period. * p i 0.05 from controls. ** p < 0.0 1 from controls.
nominal concentration ratios were 0.89, 0.76, and 0.85. Corneal opacity occurred in the 24 and 76 ppm groups at the end of the daily exposure, beginning approximately 2-3 weeks after initiation of exposures. The opacity regressed during the night-time nonexposure hours. There was also a moderate incidence (approximately 25%) of audible respiration in rats of the 76 ppm group. No animals died during the study. Body weight gains at selected intervals are presented in Table 3. The body weight gains for both sexes of the 76 ppm group were statistically significantly lower than control values for most of the latter half of the 13-week
exposure regimen. The body weight gain values for the 76 ppm group returned to control values during the recovery period. There were no exposure-related alterations of body weight gain for rats exposed to 8 or 24 ppm of DMEA. There were no exposure-related effects on the neurobehavioral, food and water consumption, hematologic, serum chemistry, or urinalysis evaluations, on organ weights, or on the gross appearance of organs. Exposure-related nasal lesions were observed histologically at the termination of exposures in both sexes of the 76 ppm group, but were generally not observed in rats of the 24 ppm group (Table 2). The lesions were
FIG. 2D. Mucous cell hyperplasia involving the ventromedial respiratory mucosa in the anterior nasal cavity from a female rat exposed 13 weeks to 76 ppm of DMEA. H&E, X440. FIG. 2E. Intraepithelial cysts and epithelial atrophy in the junctional area between respiratory and olfactory mucosa in a female rat exposed 13 weeks to 76 ppm of DMEA. H&E, X280. FIG. 2F. Comeal epithelial vacuolization together with comeal dystrophy (mineralization) (arrows) in the eye of a female rat exposed 13 weeks to 76 ppm of DMEA. H&E, X440.
520
KLONNE
limited to the anterior nasal cavity and included squamous metaplasia, microcysts (cystic intraepithelial glands) (Fig. 2C) and mucous cell hyperplasia (Fig. 2D) of the respiratory epithelium, mild rhinitis, and atrophy of the dorsal olfactory epithelium (Fig. 2E). The incidence and severity of these lesions were decreased at the end of the recovery period, indicating some degree of repair. Additionally, 4/ 10 males had laryngitis and two of these rats also had tracheitis. No similar lesions were found in female rats. Vacuolization of the corneal epithelium (Fig. 2F) was observed in 3/10 female rats of the 76 ppm group at the termination of exposures but not at the end of the recovery period. DISCUSSION Similar to other aliphatic amines, DMEA vapor acts primarily as an irritant to the eyes and respiratory tract. In these studies the potential for exogenously administered DMEA vapor to affect neurologic function or structure appears to be minimal, despite DMEA being an endogenous compound involved in the synthesis of acetylcholine. The decreased motor activity and loss of coordination observed in rats exposed acutely to 1668 ppm DMEA or greater were the only neurobehavioral changes associated with DMEA exposure. These concentrations killed 50% or greater of the exposed rats. In the 2-week study, a concentration of 586 ppm of DMEA, which resulted in complete mortality 4-8 days into the study, or a concentration of 288 ppm, which produced 27% mortality in the male rats, caused neither clinical signs nor microscopic pathology indicative of neurologic involvement. There was also no indication of neurologic toxicity from DMEA exposure in the 13-week study. It has been postulated that the water solubility of respiratory irritants affects their site of action in the respiratory tract (Casarett, 1975; Buckley et al., 1984), although other
ET AL.
factors such as reactivity of the chemical and flow rate through the nasal passages may also be important determinants in the absorption of chemicals by the nasal tissues (Stott et al., 1986). Irritants which are highly water soluble (e.g., formaldehyde, dimethylamine, ammonia) tend to produce upper respiratory tract lesions, while irritants which have limited water solubility (e.g., nitrogen dioxide, phosgene) tend to produce pulmonary damage. DMEA is a highly water-soluble compound (100% soluble at 20°C) and, thus, would be expected to produce lesions primarily in the upper nasal passages. If DMEA vapor concentrations exceed the capacity of the upper respiratory tract to “scrub” the chemical from the inhaled air, then pulmonary lesions would be expected. In the 2-week study, some rats of the 288 ppm group did have squamous metaplasia of the laryngeal epithelium, but there was no histologic evidence of pulmonary toxicity. Thus, at DMEA concentrations of 288 ppm and less, irritation and/ or toxicity was limited to the eyes and upper respiratory tract. In conclusion, the irritation of the eyes and nasal passages induced by DMEA exposure were consistent with effects observed with other alkyl amines in other animal studies and in cases of human exposure. At a concentration of 24 ppm of DMEA, biologically significant lesions were not observed in the eyes or nasal mucosa, and this concentration was considered to be the no-observable-effectlevel for the 13-week study. ACKNOWLEDGMENTS The authors thank Mssrs. F. L. Howard, D. W. Fait, and W. J. Kintigh for their technical assistance, and Ms. F. C. Wilt for her secretarial assistance.
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