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2,4-Pentanedione: 9-Day and 14-week vapor inhalation studies in Fischer-344 rats
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
( 1986)
7,329-339
2,dPentanedione: g-Day and 14-Week Vapor Inhalation Studies in Fischer-344 Rats D. E. DODD,’ R. H. GARMAN, I. M. PRITTS, C. M. TROUP, W. M. SNELLINGS, AND B. BALLANTYNE* Bushy Run Research and *Union
Center, Carbide
R.D. 4. Mellon Road, Export, Pennsylvania 15632, Corporation, Danbury, Connecticut 068I 7
2,4-Pentanedione: 9-Day and 14-Week Vapor Inhalation Studies in Fischer-344 Rats. DODD, D. E., GARMAN,
R. H., PRITTS,
I. M.,
TROUP,
C. M.,
SNELLINGS,
W.
M.,
AND
BALLANTYNE,
B. (1986). Fundam. Appf. Toxicol. 7, 329-339. Fischer-344 rats, in groups of 10 males and 10 females, were exposed for 9 days (6 hr/day) to 2,4-pentanedione (2,4-PD) vapor at mean concentrations of 805,418, 197, and 0 (control) ppm. No deaths occurred, and the only adverse signs were of sensory irritation (partial closure of eyelids, periocular and perioral wetness) at 805 ppm. Also at 805 ppm were decreased body and organ weights, lymphocytosis, and moderate inflammation of the nasal mucosa. At 4 18 ppm there was a decrease in body weight gain and mild inflammation ofthe nasal mucosa. Apart from minimal nasal mucosal inflammation, there were no effects at 197 ppm. In the subchronic (14-week) study, rats were exposed (6 hr/day; 5 days/week) to 650,307, 101, and 0 (control) ppm of 2,4-PD vapor, using groups containing 20 males and 20 females, with halfbeing sacrificed at the end ofthe exposure period and the remainder kept for a 4-week postexposure recovery period. An additional 10 males were added to the 650 and 0 ppm groups for glutaraldehyde perfusion and subsequent electron microscopic examination of sciatic nerves. At 650 ppm, all females and 10 of 30 males died between the second and sixth weeks of exposure. These animals had acute degenerative changes in the deep cerebellar nuclei, vestibular nuclei and corpora striata, and acute lymphoid degeneration in the thymus. Seven of 15 male survivors of the 650 ppm group (combined 14-week and recovery sacrifices) had gliosis and malacia in the same brain regions, minimal squamous metaplasia in the nasal mucosa, decreased body and organ weights, lymphocytosis, and minor alterations in serum and urine chemistries. No ultrastructural evidence of peripheral neuropathy was observed. Except for central neuropathy, many of the adverse effects at 650 ppm were less marked in the 4-week recovery animals. No deaths occurred at 307 ppm, but females had slightly decreased body weight gains, and in both sexesthere were minor alterations in hematology, serum chemistry, and urinalysis parameters, which were not present in the 4-week recovery animals. Rats exposed to 10 1 ppm showed no differences from the control rats. Subchronic exposure to 650 ppm of 2,4-PD vapor causes serious adverse biological effects. Under these study conditions, the minimum-effects concentration was 307 ppm, and the no-adverse effects concentration was 101 ppm. 0 1986 SocietyofToxicology.
2,4-pentanedione (2,4-PD: CAS No. 123-546), a liquid P-diketone, is used as a chemical intermediate, metal chelator, and lubricant additive (Hawley, 1977). Results of acute studies indicate 2,4-PD to be of moderately high peroral and percutaneous toxicity, ’ To whom correspondence and requests for reprints should be addressed.
mildly irritating to the skin, and moderately irritating to the eye (Ballantyne et al., 1986). With a vapor pressure of 7 mm Hg at 20°C (Sax, 1979), the 4-hr LC50 of 2,4-PD vapor for rats is 1224 (1063- 1409) ppm, and LT50 values for exposure to vapor-saturated atmospheres have been determined to be 52 min (7060 ppm) for male rats and 55 min (7912 ppm) for female rats (Ballantyne et al., 1986). 329
0272-0590186 $3.00 Copyright 0 1986 by the society of Toxicology. All rights of reproduction in any form reserved.
DODD ET AL.
Repeated dosing of 2,4-PD to rats by gavage causes gastric erosions, thymic necrosis, and central nervous system injury characterized by perivascular edema and hemorrhage, endothelial swelling, and bilateral foci of malacia and gliosis in the cerebellar peduncles, olivary nuclei and lower brain stem (Krasavage et al., 1982). In view of the vapor pressure of 2,4-PD, its known toxicity, and the potential for occupational exposure, it was appropriate to investigate the short-term repeated and subchronic inhalation toxicity of 2,4-PD vapor at ambient temperature and pressure. METHODS Test material. 2,4-PD was obtained from Union Carbide Corporation, South Charleston, West Virginia. The test samples were kept in 1-gal glass bottles and blanketed with nitrogen. Both before and after the 9-day and 14week studies, the material was analyzed by gas chromatography for composition and found to be 99% pure. Animals and exposuregroups. Male and female Fischer-344 rats, 34 to 38 days of age, were obtained from Charles River Breeding Laboratories, Inc., Kingston, New York. They were confirmed to be free from intestinal parasites, and on the basis of histologic examination of tissues from selected animals, did not have any common rodent diseases. They were clinically normal upon receipt and during a 2-week acclimatization period prior to the start of exposures. Rats were uniquely numbered by toe clipping and ear notching procedures. They were assigned to exposure groups using a computer-based randomization program. At the time of assignment, only animals having body weights within 2 SD of the group means for each sex were used. Animals were housed, two per cage, in stainless-steel wire-mesh cages. They were allowed free access to food (Purina Certified Rodent Chow #5002, Ralston Purina Co., St. Louis, MO.) and water during the nonexposure periods. Animals were kept on a 12-hr light-dark cycle. For the 9-day study, four groups, each containing 10 male and 10 female rats, were exposed for 6 hr/day to 3 different target concentrations of 2,4-PD vapor and one air control atmosphere. For the subchronic study, there were also three different target concentrations of 2,4-PD vapor and one air control group. There were 20 males and 20 females for each exposure group, with half the animals to be sacrificed after the final exposure. The remaining animals were kept as recovery animals for 4 weeks after the final exposure and sacrificed. An additional 10 males were added to the high dose and control groups for glutaraldehyde perfusion and subsequent
transmission electron microscopic examination of sciatic and tibiaf nerves. Exposure conditions. Exposures for the 9&y study were conducted in 1.3 m3 chambers with an airflow of 300 liters/min, and for the subchronic study, chambers with a volume of 4.3 m3 operated at an airflow rate of 1000 liters/min were used. The chambers were constructed of stainless steel and had glass observation ports. Temperature and relative humidity were measured at least four times during each exposure. For all nine exposures, the range of daily mean temperature and relative humidity values was 23 to 24°C and 40 to 48%, respectively. During any individual 6 hr exposure, the temperature and relative humidity did not vary more than 2°C and 8%, respectively. For the 9-day study, the target concentrations were 200,400, and 800 ppm. Exposures were for 6 hr/day for 5 consecutive days, and after a 2-day nonexposure period, exposures were resumed for 4 consecutive days. Based on the results of this study, the target concentrations for the subchronic study were chosen at 100, 300, and 650 ppm. Exposures were for 6 hr/day, 5 days a week for 14 weeks, with some animals being sacrificed during the 14th week. The number of 6-hr exposures was 67 for male rats and 68 for females rats. The position of cages in the exposure chambers was rotated on a weekly basis to compensate for any possible, but not detected, variation in chamber environment or 2.4-PD vapor concentration. 2,4-PD vapor was generated by metering the test material with a piston pump (Fluid Metering Inc., Oyster Bay, N.Y.) into a heated glass evaporator (Carpenter et al., 1975) whose temperature was maintained at the lowest level sufficient to vaporize the liquid. The vapor was carried into the exposure chamber using a diluting countercurrent airstream entering the bottom of the evaporator. Chamber concentrations of 2,4-PD vapor were analyzed approximately once every 33 min during the 6-hr exposure periods using a Perkin-Elmer Model 3920B gas chromatograph equipped with a flame ionization detector. The GC column used was a 5 ft X 4 in stainless steel tube packed with SP2 100 on 80/100 mesh Supelcoport@ and maintained at 140°C. Chamber atmosphere samples were automatically injected into the CC with the aid of a Perkin-Elmer Environmental Sampler and vacuum pumps attached to Teflon-lined, polyethylene sample lines. The CC was calibrated by either dynamically generated gas standards, using a glass diffusion tube and a VICI Metronics Dynacalibrator 450, or injection of gas bag standards, which were prepared by syringe injection of the test material into Tedlar gas bags. A series of gas standards encompassing the entire range of vapor concentrations generated were prepared. A linear calibration curve was obtained when integration counts were plotted against the vapor concentrations of the standards. The calibration was checked at least once weekly. Daily nominal concentrations of 2,4-PD vapor were calculated by
2,4-PD 9-DAY AND ICWEEK taking the amount of 2,4-PD delivered and dividing it by the total volume of chamber air. Biological monitoring. In the 9-day study, animals were observed for toxicologic and pharmacologic signs prior to, during, and following each exposure. Body weights were measured just before the first exposure, preceding the second, fifth, sixth, and seventh exposures, and just before sacrifice. Blood for hematologic evaluations was obtained from the orbital sinus on the day of sacrifice. Standard laboratory procedures were used for the following determinations: total white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, and differential leukocyte count. All surviving animals were sacrificed the day following the final exposure by exsanguination following methoxyflurane anesthesia. Necropsy was performed, and weights of the brain, liver, kidneys, lungs, heart, thymus, and testes were determined. The following tissues were removed and fixed in 10% neutral buffered Formalin: nasal turbinates, larynx, trachea, lungs, spleen, liver. kidneys, ovaries, testes, thymus, brain (5 levels), and all gross lesions. Histologic examination was performed on all tissues from the high concentration and control groups. In addition, nasal turbinates from the intermediate and low concentration groups were examined microscopically. In the subchronic inhalation study, animals were observed daily for signs of toxicity. Prior to the first exposure and at sacrifice, the anterior segment of the eye was examined ophthalmoscopically. An Irwin screen (Irwin, 1968) was performed prior to the tint exposure and monthly thereafter, including just before sacrifice of the recovery animals. The neurobehavioral screen did not include all the assessments discussed by Irwin (1968), and evaluations were not scored. Body weights were measured the morning before the first exposure, weekly during the exposure period, and immediately before sacrifice. Animals ofthe 4-week recovery period were weighed weekly and immediately before sacrifice. Individual animal food and water consumption was measured for approximately 15 hr in metabolism cages with 10 males and 10 females from each exposure concentration; measurements were made during exposure week 14. Urine was collected while animals were in the metabolism cages. Urine volume, color, and turbidity were recorded, and semiquantitative measurements were made of pH, glucose, ketones, protein, bilirubin, and urobilinogen using Ames Multi-stix@ Reagent Strips read on a Clini-tek (Ames Division, Miles Laboratories, Elkhart, Ind.). Urine osmolality was determined with an Advanced Cryomatic Osmometer (Advanced Instruments Inc., Needham Heights, Mass.). Serum chemistry and hematologic evaluations were performed on blood samples collected from survivors at the end of the 14-week exposure or 4week recovery periods. Blood was obtained from the or-
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bital sinus of methoxyflurane-anesthetized animals. Hematologic evaluations were the same as for the 9-day animals. Serum chemistry determinations, performed on an Astra Automated Stat/Routine Analyzer (Beckman Instruments Inc., Brea, Cahf.), were glucose, urea nitrogen, creatinine, total and direct bilirubin, calcium, sodium, potassium, chloride, and carbon dioxide. A Centrifichem centrifugal analyzer (Baker Instruments, Pleasantville, N.Y.) was used to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase, creatine phosphokinase, y-glutamyl transferase, sorbitol dehydrogenase, alkaline phosphatase, total protein, albumin, and phosphorus. Animals that were moribund or found dead and survivors sacrificed at the end of the exposure regimen and recovery periods were subjected to necropsy. Organ weights were measured for liver, lungs, brain, heart, thymus, kidneys, and testes in survivors at scheduled sacrifices. Selected tissues for histologic evaluation were as follows: nasal turbinates, larynx, trachea, lungs, epididymides, testes, spleen, thymus, urinary bladder, adrenal glands, brain (5 sections), thyroid, parathyroids, heart, kidney, pituitary, skeietai muscle (gastrocnemius), sternal bone, spinal cord (lumhosacral region), and liver. Turbinates were decalcified for approximately 24 hr in 10% nitric acid before paraffin embedding. For ultrastructural evaluation of the sciatic and tibia1 nerves, five high concentration and five control male rats were sactificed at the end of the 14-week exposure and perfused through the left ventricle with saline followed by a buffered mixture of 4% formaldehyde and 1% glutaraldehyde. Because of the incidence of mortality in the high concentration group, animals designated for perfusion were not available at the 4-week recovery sacrifice. However, peripheral nerves were removed from five high concentration and five control male rats and immemionfixed in the 4% formaldehyde/l% glutaraldehyde solution for electron microscopy evaluation, Brains were paraffin embedded, sections were cut at 4 to 6 pm and stained with hematoxylin and eosin or a Bodian Silver Protargol-Luxol fast blue method. Histologic examination was performed on tissues from animals in the highest concentration and control groups. In addition, all selected tissues from females and brains from males of the intermediate concentration group were examined microscopically. Statisticalprocedures. Results of quantitative continuous variables were intercompared among the three concentration groups and the one control group by use of analysis of variance (Sokal and R&If, 1969), Bartlett’s homogeneity of variance (Sokal and Rohlf, 1969), and Duncan’s multiple range tests (Snedecor and Cochran, 1967). The latter was used to delineate which exposure groups differed from the control, when F from the analysis of variance was significant. If Bartlett’s test indicated heterogenous variances, all groups were compared by an analysis of variance for unequal variances (Brown and
332
DODD
Forsythe, 1974) followed, if necessary, by t tests. Corrected Bonferroni probabilities were used for t test comparisons. A fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.
ET AL. ‘90-l
IW-
RESULTS Nine-Day Study Gas chromatographic analysis of chamber atmosphere samples indicated the mean (*SD) of the daily average 2,4-PD concentrations to be 197 (+-2. l), 418 (+4.2), and 805 (k15.2) ppm. No 2,4-PD was observed in the control chamber. The analytical/nominal concentration ratios for the three test atmospheres ranged from 0.85 to 1.06, indicating no decompositional changes or significant chamber losses of metered 2,4-PD. No animals died, and the only signs were of sensory irritancy, presented as partial eyelid closure with periocular and perioral wetness, in three females of the 805ppm group. No signs of sensory irritancy were seen in female rats of the intermediate and low concentration groups, or in males of any group. Mean body weights are illustrated in Fig. 1. A decrease in body weight was observed during the exposure period for male and female rats of the 805-ppm group. However, during the 2-day rest period, body weights increased substantially. Male rats of the 4 18-ppm group had statistically significantly decreased body weight gains throughout the study (data not shown). This effect was only transient during the first week of exposure in the 4 18-ppm female rats (data not shown). Body weight was unaffected in the 197-ppm group. The hematology results indicated a mild (approximately 20% above control value) leukocytosis for rats of the 805-ppm group, which was mainly the result of an increase in lymphocytes. The only other statistically significant alterations in hematologic indices were slight increases in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration (male rats only). However, these alterations were not considered tox-
197 ppm .-418ppm ’ *. . 805 ppm
100
,2,4-PD 0
I 2
1-r 4
Study
Expooure
\ I 6
6
(1 10
12
Day
FIG. 1. Mean body weights of Fischer-344 rats during the 2,4-pentanedione 9-day study.
icologically significant because there was no effect in red blood cell count or in hemoglobin concentration. No hematological changes were found in the animals of the 197or 4 18-ppm groups. No treatment-related gross lesions were seen in any group at necropsy. Due to the decrease in body weight gain at 805 ppm, the weights of several organs (brain, liver, kidney, lung, and thymus) were lower than those of the controls. However, relative weights of most ofthese organs from the 805-ppm group were higher than control values, indicating that body weight decrease was more marked than decreases in absolute organ weights. A noteworthy exception to this trend was the relative thymus gland weights of the 805-ppm male and female rats, where a 25 to 43% decrease from respective mean control values was observed. In addition, the absolute weight of the thymus of the 418-ppm male rats was reduced in comparison with the controls. No treatment-related differences in organ weights were noted at 197 ppm.
2,4-PD 9-DAY AND ICWEEK TABLE 1 ANALYTICALCHAMBERCONCENTRATIONSFORTHE .&@%NTANEDIONE14-WEEK STUDY
Target concentration (ppm) Exposure week
100
9 10 II 12 13 14
103 + 101 f 103+ 102* 103+ 104+ 99* 99* 99& 98+ 99* 99+99? 99+
10” 2 1 2 1 2 2 1 1 0 1 2 2 1
Mean f SD of weekly means
101 f
2
2 3 4 6
8
300
650
282 f 23 3082 4 316~ 2 310* 2 3132 6 311? 8 3132 4 3122 3 306+ 3 307* 3 307+ 2 305 Ik 3 302 _t 1 302 -+ 2
605 f 39 651+ 5 663 + 7 654+ 4 661k 2 663 f 9 653 + 24 661+ 4 650~ 4 644+ 5 660 rf: 15 645-c 4 64lk 5 640 f 6
3072
650 + 15
8
a Weekly mean f SD concentration in ppm.
An exposure-related inflammation of the nasal mucosa, seen as multifocal areas of congestion, epithelial vacuolization, and lymphocyte and neutrophil infiltration of the submucosa, was present in all 2,4-PD exposed animals. Necrosis of the nasal mucosa was observed frequently in the 805-ppm rats, and occasionally in the 418-ppm animals, but was not observed in the 197-ppm group. Nasal mucosal lesions were most marked in the maxillary and nasal turbinates. Two male rats of the 805-ppm group had mild laryngitis, but no inflammatory changes were seen in the lower respiratory tract. Another two male rats of the 805ppm group had mild vacuolization of the brain stem. Fourteen- Week Study Analytically determined concentrations of 2,4-PD vapor in the exposure chambers were close to target values (Table 1). No 2,4-PD
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was detected in the air control chamber. The analytical/nominal concentration ratios for the three test atmospheres ranged from 0.89 to 0.95, indicating no decompositional changes or significant chamber losses of metered 2,4-PD. Deaths occurred only at 650 ppm, with all females and 10 of the 30 males dying between the second and sixth week of exposure. The earliest death was after 9 days of exposure. General signs of toxicity and sensory irritancy, seen at 650 ppm, were complete or partial eyelid closure, periocular, perinasal, and perioral encrustation, wetness around the urogenital area, hypoactivity, lack of coordination, paresis, ataxia, irregular gait, hypothermia, and emaciation. There were no exposure-related ophthalmic findings. In the Irwin neurobehavioral screen, effects were noted only at 650 ppm (Table 2). Due to early mortality, only two female rats had the first monthly examination (data not shown). Although neurobehavioral effects were observed in several of the 650-ppm exposed males, the majority of those surviving the first month of exposure did not exhibit neurobehavioral abnormalities when retested at 2 and 3 months. Also, there were no treatment-related signs following the 4-week postexposure recovery period. Body weights of the 650-ppm males were reduced throughout the 14-week exposure period, but there was considerable weight increase during the 4-week recovery period (Fig. 2). However, final weights were still statistically significantly below those for the control males. Before death, body weights of the female rats of the 650-ppm group were considerably reduced (Fig. 2). Also, females of the 307-ppm group had slight, but statistically significant, decreased body weight gains for study Days 45 through 121. However, at the conclusion of the 4-week recovery period, the difference between the absolute body weight means for the control and 307-ppm female rats was not statistically significant. Body weights of the lOl-ppm-exposed rats were similar to the controls. No treatment-
334
DODD ET AL.
related alterations in food and water intake were observed at the conclusion of the 14week exposure regimen. Thus, the 650-ppm male survivors had normal appetites and gained weight rapidly during the 4-week recovery period (Fig. 2). Only slight changes in some hematologic parameters were found at the end of the 14week exposure period. For the 650-ppm males and the 307-ppm females, the respective mean erythrocyte counts were 11 and 4% below control values (Fig. 3). The decrease in red blood cell count was accompanied with slight increases in MCV and MCH and a mild decrease in hematocrit. The white blood cell count was increased in the 650-ppm male rats (Fig. 3) due to an increase in lymphocytes. There was recovery from these effects by the end of the 4-week postexposure period. Clinical chemistry determinations which deviated from controls were an increase in urea nitrogen and alkaline phosphatase activity, and a decrease in creatinine, calcium, and aspartate
aminotransferase (AST) activity in the 650ppm males (Fig. 4). Serum calcium was also decreased in males and females of the 307ppm group. Although a mild increase in alkaline phosphatase activity persisted in the 650ppm recovery male rats, the remaining serum chemistry values were similar to control values, indicating reversibility of effects. Urinalysis showed minimal alterations in males, which included low pH (6.0 vs 7.0 in controls) at 650 ppm, and slightly increased bilirubin and urobilinogen at 650 and 307 ppm. These alterations in urine parameters were not found at the end of the postexposure observation period. After 1Cweeks of 2,4-PD vapor exposure, a statistically significant decrease in most absolute organ weights was observed for the 650-ppm males, but organ weights relative to body weight had increased (Table 3). This suggests that organ weight alterations were a reflection of decreased body weight. Organ weights of the 10 1- and 307-ppm groups were
TABLE 2 NEUROBEHAVIORALFINDINGSINMALERATSEXPOSEDTO~~O
ppm OF&~-PENTANEDIONEVAPOR~
Duration of exposure regimen (months) Observation
1
2
3
3 + 1 month recovery
Tremors Impaired gait Paresis Lacrimation Hypothermia Abnormal surface righting Abnormal midair righting Abnormal wire grasping Abnormal body tone Abnormal limb rotation Dilated pupils Decreased breathing rate Decreased locomotor activity Absence of comeal reflex Absence of tail pinch response Absence of auditory startle response
l/26b 4126 4126 2126 3126 2126
o/20 o/20 o/20 l/20 o/20 o/20
O/20 2120 o/20 O/20 O/20 O/20
015 015 015 015 O/5 O/5
6126 4126 6126 3126 6126 2126 6126 2126 3126 6126
3120 0120 0120 0120 0120 0120 0120 0120 0120 0120
4120 O/20 0120 0120 0120 0120 0120 O/20 O/20 0120
O/5 O/5 O/5 O/5 O/5 O/5 o/5 o/5 O/5 o/5
’ No effectswere observed in the control rats or rats exposed to 10 1 or 307 ppm. b No. affected/No. examined.
2,4-PD 9-DAY AND 1CWEEK 370
270
250 E
1
6,
210
190
170
Isa
-
-101 ---307ppm . . ..osoppm
0 wm ppm
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335
thy histopathologic features in brain, thymus, skin, and nasal mucosa. The brain lesions observed in 7 of 10 male and 18 of 20 female rats that died during the exposure regimen took the form of acute degenerative changes in the vestibular nuclei, deep cerebellar nuclei, and corpora striata. The lesions consisted of peracute to acute areas of pallor and vacuolation in the neuropil. Within these foci were nuclear pyknosis and karyorrhexis, and the presence of macrophages with vacuolated cytoplasm. The degree of vacuolation and macrophage infiltration was greatest in the deep cerebellar and vestibular nuclei. These brain lesions were bilaterally symmetrical. Of the surviving males of the 650-ppm group, 7 of 15 had gliosis with or without malacia in the deep cerebellar nuclei, vestibular nuclei, and corpus striatum. Four of the 7 affected surviving males were from the 10 sacrificed at 14 weeks, and the other 3 were from the 5 sacrificed following the 4-week re-
130
iia
FIG. 2. Mean body weights of Fischer-344 rats during the 2,4pentanedione 1Cweek exposure plus 4-week recovery study.
unaltered, except for absolute lung weights of the females which were slightly increased at 101 and slightly decreased at 307 ppm; this was not considered to be of biological importance. Organ weights of the 4-week recovery animals followed a pattern generally similar to that of the 14-week sacrifice. However, due to the substantial body weight gain in the 650-ppm males during the postexposure period, the magnitude of the increases in relative organ weights when compared to control values was much reduced in the recovery animals (Table 3). Light microscopy did not reveal any abnormalities in tissues removed from the animals of the 10 1- and 307-ppm groups. However, for the 650-ppm animals there were notewor-
FIG. 3. Hematologic values of Fischer-344 rats following the 2,4pentanedione 14-week study. WBC = white blood cells; RBC = red blood cells; HCT = hematocrit; MCH = mean corpuscular hemoglobin; MCV = mean corpuscular volume.
336
DODD ET AL.
Km
80
60
Ak
Phce.
AST
FIG. 4. Serum chemistry values of Fischer-344 rats following the 2,4-pentanedione 1Cweek study. Urea N = urea nitrogen; Alk. Phos. = alkaline phosphatase; Ca = calcium; AST = aspartate aminotransferase.
cover-y period. (Another five 650-ppm male survivors had been sacrificed at 14 weeks for glutaraldehyde perfusion and subsequent electron microscopic examination of sciatic nerves.) Gliosis was most extensive in the deep cerebellar and vestibular nuclei. Many of the cells had irregular or rod-shaped nuclei typical of microglia, but others resembled astrocytes. Multifocal areas of microscopic mineralization, hemosiderosis, and neuronal degeneration were evident. No degenerative changes were seen in the spinal cords of the 650-ppm exposed animals. Transmission electron microscopic evaluation of sciatic nerves from the 650-ppm group did not produce any evidence of a peripheral neuropathy. There was excellent agreement between the results of the Irwin neurobehavioral screen (Table 2) and the microscopic findings in the brain. For example, the location of the brain lesion (cerebellar and vestibular nuclei) cor-
related with those Irwin screen assessments related to equilibrium (abnormal midair righting reflex and impaired gait). Furthermore, every animal that exhibited an abnormality during one of the examination periods had brain lesions. Seven of fifteen 650-ppm male survivors had brain lesions, and five of these showed either abnormal midair righting reflex or impaired gait at the conclusion of the 14-week exposure regimen. The failure of some rats to show neurologic deficits during the Irwin screen probably reflects the insensitivity of the test. Thymic lesions, consisting of acute lymphoid degeneration and atrophy, were observed in 7 of 10 male and 13 of 20 female rats that died during the lbweek exposure regimen. They were not present in survivors. Dermatitis, dermal necrosis, and in a few cases, cellulitis were seen in the skin and subcutis of the heads of 5 male and 8 female rats that died, but not in survivors. The inflammatory infiltrate was predominately of polymorphonuclear leukocytes and mast cells. The nasal mucosa showed mild multifocal squamous metaplasia at the anterior portion of the nasal cavity, in both the maxillary and nasal turbinates. This change was seen in 9 of the 10 animals exposed to 650 ppm which were sacrificed at the end of the 1Cweek exposure period, but was present in only one of five and with reduced severity following the 4-week recovery period. The nasal mucosa was normal in female rats exposed to 307 ppm of 2,4-PD vapor. DISCUSSION In the 1Cweek study, death in the 650ppm-exposed rats was attributed to brain lesions in the majority of cases. The exposure conditions to cause death by inhalation of 2,4-PD vapor appear to be well demarcated with respect to concentration and exposure time. For example, malacia within the brain was not observed in the 1Cweek study until study Day 16. Although one female rat ex-
2,4-PD 9-DAY AND ICWEEK
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337
TABLE 3 ORGANWEIGHTSASAPERCENTAGEOFBODYWTFORMALEFISCHER-~~~RATS FOLLOWINGTHE2,4-&NTANEDIONEl4-WEEKSTUDY
2,4PD concentration (ppm) Organ Brain Liver Kidney Heart Lungs Thymus Testes
Time (Weeks)
0
101
307
6.50
14 18 14 18 14 18 14 18 14 18 14 18 14 18
0.57 (0.03)b 0.55 (0.02) 3.07 (0.12) 2.90 (0.12) 0.64 (0.02) 0.65 (0.03) 0.27 (0.02) 0.28 (0.02) 0.43 (0.03) 0.37 (0.04) 0.10 (0.02) 0.10(0.01) 0.94 (0.03) 0.9 l(O.04)
0.56 (0.02) 0.53 (0.03) 3.02 (0.09) 2.99 (0.16) 0.64 (0.03) 0.64 (0.02) 0.27 (0.01) 0.28 (0.01) 0.4 1 (0.02) 0.36 (0.04) 0.11 (0.03) 0.10 (0.02) 0.93 (0.04) 0.88 (0.02)
0.57 (0.03) 0.54 (0.03) 3.20 (0.15) 3.01 (0.11) 0.65 (0.03) 0.64 (0.02) 0.28 (0.01) 0.28 (0.0 1) 0.42 (0.04) 0.39 (0.04) 0.10 (0.02) 0.09 (0.01) 0.95 (0.05) 0.89 (0.03)
0.73 (0.05)c 0.61 (0.02) 3.64 (0.14) 3.27 (0.07)c 0.75 (0.05) 0.70 (0.04) 0.32 (0.02) 0.29 (0.01) 0.48 (0.05) 0.4 1 (0.04) 0.10 (0.04) 0.11 (0.02) 1.26 (0.10) 1.09 (0.08)
D 14 weeks = end of exposure period; 18 weeks = end of recovery period. b All values are means (SD); units are in %. ‘P < 0.05 compared to control value.
posed to 650 ppm of 2,4-PD died on Day 9, no lesions were seen microscopically in the brain of this animal. Consistent with these results was the lack of mortality and the absence of malacia within the brain in any of the 9-day study animals. However, two male rats of the 805-ppm group had mild vacuolization of the brain stem, which may indicate that had 2,4-PD exposures continued into a third week, more prominent brain lesions may have developed. Thus, the mechanism(s) of lethal toxicity appear to have a well-dehned threshold and may be based on a saturation process. The most noticeable feature in this study was the presence of degenerative changes in the deep cerebellar and vestibular nuclei and the corpora striata in animals that died following repeated exposures to 650 ppm of 2,4PD vapor. Additionally, gliosis and malacia were seen in the brains of about half the 650ppm animals that survived. Central neuropathologic lesions have also been described by Krasavage et al. (1982) who gave rats 100
to 150 mg/kg of 2,4-PD twice daily by gavage for periods from 3 to 6 1 days. Animals developed weakness, ataxia, tremors, paresis, and rolling movements of the head. Histologic changes induced by the shorter term dosing procedures were perivascular edema, hemorrhage into the Virchow-Robin spaces, and endothelial cell swelling, all primarily localized in the cerebellum and the brainstem. For the more prolonged dosing schedules there were bilateral, symmetrical foci of malacia and gliosis in the cerebellar peduncles, olivary nuclei and lower brainstem. Lesions were not seen in the basal ganglia, and there was no evidence of peripheral nerve involvement. Likewise, in the current studies by respiratory exposure, there was no ultrastructural evidence for a peripheral neuropathy. Thus, the changes seen in the rats following repeated gavage with 2,4-PD are similar to those seen by recurrent exposure to high concentrations of 2,4-PD vapor, and are clinically and morphologically distinct from those produced by y-diketones (O’Donoghue,
338
DODD ET AL.
1985). Furthermore, the central neuropathologic lesions induced by 2,4-PD appear species dependent since rabbits did not develop these lesions following repeated gavage administration (Krasavage et al., 1982). The central neuropathologic lesions resulting from repeated exposure to 2,4-PD have been compared with those produced by acute vitamin B complex deficiency (Krasavage et al., 1982) and related to the known ability of 2,4-PD to inactivate the lysyl residue of enzymes, some of which are related to coenzyme formation (Gilbert and O’Leary, 1975; Otwell et al., 1979). Gilbert and O’Leary (1977) have observed that, in vitro, 2,4-PD is highly specific for lysine residues of porcine aspartate aminotransferase (AST). The decrease in AST activity in the present investigation (Fig. 4) may indicate in vivo modification of AST following 2,4-PD exposure. The primary region of brain damage in rats with experimentally induced thiamine deficiency is the lateral vestibular nucleus (Dreyfus, 1973; McCandless, 1982), one of the areas in which major damage is seen after 2,4PD exposure. Although degenerative lesions have not been described in the basal ganglia of thiamine deficient rats, such lesions have been seen in rhesus monkeys given diets deficient in thiamine (Blank et al., 1975). The similarity of the lesions induced in rat brains by thiamine deficiency, together with the enzyme inactivation effects of 2,4-PD, indicate that the 2,CPD-induced brain lesion is likely to be due, in part at least, to an interruption of normal thiamine biochemistry in specific regions of the brain. This would accord with the sharply defined exposure conditions for the neurological toxicity of 2,4-PD. Furthermore, interanimal variation of thiamine concentration, turnover, and/or requirement in specific brain regions may explain the sex-related differences in mortality observed in the current study and the absence of brain lesions in eight of fifteen 650-ppm-exposed male rats that survived the 1Cweek exposure regimen. The changes observed in the thymus gland (a decrease in weight in the 9-day study and
acute lymphoid degeneration in the 14-week study) were probably stress related because they were observed only in animals exposed to high concentrations of 2,4-PD (650 to 805 ppm) and that died during the exposure regimen ( 14-week study). No thymic lesions were observed in the 9-day 805-ppm-exposed rats or in the 650 ppm survivors of the 1Cweek study. Dose-related inflammatory lesions of the nasal mucosa were noted at all concentrations of 2,4-PD in the 9-day study, although the effects were marginal at 197 ppm. With the subchronic study, a mild squamous metaplasia, reversible on cessation of exposure, was seen at 650 ppm, but no nasal mucosal lesions were observed at 307 ppm. These findings suggest that nasal mucosal inflammation is not a significant biological effect until concentrations greater than 300 ppm are attained. The pattern of toxicity in the subchronic study indicates a steep slope on the dose response for 2,4-PD. Thus, although 650 ppm was a concentration by which repeated exposure caused severe toxicity, at 307 ppm there were no histopathologic changes or clinical abnormalities, and the only effects were minor alterations in body weight and serum chemistry which were reversible during the 4week postexposure recovery period. No effects were seen at 10 1 ppm. In the context of possible human exposures, adverse effects are likely to occur only at concentrations significantly in excess of the odor threshold of 0.01 ppm (Ballantyne et al., 1986). Thus, potentially hazardous atmospheres would be intolerable to humans. The above considerations indicate that by subchronic exposure to 2,4-PD vapor the minimum-effects concentration is 307 ppm and the no-adverse effects concentration is 101 ppm. ACKNOWLEDGMENTS The authors are grateful to P. E. Losco for her participation in this study and to F. C. Wilt for her assistance in typing this manuscript.
2,4-PD 9-DAY AND ICWEEK
REFERENCES BALLANTYNE, B., DODD, D. E., MYERS, R. C., AND NACHREINER, D. J. (1986). The acute toxicity and primary initancy of 2,4pentanedione. Drug. Chem. Toxicoi., in press. BLANK, N. K., VICK, N. A., AND SCHULMAN, S. (1975). Wenicke’s encephalopathy, an experimental study in the rhesus monkey. Acta Neuropathol. 31, 137-150. BROWN, M. B., AND FORSYTHE, A. B. (1974). The smaII sample behavior ofsome statistics which test the equality of several means. Technometrics 16, 129- 132. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., SULLIVAN, L. J., AND KING, J. M. (1975). Petroleum hydrocarbon toxicity studies. I _ Methodology. Toxicol. Appl. Pharmacol.
32,246-262.
DREYFUS, P. M. (1973). Thoughts on the pathophysiology of Wemicke’s disease. Ann. N. Y. Acad. Sci. 215, 367-369.
GILBERT, H. F. III, AND O’LEARY, M. H. (1975). Modification of arginine and Iysine in proteins with 2,4-pentanedione. Biochemistry 14,5 194-5 198. GILBERT, H. F. III, AND O’LEARY, M. H. (1977). Reversible modification of amino groups in aspartate aminotransferase. Biochim. Biophys. Acta 483,79-89. HAWLEY, G. G. (1977). The Condensed Chemical Dictionary, pp. 7-8. Van Nostrand-Reinhold, New York.
INHALATION
339
STUDIES
IRWIN, S. (I 968). Comprehensive observation assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. PsychopharmacoIogia 13,222-257. KRASAVAGE, W. J., G’DONOGHUE, J. L., AND DrVINCENZO, G. D. (1982). 2,CPentandione. In Patty’s Industrial Hygiene and Toxicology (G. D. Clayton and F. E. Clayton, eds.), Vol. 2C, pp. 4773-4776. Wiley, New York. MCCANDLESS, D. W. (I 982). Energy metabolism in the lateral vestibular nucleus in pyrithiamin-induced thiamin deficiency. Ann. N. Y. Acad. Sci. 378,335-364. G’DONOGHUE, J. L. (1985). Diketone neurotoxicity. In Neurotoxicity cals (J. L.
of Industrial
and Commercial
Chemi-
G’Donoghue, ed.), Vol. 2, pp. 77-78. CRC Press, Boca Raton, FIa. OTWELL, H. B., CIPOLLO, K. L., AND DUNLAP, R. B. (1979). Modification of Iysyl residues of dihydrofolate reductase with 2,4pentanedione. Biochim. Biophys. Acta 568,297-306.
SAX, N. I. (1979). Dangerous Properties of Industrial Materials, p. 890. Van Nostrand-Reinhold, New York. SNEDECOR,G. W., AND COCHRAN, W. G. (1967). Statistical Methods, pp. 271-275. Iowa State Univ. Press, Ames. SOKAL, R. R., AND ROHLF, F. J. (1969). Biometry, pp. 204-2 16,369-376. Freeman, San Francisco.
AND
APPLIED
TOXICOLOGY
( 1986)
7,329-339
2,dPentanedione: g-Day and 14-Week Vapor Inhalation Studies in Fischer-344 Rats D. E. DODD,’ R. H. GARMAN, I. M. PRITTS, C. M. TROUP, W. M. SNELLINGS, AND B. BALLANTYNE* Bushy Run Research and *Union
Center, Carbide
R.D. 4. Mellon Road, Export, Pennsylvania 15632, Corporation, Danbury, Connecticut 068I 7
2,4-Pentanedione: 9-Day and 14-Week Vapor Inhalation Studies in Fischer-344 Rats. DODD, D. E., GARMAN,
R. H., PRITTS,
I. M.,
TROUP,
C. M.,
SNELLINGS,
W.
M.,
AND
BALLANTYNE,
B. (1986). Fundam. Appf. Toxicol. 7, 329-339. Fischer-344 rats, in groups of 10 males and 10 females, were exposed for 9 days (6 hr/day) to 2,4-pentanedione (2,4-PD) vapor at mean concentrations of 805,418, 197, and 0 (control) ppm. No deaths occurred, and the only adverse signs were of sensory irritation (partial closure of eyelids, periocular and perioral wetness) at 805 ppm. Also at 805 ppm were decreased body and organ weights, lymphocytosis, and moderate inflammation of the nasal mucosa. At 4 18 ppm there was a decrease in body weight gain and mild inflammation ofthe nasal mucosa. Apart from minimal nasal mucosal inflammation, there were no effects at 197 ppm. In the subchronic (14-week) study, rats were exposed (6 hr/day; 5 days/week) to 650,307, 101, and 0 (control) ppm of 2,4-PD vapor, using groups containing 20 males and 20 females, with halfbeing sacrificed at the end ofthe exposure period and the remainder kept for a 4-week postexposure recovery period. An additional 10 males were added to the 650 and 0 ppm groups for glutaraldehyde perfusion and subsequent electron microscopic examination of sciatic nerves. At 650 ppm, all females and 10 of 30 males died between the second and sixth weeks of exposure. These animals had acute degenerative changes in the deep cerebellar nuclei, vestibular nuclei and corpora striata, and acute lymphoid degeneration in the thymus. Seven of 15 male survivors of the 650 ppm group (combined 14-week and recovery sacrifices) had gliosis and malacia in the same brain regions, minimal squamous metaplasia in the nasal mucosa, decreased body and organ weights, lymphocytosis, and minor alterations in serum and urine chemistries. No ultrastructural evidence of peripheral neuropathy was observed. Except for central neuropathy, many of the adverse effects at 650 ppm were less marked in the 4-week recovery animals. No deaths occurred at 307 ppm, but females had slightly decreased body weight gains, and in both sexesthere were minor alterations in hematology, serum chemistry, and urinalysis parameters, which were not present in the 4-week recovery animals. Rats exposed to 10 1 ppm showed no differences from the control rats. Subchronic exposure to 650 ppm of 2,4-PD vapor causes serious adverse biological effects. Under these study conditions, the minimum-effects concentration was 307 ppm, and the no-adverse effects concentration was 101 ppm. 0 1986 SocietyofToxicology.
2,4-pentanedione (2,4-PD: CAS No. 123-546), a liquid P-diketone, is used as a chemical intermediate, metal chelator, and lubricant additive (Hawley, 1977). Results of acute studies indicate 2,4-PD to be of moderately high peroral and percutaneous toxicity, ’ To whom correspondence and requests for reprints should be addressed.
mildly irritating to the skin, and moderately irritating to the eye (Ballantyne et al., 1986). With a vapor pressure of 7 mm Hg at 20°C (Sax, 1979), the 4-hr LC50 of 2,4-PD vapor for rats is 1224 (1063- 1409) ppm, and LT50 values for exposure to vapor-saturated atmospheres have been determined to be 52 min (7060 ppm) for male rats and 55 min (7912 ppm) for female rats (Ballantyne et al., 1986). 329
0272-0590186 $3.00 Copyright 0 1986 by the society of Toxicology. All rights of reproduction in any form reserved.
DODD ET AL.
Repeated dosing of 2,4-PD to rats by gavage causes gastric erosions, thymic necrosis, and central nervous system injury characterized by perivascular edema and hemorrhage, endothelial swelling, and bilateral foci of malacia and gliosis in the cerebellar peduncles, olivary nuclei and lower brain stem (Krasavage et al., 1982). In view of the vapor pressure of 2,4-PD, its known toxicity, and the potential for occupational exposure, it was appropriate to investigate the short-term repeated and subchronic inhalation toxicity of 2,4-PD vapor at ambient temperature and pressure. METHODS Test material. 2,4-PD was obtained from Union Carbide Corporation, South Charleston, West Virginia. The test samples were kept in 1-gal glass bottles and blanketed with nitrogen. Both before and after the 9-day and 14week studies, the material was analyzed by gas chromatography for composition and found to be 99% pure. Animals and exposuregroups. Male and female Fischer-344 rats, 34 to 38 days of age, were obtained from Charles River Breeding Laboratories, Inc., Kingston, New York. They were confirmed to be free from intestinal parasites, and on the basis of histologic examination of tissues from selected animals, did not have any common rodent diseases. They were clinically normal upon receipt and during a 2-week acclimatization period prior to the start of exposures. Rats were uniquely numbered by toe clipping and ear notching procedures. They were assigned to exposure groups using a computer-based randomization program. At the time of assignment, only animals having body weights within 2 SD of the group means for each sex were used. Animals were housed, two per cage, in stainless-steel wire-mesh cages. They were allowed free access to food (Purina Certified Rodent Chow #5002, Ralston Purina Co., St. Louis, MO.) and water during the nonexposure periods. Animals were kept on a 12-hr light-dark cycle. For the 9-day study, four groups, each containing 10 male and 10 female rats, were exposed for 6 hr/day to 3 different target concentrations of 2,4-PD vapor and one air control atmosphere. For the subchronic study, there were also three different target concentrations of 2,4-PD vapor and one air control group. There were 20 males and 20 females for each exposure group, with half the animals to be sacrificed after the final exposure. The remaining animals were kept as recovery animals for 4 weeks after the final exposure and sacrificed. An additional 10 males were added to the high dose and control groups for glutaraldehyde perfusion and subsequent
transmission electron microscopic examination of sciatic and tibiaf nerves. Exposure conditions. Exposures for the 9&y study were conducted in 1.3 m3 chambers with an airflow of 300 liters/min, and for the subchronic study, chambers with a volume of 4.3 m3 operated at an airflow rate of 1000 liters/min were used. The chambers were constructed of stainless steel and had glass observation ports. Temperature and relative humidity were measured at least four times during each exposure. For all nine exposures, the range of daily mean temperature and relative humidity values was 23 to 24°C and 40 to 48%, respectively. During any individual 6 hr exposure, the temperature and relative humidity did not vary more than 2°C and 8%, respectively. For the 9-day study, the target concentrations were 200,400, and 800 ppm. Exposures were for 6 hr/day for 5 consecutive days, and after a 2-day nonexposure period, exposures were resumed for 4 consecutive days. Based on the results of this study, the target concentrations for the subchronic study were chosen at 100, 300, and 650 ppm. Exposures were for 6 hr/day, 5 days a week for 14 weeks, with some animals being sacrificed during the 14th week. The number of 6-hr exposures was 67 for male rats and 68 for females rats. The position of cages in the exposure chambers was rotated on a weekly basis to compensate for any possible, but not detected, variation in chamber environment or 2.4-PD vapor concentration. 2,4-PD vapor was generated by metering the test material with a piston pump (Fluid Metering Inc., Oyster Bay, N.Y.) into a heated glass evaporator (Carpenter et al., 1975) whose temperature was maintained at the lowest level sufficient to vaporize the liquid. The vapor was carried into the exposure chamber using a diluting countercurrent airstream entering the bottom of the evaporator. Chamber concentrations of 2,4-PD vapor were analyzed approximately once every 33 min during the 6-hr exposure periods using a Perkin-Elmer Model 3920B gas chromatograph equipped with a flame ionization detector. The GC column used was a 5 ft X 4 in stainless steel tube packed with SP2 100 on 80/100 mesh Supelcoport@ and maintained at 140°C. Chamber atmosphere samples were automatically injected into the CC with the aid of a Perkin-Elmer Environmental Sampler and vacuum pumps attached to Teflon-lined, polyethylene sample lines. The CC was calibrated by either dynamically generated gas standards, using a glass diffusion tube and a VICI Metronics Dynacalibrator 450, or injection of gas bag standards, which were prepared by syringe injection of the test material into Tedlar gas bags. A series of gas standards encompassing the entire range of vapor concentrations generated were prepared. A linear calibration curve was obtained when integration counts were plotted against the vapor concentrations of the standards. The calibration was checked at least once weekly. Daily nominal concentrations of 2,4-PD vapor were calculated by
2,4-PD 9-DAY AND ICWEEK taking the amount of 2,4-PD delivered and dividing it by the total volume of chamber air. Biological monitoring. In the 9-day study, animals were observed for toxicologic and pharmacologic signs prior to, during, and following each exposure. Body weights were measured just before the first exposure, preceding the second, fifth, sixth, and seventh exposures, and just before sacrifice. Blood for hematologic evaluations was obtained from the orbital sinus on the day of sacrifice. Standard laboratory procedures were used for the following determinations: total white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, and differential leukocyte count. All surviving animals were sacrificed the day following the final exposure by exsanguination following methoxyflurane anesthesia. Necropsy was performed, and weights of the brain, liver, kidneys, lungs, heart, thymus, and testes were determined. The following tissues were removed and fixed in 10% neutral buffered Formalin: nasal turbinates, larynx, trachea, lungs, spleen, liver. kidneys, ovaries, testes, thymus, brain (5 levels), and all gross lesions. Histologic examination was performed on all tissues from the high concentration and control groups. In addition, nasal turbinates from the intermediate and low concentration groups were examined microscopically. In the subchronic inhalation study, animals were observed daily for signs of toxicity. Prior to the first exposure and at sacrifice, the anterior segment of the eye was examined ophthalmoscopically. An Irwin screen (Irwin, 1968) was performed prior to the tint exposure and monthly thereafter, including just before sacrifice of the recovery animals. The neurobehavioral screen did not include all the assessments discussed by Irwin (1968), and evaluations were not scored. Body weights were measured the morning before the first exposure, weekly during the exposure period, and immediately before sacrifice. Animals ofthe 4-week recovery period were weighed weekly and immediately before sacrifice. Individual animal food and water consumption was measured for approximately 15 hr in metabolism cages with 10 males and 10 females from each exposure concentration; measurements were made during exposure week 14. Urine was collected while animals were in the metabolism cages. Urine volume, color, and turbidity were recorded, and semiquantitative measurements were made of pH, glucose, ketones, protein, bilirubin, and urobilinogen using Ames Multi-stix@ Reagent Strips read on a Clini-tek (Ames Division, Miles Laboratories, Elkhart, Ind.). Urine osmolality was determined with an Advanced Cryomatic Osmometer (Advanced Instruments Inc., Needham Heights, Mass.). Serum chemistry and hematologic evaluations were performed on blood samples collected from survivors at the end of the 14-week exposure or 4week recovery periods. Blood was obtained from the or-
INHALATION
STUDIES
331
bital sinus of methoxyflurane-anesthetized animals. Hematologic evaluations were the same as for the 9-day animals. Serum chemistry determinations, performed on an Astra Automated Stat/Routine Analyzer (Beckman Instruments Inc., Brea, Cahf.), were glucose, urea nitrogen, creatinine, total and direct bilirubin, calcium, sodium, potassium, chloride, and carbon dioxide. A Centrifichem centrifugal analyzer (Baker Instruments, Pleasantville, N.Y.) was used to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase, creatine phosphokinase, y-glutamyl transferase, sorbitol dehydrogenase, alkaline phosphatase, total protein, albumin, and phosphorus. Animals that were moribund or found dead and survivors sacrificed at the end of the exposure regimen and recovery periods were subjected to necropsy. Organ weights were measured for liver, lungs, brain, heart, thymus, kidneys, and testes in survivors at scheduled sacrifices. Selected tissues for histologic evaluation were as follows: nasal turbinates, larynx, trachea, lungs, epididymides, testes, spleen, thymus, urinary bladder, adrenal glands, brain (5 sections), thyroid, parathyroids, heart, kidney, pituitary, skeietai muscle (gastrocnemius), sternal bone, spinal cord (lumhosacral region), and liver. Turbinates were decalcified for approximately 24 hr in 10% nitric acid before paraffin embedding. For ultrastructural evaluation of the sciatic and tibia1 nerves, five high concentration and five control male rats were sactificed at the end of the 14-week exposure and perfused through the left ventricle with saline followed by a buffered mixture of 4% formaldehyde and 1% glutaraldehyde. Because of the incidence of mortality in the high concentration group, animals designated for perfusion were not available at the 4-week recovery sacrifice. However, peripheral nerves were removed from five high concentration and five control male rats and immemionfixed in the 4% formaldehyde/l% glutaraldehyde solution for electron microscopy evaluation, Brains were paraffin embedded, sections were cut at 4 to 6 pm and stained with hematoxylin and eosin or a Bodian Silver Protargol-Luxol fast blue method. Histologic examination was performed on tissues from animals in the highest concentration and control groups. In addition, all selected tissues from females and brains from males of the intermediate concentration group were examined microscopically. Statisticalprocedures. Results of quantitative continuous variables were intercompared among the three concentration groups and the one control group by use of analysis of variance (Sokal and R&If, 1969), Bartlett’s homogeneity of variance (Sokal and Rohlf, 1969), and Duncan’s multiple range tests (Snedecor and Cochran, 1967). The latter was used to delineate which exposure groups differed from the control, when F from the analysis of variance was significant. If Bartlett’s test indicated heterogenous variances, all groups were compared by an analysis of variance for unequal variances (Brown and
332
DODD
Forsythe, 1974) followed, if necessary, by t tests. Corrected Bonferroni probabilities were used for t test comparisons. A fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.
ET AL. ‘90-l
IW-
RESULTS Nine-Day Study Gas chromatographic analysis of chamber atmosphere samples indicated the mean (*SD) of the daily average 2,4-PD concentrations to be 197 (+-2. l), 418 (+4.2), and 805 (k15.2) ppm. No 2,4-PD was observed in the control chamber. The analytical/nominal concentration ratios for the three test atmospheres ranged from 0.85 to 1.06, indicating no decompositional changes or significant chamber losses of metered 2,4-PD. No animals died, and the only signs were of sensory irritancy, presented as partial eyelid closure with periocular and perioral wetness, in three females of the 805ppm group. No signs of sensory irritancy were seen in female rats of the intermediate and low concentration groups, or in males of any group. Mean body weights are illustrated in Fig. 1. A decrease in body weight was observed during the exposure period for male and female rats of the 805-ppm group. However, during the 2-day rest period, body weights increased substantially. Male rats of the 4 18-ppm group had statistically significantly decreased body weight gains throughout the study (data not shown). This effect was only transient during the first week of exposure in the 4 18-ppm female rats (data not shown). Body weight was unaffected in the 197-ppm group. The hematology results indicated a mild (approximately 20% above control value) leukocytosis for rats of the 805-ppm group, which was mainly the result of an increase in lymphocytes. The only other statistically significant alterations in hematologic indices were slight increases in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration (male rats only). However, these alterations were not considered tox-
197 ppm .-418ppm ’ *. . 805 ppm
100
,2,4-PD 0
I 2
1-r 4
Study
Expooure
\ I 6
6
(1 10
12
Day
FIG. 1. Mean body weights of Fischer-344 rats during the 2,4-pentanedione 9-day study.
icologically significant because there was no effect in red blood cell count or in hemoglobin concentration. No hematological changes were found in the animals of the 197or 4 18-ppm groups. No treatment-related gross lesions were seen in any group at necropsy. Due to the decrease in body weight gain at 805 ppm, the weights of several organs (brain, liver, kidney, lung, and thymus) were lower than those of the controls. However, relative weights of most ofthese organs from the 805-ppm group were higher than control values, indicating that body weight decrease was more marked than decreases in absolute organ weights. A noteworthy exception to this trend was the relative thymus gland weights of the 805-ppm male and female rats, where a 25 to 43% decrease from respective mean control values was observed. In addition, the absolute weight of the thymus of the 418-ppm male rats was reduced in comparison with the controls. No treatment-related differences in organ weights were noted at 197 ppm.
2,4-PD 9-DAY AND ICWEEK TABLE 1 ANALYTICALCHAMBERCONCENTRATIONSFORTHE .&@%NTANEDIONE14-WEEK STUDY
Target concentration (ppm) Exposure week
100
9 10 II 12 13 14
103 + 101 f 103+ 102* 103+ 104+ 99* 99* 99& 98+ 99* 99+99? 99+
10” 2 1 2 1 2 2 1 1 0 1 2 2 1
Mean f SD of weekly means
101 f
2
2 3 4 6
8
300
650
282 f 23 3082 4 316~ 2 310* 2 3132 6 311? 8 3132 4 3122 3 306+ 3 307* 3 307+ 2 305 Ik 3 302 _t 1 302 -+ 2
605 f 39 651+ 5 663 + 7 654+ 4 661k 2 663 f 9 653 + 24 661+ 4 650~ 4 644+ 5 660 rf: 15 645-c 4 64lk 5 640 f 6
3072
650 + 15
8
a Weekly mean f SD concentration in ppm.
An exposure-related inflammation of the nasal mucosa, seen as multifocal areas of congestion, epithelial vacuolization, and lymphocyte and neutrophil infiltration of the submucosa, was present in all 2,4-PD exposed animals. Necrosis of the nasal mucosa was observed frequently in the 805-ppm rats, and occasionally in the 418-ppm animals, but was not observed in the 197-ppm group. Nasal mucosal lesions were most marked in the maxillary and nasal turbinates. Two male rats of the 805-ppm group had mild laryngitis, but no inflammatory changes were seen in the lower respiratory tract. Another two male rats of the 805ppm group had mild vacuolization of the brain stem. Fourteen- Week Study Analytically determined concentrations of 2,4-PD vapor in the exposure chambers were close to target values (Table 1). No 2,4-PD
INHALATION
STUDIES
333
was detected in the air control chamber. The analytical/nominal concentration ratios for the three test atmospheres ranged from 0.89 to 0.95, indicating no decompositional changes or significant chamber losses of metered 2,4-PD. Deaths occurred only at 650 ppm, with all females and 10 of the 30 males dying between the second and sixth week of exposure. The earliest death was after 9 days of exposure. General signs of toxicity and sensory irritancy, seen at 650 ppm, were complete or partial eyelid closure, periocular, perinasal, and perioral encrustation, wetness around the urogenital area, hypoactivity, lack of coordination, paresis, ataxia, irregular gait, hypothermia, and emaciation. There were no exposure-related ophthalmic findings. In the Irwin neurobehavioral screen, effects were noted only at 650 ppm (Table 2). Due to early mortality, only two female rats had the first monthly examination (data not shown). Although neurobehavioral effects were observed in several of the 650-ppm exposed males, the majority of those surviving the first month of exposure did not exhibit neurobehavioral abnormalities when retested at 2 and 3 months. Also, there were no treatment-related signs following the 4-week postexposure recovery period. Body weights of the 650-ppm males were reduced throughout the 14-week exposure period, but there was considerable weight increase during the 4-week recovery period (Fig. 2). However, final weights were still statistically significantly below those for the control males. Before death, body weights of the female rats of the 650-ppm group were considerably reduced (Fig. 2). Also, females of the 307-ppm group had slight, but statistically significant, decreased body weight gains for study Days 45 through 121. However, at the conclusion of the 4-week recovery period, the difference between the absolute body weight means for the control and 307-ppm female rats was not statistically significant. Body weights of the lOl-ppm-exposed rats were similar to the controls. No treatment-
334
DODD ET AL.
related alterations in food and water intake were observed at the conclusion of the 14week exposure regimen. Thus, the 650-ppm male survivors had normal appetites and gained weight rapidly during the 4-week recovery period (Fig. 2). Only slight changes in some hematologic parameters were found at the end of the 14week exposure period. For the 650-ppm males and the 307-ppm females, the respective mean erythrocyte counts were 11 and 4% below control values (Fig. 3). The decrease in red blood cell count was accompanied with slight increases in MCV and MCH and a mild decrease in hematocrit. The white blood cell count was increased in the 650-ppm male rats (Fig. 3) due to an increase in lymphocytes. There was recovery from these effects by the end of the 4-week postexposure period. Clinical chemistry determinations which deviated from controls were an increase in urea nitrogen and alkaline phosphatase activity, and a decrease in creatinine, calcium, and aspartate
aminotransferase (AST) activity in the 650ppm males (Fig. 4). Serum calcium was also decreased in males and females of the 307ppm group. Although a mild increase in alkaline phosphatase activity persisted in the 650ppm recovery male rats, the remaining serum chemistry values were similar to control values, indicating reversibility of effects. Urinalysis showed minimal alterations in males, which included low pH (6.0 vs 7.0 in controls) at 650 ppm, and slightly increased bilirubin and urobilinogen at 650 and 307 ppm. These alterations in urine parameters were not found at the end of the postexposure observation period. After 1Cweeks of 2,4-PD vapor exposure, a statistically significant decrease in most absolute organ weights was observed for the 650-ppm males, but organ weights relative to body weight had increased (Table 3). This suggests that organ weight alterations were a reflection of decreased body weight. Organ weights of the 10 1- and 307-ppm groups were
TABLE 2 NEUROBEHAVIORALFINDINGSINMALERATSEXPOSEDTO~~O
ppm OF&~-PENTANEDIONEVAPOR~
Duration of exposure regimen (months) Observation
1
2
3
3 + 1 month recovery
Tremors Impaired gait Paresis Lacrimation Hypothermia Abnormal surface righting Abnormal midair righting Abnormal wire grasping Abnormal body tone Abnormal limb rotation Dilated pupils Decreased breathing rate Decreased locomotor activity Absence of comeal reflex Absence of tail pinch response Absence of auditory startle response
l/26b 4126 4126 2126 3126 2126
o/20 o/20 o/20 l/20 o/20 o/20
O/20 2120 o/20 O/20 O/20 O/20
015 015 015 015 O/5 O/5
6126 4126 6126 3126 6126 2126 6126 2126 3126 6126
3120 0120 0120 0120 0120 0120 0120 0120 0120 0120
4120 O/20 0120 0120 0120 0120 0120 O/20 O/20 0120
O/5 O/5 O/5 O/5 O/5 O/5 o/5 o/5 O/5 o/5
’ No effectswere observed in the control rats or rats exposed to 10 1 or 307 ppm. b No. affected/No. examined.
2,4-PD 9-DAY AND 1CWEEK 370
270
250 E
1
6,
210
190
170
Isa
-
-101 ---307ppm . . ..osoppm
0 wm ppm
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thy histopathologic features in brain, thymus, skin, and nasal mucosa. The brain lesions observed in 7 of 10 male and 18 of 20 female rats that died during the exposure regimen took the form of acute degenerative changes in the vestibular nuclei, deep cerebellar nuclei, and corpora striata. The lesions consisted of peracute to acute areas of pallor and vacuolation in the neuropil. Within these foci were nuclear pyknosis and karyorrhexis, and the presence of macrophages with vacuolated cytoplasm. The degree of vacuolation and macrophage infiltration was greatest in the deep cerebellar and vestibular nuclei. These brain lesions were bilaterally symmetrical. Of the surviving males of the 650-ppm group, 7 of 15 had gliosis with or without malacia in the deep cerebellar nuclei, vestibular nuclei, and corpus striatum. Four of the 7 affected surviving males were from the 10 sacrificed at 14 weeks, and the other 3 were from the 5 sacrificed following the 4-week re-
130
iia
FIG. 2. Mean body weights of Fischer-344 rats during the 2,4pentanedione 1Cweek exposure plus 4-week recovery study.
unaltered, except for absolute lung weights of the females which were slightly increased at 101 and slightly decreased at 307 ppm; this was not considered to be of biological importance. Organ weights of the 4-week recovery animals followed a pattern generally similar to that of the 14-week sacrifice. However, due to the substantial body weight gain in the 650-ppm males during the postexposure period, the magnitude of the increases in relative organ weights when compared to control values was much reduced in the recovery animals (Table 3). Light microscopy did not reveal any abnormalities in tissues removed from the animals of the 10 1- and 307-ppm groups. However, for the 650-ppm animals there were notewor-
FIG. 3. Hematologic values of Fischer-344 rats following the 2,4pentanedione 14-week study. WBC = white blood cells; RBC = red blood cells; HCT = hematocrit; MCH = mean corpuscular hemoglobin; MCV = mean corpuscular volume.
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Km
80
60
Ak
Phce.
AST
FIG. 4. Serum chemistry values of Fischer-344 rats following the 2,4-pentanedione 1Cweek study. Urea N = urea nitrogen; Alk. Phos. = alkaline phosphatase; Ca = calcium; AST = aspartate aminotransferase.
cover-y period. (Another five 650-ppm male survivors had been sacrificed at 14 weeks for glutaraldehyde perfusion and subsequent electron microscopic examination of sciatic nerves.) Gliosis was most extensive in the deep cerebellar and vestibular nuclei. Many of the cells had irregular or rod-shaped nuclei typical of microglia, but others resembled astrocytes. Multifocal areas of microscopic mineralization, hemosiderosis, and neuronal degeneration were evident. No degenerative changes were seen in the spinal cords of the 650-ppm exposed animals. Transmission electron microscopic evaluation of sciatic nerves from the 650-ppm group did not produce any evidence of a peripheral neuropathy. There was excellent agreement between the results of the Irwin neurobehavioral screen (Table 2) and the microscopic findings in the brain. For example, the location of the brain lesion (cerebellar and vestibular nuclei) cor-
related with those Irwin screen assessments related to equilibrium (abnormal midair righting reflex and impaired gait). Furthermore, every animal that exhibited an abnormality during one of the examination periods had brain lesions. Seven of fifteen 650-ppm male survivors had brain lesions, and five of these showed either abnormal midair righting reflex or impaired gait at the conclusion of the 14-week exposure regimen. The failure of some rats to show neurologic deficits during the Irwin screen probably reflects the insensitivity of the test. Thymic lesions, consisting of acute lymphoid degeneration and atrophy, were observed in 7 of 10 male and 13 of 20 female rats that died during the lbweek exposure regimen. They were not present in survivors. Dermatitis, dermal necrosis, and in a few cases, cellulitis were seen in the skin and subcutis of the heads of 5 male and 8 female rats that died, but not in survivors. The inflammatory infiltrate was predominately of polymorphonuclear leukocytes and mast cells. The nasal mucosa showed mild multifocal squamous metaplasia at the anterior portion of the nasal cavity, in both the maxillary and nasal turbinates. This change was seen in 9 of the 10 animals exposed to 650 ppm which were sacrificed at the end of the 1Cweek exposure period, but was present in only one of five and with reduced severity following the 4-week recovery period. The nasal mucosa was normal in female rats exposed to 307 ppm of 2,4-PD vapor. DISCUSSION In the 1Cweek study, death in the 650ppm-exposed rats was attributed to brain lesions in the majority of cases. The exposure conditions to cause death by inhalation of 2,4-PD vapor appear to be well demarcated with respect to concentration and exposure time. For example, malacia within the brain was not observed in the 1Cweek study until study Day 16. Although one female rat ex-
2,4-PD 9-DAY AND ICWEEK
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TABLE 3 ORGANWEIGHTSASAPERCENTAGEOFBODYWTFORMALEFISCHER-~~~RATS FOLLOWINGTHE2,4-&NTANEDIONEl4-WEEKSTUDY
2,4PD concentration (ppm) Organ Brain Liver Kidney Heart Lungs Thymus Testes
Time (Weeks)
0
101
307
6.50
14 18 14 18 14 18 14 18 14 18 14 18 14 18
0.57 (0.03)b 0.55 (0.02) 3.07 (0.12) 2.90 (0.12) 0.64 (0.02) 0.65 (0.03) 0.27 (0.02) 0.28 (0.02) 0.43 (0.03) 0.37 (0.04) 0.10 (0.02) 0.10(0.01) 0.94 (0.03) 0.9 l(O.04)
0.56 (0.02) 0.53 (0.03) 3.02 (0.09) 2.99 (0.16) 0.64 (0.03) 0.64 (0.02) 0.27 (0.01) 0.28 (0.01) 0.4 1 (0.02) 0.36 (0.04) 0.11 (0.03) 0.10 (0.02) 0.93 (0.04) 0.88 (0.02)
0.57 (0.03) 0.54 (0.03) 3.20 (0.15) 3.01 (0.11) 0.65 (0.03) 0.64 (0.02) 0.28 (0.01) 0.28 (0.0 1) 0.42 (0.04) 0.39 (0.04) 0.10 (0.02) 0.09 (0.01) 0.95 (0.05) 0.89 (0.03)
0.73 (0.05)c 0.61 (0.02) 3.64 (0.14) 3.27 (0.07)c 0.75 (0.05) 0.70 (0.04) 0.32 (0.02) 0.29 (0.01) 0.48 (0.05) 0.4 1 (0.04) 0.10 (0.04) 0.11 (0.02) 1.26 (0.10) 1.09 (0.08)
D 14 weeks = end of exposure period; 18 weeks = end of recovery period. b All values are means (SD); units are in %. ‘P < 0.05 compared to control value.
posed to 650 ppm of 2,4-PD died on Day 9, no lesions were seen microscopically in the brain of this animal. Consistent with these results was the lack of mortality and the absence of malacia within the brain in any of the 9-day study animals. However, two male rats of the 805-ppm group had mild vacuolization of the brain stem, which may indicate that had 2,4-PD exposures continued into a third week, more prominent brain lesions may have developed. Thus, the mechanism(s) of lethal toxicity appear to have a well-dehned threshold and may be based on a saturation process. The most noticeable feature in this study was the presence of degenerative changes in the deep cerebellar and vestibular nuclei and the corpora striata in animals that died following repeated exposures to 650 ppm of 2,4PD vapor. Additionally, gliosis and malacia were seen in the brains of about half the 650ppm animals that survived. Central neuropathologic lesions have also been described by Krasavage et al. (1982) who gave rats 100
to 150 mg/kg of 2,4-PD twice daily by gavage for periods from 3 to 6 1 days. Animals developed weakness, ataxia, tremors, paresis, and rolling movements of the head. Histologic changes induced by the shorter term dosing procedures were perivascular edema, hemorrhage into the Virchow-Robin spaces, and endothelial cell swelling, all primarily localized in the cerebellum and the brainstem. For the more prolonged dosing schedules there were bilateral, symmetrical foci of malacia and gliosis in the cerebellar peduncles, olivary nuclei and lower brainstem. Lesions were not seen in the basal ganglia, and there was no evidence of peripheral nerve involvement. Likewise, in the current studies by respiratory exposure, there was no ultrastructural evidence for a peripheral neuropathy. Thus, the changes seen in the rats following repeated gavage with 2,4-PD are similar to those seen by recurrent exposure to high concentrations of 2,4-PD vapor, and are clinically and morphologically distinct from those produced by y-diketones (O’Donoghue,
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DODD ET AL.
1985). Furthermore, the central neuropathologic lesions induced by 2,4-PD appear species dependent since rabbits did not develop these lesions following repeated gavage administration (Krasavage et al., 1982). The central neuropathologic lesions resulting from repeated exposure to 2,4-PD have been compared with those produced by acute vitamin B complex deficiency (Krasavage et al., 1982) and related to the known ability of 2,4-PD to inactivate the lysyl residue of enzymes, some of which are related to coenzyme formation (Gilbert and O’Leary, 1975; Otwell et al., 1979). Gilbert and O’Leary (1977) have observed that, in vitro, 2,4-PD is highly specific for lysine residues of porcine aspartate aminotransferase (AST). The decrease in AST activity in the present investigation (Fig. 4) may indicate in vivo modification of AST following 2,4-PD exposure. The primary region of brain damage in rats with experimentally induced thiamine deficiency is the lateral vestibular nucleus (Dreyfus, 1973; McCandless, 1982), one of the areas in which major damage is seen after 2,4PD exposure. Although degenerative lesions have not been described in the basal ganglia of thiamine deficient rats, such lesions have been seen in rhesus monkeys given diets deficient in thiamine (Blank et al., 1975). The similarity of the lesions induced in rat brains by thiamine deficiency, together with the enzyme inactivation effects of 2,4-PD, indicate that the 2,CPD-induced brain lesion is likely to be due, in part at least, to an interruption of normal thiamine biochemistry in specific regions of the brain. This would accord with the sharply defined exposure conditions for the neurological toxicity of 2,4-PD. Furthermore, interanimal variation of thiamine concentration, turnover, and/or requirement in specific brain regions may explain the sex-related differences in mortality observed in the current study and the absence of brain lesions in eight of fifteen 650-ppm-exposed male rats that survived the 1Cweek exposure regimen. The changes observed in the thymus gland (a decrease in weight in the 9-day study and
acute lymphoid degeneration in the 14-week study) were probably stress related because they were observed only in animals exposed to high concentrations of 2,4-PD (650 to 805 ppm) and that died during the exposure regimen ( 14-week study). No thymic lesions were observed in the 9-day 805-ppm-exposed rats or in the 650 ppm survivors of the 1Cweek study. Dose-related inflammatory lesions of the nasal mucosa were noted at all concentrations of 2,4-PD in the 9-day study, although the effects were marginal at 197 ppm. With the subchronic study, a mild squamous metaplasia, reversible on cessation of exposure, was seen at 650 ppm, but no nasal mucosal lesions were observed at 307 ppm. These findings suggest that nasal mucosal inflammation is not a significant biological effect until concentrations greater than 300 ppm are attained. The pattern of toxicity in the subchronic study indicates a steep slope on the dose response for 2,4-PD. Thus, although 650 ppm was a concentration by which repeated exposure caused severe toxicity, at 307 ppm there were no histopathologic changes or clinical abnormalities, and the only effects were minor alterations in body weight and serum chemistry which were reversible during the 4week postexposure recovery period. No effects were seen at 10 1 ppm. In the context of possible human exposures, adverse effects are likely to occur only at concentrations significantly in excess of the odor threshold of 0.01 ppm (Ballantyne et al., 1986). Thus, potentially hazardous atmospheres would be intolerable to humans. The above considerations indicate that by subchronic exposure to 2,4-PD vapor the minimum-effects concentration is 307 ppm and the no-adverse effects concentration is 101 ppm. ACKNOWLEDGMENTS The authors are grateful to P. E. Losco for her participation in this study and to F. C. Wilt for her assistance in typing this manuscript.
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SAX, N. I. (1979). Dangerous Properties of Industrial Materials, p. 890. Van Nostrand-Reinhold, New York. SNEDECOR,G. W., AND COCHRAN, W. G. (1967). Statistical Methods, pp. 271-275. Iowa State Univ. Press, Ames. SOKAL, R. R., AND ROHLF, F. J. (1969). Biometry, pp. 204-2 16,369-376. Freeman, San Francisco.