Isopropanol: Summary of TSCA Test Rule Studies and Relevance to Hazard Identification

REGULATORY TOXICOLOGY AND PHARMACOLOGY ARTICLE NO.

23, 183–192 (1996)

0042

Isopropanol: Summary of TSCA Test Rule Studies and Relevance to Hazard Identification ROBERT W. KAPP, JR.,1,* CHRISTOPHER BEVAN,2,† THOMAS H. GARDINER,‡ MARCY I. BANTON,‡ TIPTON R. TYLER,§ AND GARY A. WRIGHTØ *BioTox for BP Chemicals, Inc., Richmond, Virginia 23236-3465; †Exxon Biomedical Sciences, Inc., East Millstone, NJ 08873; ‡Shell Chemical Company, Houston, TX 77210; §Union Carbide Corporation, Danbury, CT 06817; and Ø ARCO Chemical Company, Newtown Square, PA 19073 Received October 4, 1995

The toxicity of isopropanol (IPA) has been extensively studied as a result of a Test Rule under Section 4 of the Toxic Substances Control Act. In general, the data showed that IPA has a low order of acute and chronic toxicity; does not produce adverse effects on reproduction; is neither a teratogen, a selective developmental toxicant, nor a developmental neurotoxicant; and is not genotoxic or an animal carcinogen. IPA is, however, a potential hazard for transient central nervous system depression at high exposure levels. In addition, IPA produced effects to several rodent toxicity endpoints at high dose levels (i.e., motor activity, male mating index, and exacerbated renal disease) which are of unclear relevance to human health. The data generated by these studies confirmed that IPA acts as a typical short-chain alcohol in mammalian biological systems. It produces a significant narcotic effect upon exposure at high levels for extended periods of time, with no irreversible effects even after repeated exposure, which is consistent with other short-chain alcohols. The metabolism of IPA appears equivalent across species with rapid conversion to acetone and carbon dioxide. Overall, these studies demonstrate IPA exposure is a low potential hazard to human health. This information will allow for an improved assessment of the human health risks from IPA exposure. q 1996 Academic Press, Inc.

INTRODUCTION

Isopropanol (IPA), also known as isopropyl alcohol and 2-propanol, is a three-carbon, branched alcohol. It is a flammable liquid, miscible with water and organic solvents. Current production of IPA is by the oxidation of propylene using weak-acid catalysis and by a non1

To whom correspondence should be addressed at Bio Tox, 300 Arboretum Place, Suite 140, Richmond, VA 23236. 2 Present address: Amoco Corporation, Chicago, IL 60601.

acid process. (Chemical Manufacturers Association, unpublished data). Significant uses for IPA include use as a solvent, as a component of numerous industrial and consumer products, and in the production of acetone and acetone derivatives (Papa, 1982). In 1994, approximately 1.4 billion pounds of IPA was produced in the United States (Chemical and Engineering News, 1995), with a potential for widespread exposure to workers and consumers. The major route of occupational exposure is by inhalation, whereas consumer exposure is primarily dermal from the use of rubbing alcohol (70% isopropanol). Despite the widespread use of IPA, there are few reports of adverse effects not associated with abuse (Lington and Bevan, 1994). IPA has a low order of acute toxicity (for review, see Lington and Bevan, 1994). It is irritating to the eyes, but not to the skin. The Environmental Protection Agency (EPA) promulgated a Final Test Rule in 1989 (U.S. EPA, 1989) specifying that IPA be tested for pharmacokinetics, subchronic toxicity, acute and subchronic neurotoxicity, genotoxicity, chronic toxicity including oncogenicity, developmental, and reproductive toxicity, and developmental neurotoxicity. The EPA specifically stated that the decision to test IPA was based on the widespread exposure and use of IPA, and not because of any specific toxicity concerns. The agency acknowledged that IPA presented no concerns for adverse environmental effects. This article will review the findings of the Toxic Substances Control Act (TSCA) Section 4 Test Rule studies and evaluate the potential health hazards associated with IPA exposure. It is not intended to be a comprehensive review of the toxicity of IPA. METHODS AND RESULTS

Pharmacokinetics/Disposition IPA is metabolized to acetone primarily by the enzyme alcohol dehydrogenase in both animals and humans (Abshagen and Rietbock, 1969; Nordman et al.,

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TABLE 1 Cumulative Excretion of Radiolabel 72 hr after Administration of [14C]Isopropanola,b Species

Route

Rat Rat Rat Rat Rat Rat Mouse Mouse Mouse

Intravenous Oral Oral Oral 1 8 days Inhalation Inhalation Intravenous Inhalation Inhalation

a b

Dose 300 300 3000 300 500 5000 300 500 5000

Volatile breath

CO2

Urine

Feces

Total

55 56 70 54 34 66 45 50 73

30 26 16 28 49 21 30 35 20

5 5 8 5 7 8 3 6 6

1.3 0.7 0.7 1.0 1.5 0.5 1.5 1.7 1.2

91 88 93 88 91 95 80 92 97

mg/kg mg/kg mg/kg mg/kg ppm ppm mg/kg ppm ppm

Values represented as percentages of administered dose. Taken from Slauter et al. (1994).

1973; Laham et al., 1980; Brugnone et al., 1983). Acetone is further metabolized to CO2 via several pathways. For endogenous (as well as exogenous) acetone, the first step in the enzymatic biotransformation of acetone is the cytochrome P-450-dependent oxidation to acetol by acetone monooxygenase. The acetol is then biotransformed by either of two pathways—an extrahepatic propanediol pathway or an intrahepatic methylgloxal pathway. The oxidation of acetol to methylgloxal is also cytochrome P-450-dependent and is catalyzed by acetol monooxygenase. Ultimately, pyruvate is formed, which by gluconeogenesis, results in glucose production. There appears to be another metabolic pathway leading to acetate and formate, which is recruited when the other pathway is overloaded. Since the study by Slauter et al. (1994) used [2-14C]IPA, 14CO2 would only occur if the carbon chain was cleaved. Presumably, it is the pathway involving acetate and formate which results in CO2 formation (Morgott, 1993). As a minor pathway, IPA is conjugated by glucuronic acid and excreted in the urine (Kamil et al., 1958). A cytochrome P-450-dependent pathway is also present in rat liver (Cederbaum et al., 1981), the relative importance of which has yet to be determined. In the pharmacokinetic studies specified in the Test Rule, Fischer 344 rats and B6C3F1 mice were given an intravenous dose of 300 mg/kg [2-14C]IPA or were exposed by inhalation to 500 or 5000 ppm [2-14C]IPA for 6 hr (Slauter et al., 1994). Rats were also given [214 C]IPA by oral gavage as a single dose of 300 or 3000 mg/kg or as multiple doses of 300 mg/kg/day for 8 days. Table 1 summarizes the cumulative excretion of radiolabel by the various routes of administration, and Table 2 summarizes peak blood levels and the half-life of the disappearance of IPA from the blood. IPA was readily absorbed by the oral and inhalation routes of exposure, and excretion was rapid. The major portion of excreted radioactivity (81–89% of the administered dose) was recovered in expired air as acetone, CO2 , and unmetabolized IPA. A small amount of the adminis-

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tered dose (3–8%) was eliminated in the urine as IPA, acetone, and as isopropyl glucuronic acid. Only 0.5 to 1.7% of the dose was eliminated in the feces. The uptake of IPA in mice, when expressed on a mg/kg basis, was over twice that in rats when exposed to the same IPA vapor concentration. This accounted for the twofold difference observed in peak blood concentrations (Table 2). This species difference in IPA uptake is likely the result of the more rapid ventilation rate of the mouse, approximately twofold higher, on a mg/kg basis, than that of the rat. It should also be noted that since exhalation of radiolabel as volatile organic compounds in the breath occurred very rapidly, it is possible that material was lost from the intravenously dosed animals during the period from dosing to actual placement in the metabolism chamber. This may explain the somewhat lower recovery of 14C in the mouse intravenous study compared to the other routes of exposure. IPA and its metabolites were widely distributed to all tissues, with no accumulation in any particular organ. Generally, the half-life for IPA elimination from blood ranged from 0.7 to 2 hr, however, a value of 5.4 hr was obtained for the high oral dose in rats (Table 2). At an oral dose of 3000 mg/kg, saturation of elimination pathways became apparent as evidenced by an increase in the half-life to 5.4 hr. There were no qualitative differences in the pattern of elimination of radioactivity between males and females of either species or when administered by different routes of administration. The elimination of IPA from the blood fits a one-compartment open pharmacokinetic model, similar to that reported in humans (Pappas et al., 1991). Repeated administration of IPA had no substantial effect on the rates or routes of excretion (Tables 1 and 2). This is in contrast with ethanol, in which chronic ethanol treatment increases in vivo ethanol metabolism (Alderman et al., 1989). Subchronic Toxicity The subchronic toxicity of IPA was evaluated in Fischer 344 rats and CD-1 mice exposed to 0, 100, 500,

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TABLE 2 Pharmacokinetic Data after Administration of [1C]Isopropanola Species

Route

Rat Rat Rat Rat Rat Rat Mouse Mouse Mouse

Intravenous Oral Oral Oral 1 8 days Inhalation Inhalation Intravenous Inhalation Inhalation

a

Dose 300 300 3000 300 500 5000 300 500 5000

Peak blood level (mg eq/g)

Elimination rate constant (hr)

Halflife (hr)

309 276 1341 238 32 876 203 65 1885

0.574 0.552 0.159 0.423 0.862 0.366 0.848 1.075 0.414

1.3 1.3 5.4 1.7 0.9 2.0 0.9 0.7 1.7

mg/kg mg/kg mg/kg mg/kg ppm ppm mg/kg ppm ppm

Taken from Slauter et al. (1994).

1500, or 5000 ppm IPA vapor 6 hr/day, 5 days/week for 13 weeks (Burleigh-Flayer et al., 1994a). There were no deaths during the study. During and immediately following exposure to 5000 ppm, ataxia, narcosis, hypoactivity, and a lack of a startle reflex were observed in some rats and mice exposed to 5000 ppm IPA. Narcosis was not observed in rats during exposure following Week 2, suggesting some adaptation to IPA. Tolerance to the narcotic effects of IPA have been previously reported by Lehman et al. (1945) in dogs receiving IPA in drinking water. During exposures to 1500 ppm, narcosis, ataxia, and hypoactivity were observed in a few mice, while only hypoactivity was observed in rats. Immediately following exposures, ataxia and/or hypoactivity were observed in a few rats or mice exposed to 5000 ppm. These clinical signs appear indicative of a reversible central nervous system (CNS) depression (see next section). Overall, the 1500- and 5000-ppm rats and the 5000ppm female mice exhibited increased body weights and/ or body weight gain during the study. The only organ weight affected by IPA exposure was that of the liver. An increase in liver weight relative to body weight was observed in rats of both sexes and female mice exposed to 5000 ppm. No corresponding microscopic changes were noted in the liver. Histopathological evaluation showed a slight increase in size and frequency of hyaline droplets in the kidneys of the IPA-exposed male rats. The increased size and frequency of hyaline droplets within the kidneys of the male rats were not clearly exposure-related, although the changes were most pronounced in the highest exposure group (5000 ppm). There was no cytotoxicity of the tubular epithelium associated with the increase in hyaline droplet formation. Thus, repeated inhalation exposure to IPA produced no biologically significant systemic toxicity in rodents. Excluding the clinical signs of CNS depression, which will be discussed in the next section, the no observed adverse effect level (NOAEL) for subchronic toxicity in this study was 5000 ppm.

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Neurotoxicity An acute neurotoxicity study was conducted with Fischer 344 rats exposed to 0, 100, 500, 1500, 5000, or 10,000 ppm IPA vapor for 6 hr (Gill, 1994; Gill et al., 1995). Behavioral function was evaluated before and 1, 6, and 24 hr after exposure using a functional observation battery (FOB). Motor activity was evaluated before and immediately after exposure. Exposure-related FOB findings were observed in the 5000and 10,000-ppm groups. All animals in the 10,000ppm group were prostrate 1 hr postexposure. Some FOB measurements could not be made for these animals and other FOB parameters were omitted to prevent injury. Partial recovery was apparent by the 6-hr postexposure evaluation in the 10,000-ppm animals. Most of the animals were able to walk, albeit with severe ataxia, and effects on arousal, gait, and surface righting tended to be less severe at 6 hr postexposure than at the 1-hr evaluation. Except for increased mean hindleg splay, complete recovery was observed by the 24-hr postexposure evaluation for the 10,000-ppm-exposed animals. The 5000-ppm-exposed animals showed exposure-related behavioral alterations 1 hr after evaluation, which included altered gait, decreased toe and nail withdrawal reflexes, decreased mean rearing events, decreased rectal temperature, and grip strength and increased mean hindlimb splay. Complete recovery, with the exception of decreased mean rectal temperatures of males, was observed by the 6-hr postexposure evaluation. Motor activity was decreased 40 and 90% for the 5000- and 10,000-ppm groups, respectively. Five hours after exposure to IPA there was also a small, but statistically significant, decrease in motor activity in the 1500-ppm males. Reversibility could not be determined since motor activity measurements were not conducted at later timepoints. The NOAEL for acute neurotoxicity in this study was 500 ppm. In a subchronic neurotoxicity study, Fischer 344 rats

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were exposed by inhalation to 0, 100, 500, 1500, or 5000 ppm IPA vapor, 6 hr/day, 5 days/week for 13 weeks (Burleigh-Flayer et al., 1994a; Gill, 1994). Neurobehavioral evaluations included a FOB, motor activity, and neuropathology. Testing of the FOB occurred prior to the first exposure and on the weekend following Weeks 1, 2, 4, 9, and 13. Approximately 42 hr elapsed between the end of the exposure and the beginning of the FOB testing. Motor activity evaluations were conducted prior to the first exposure and on the weekend following Weeks 4, 9, and 13. The time between the end of the exposure and the beginning of the motor activity testing was approximately 20 and 24 hr for females and males, respectively. The neuropathology included both the central and the peripheral nervous systems, prepared by in situ perfusion, plastic embedding, and the use of specific nerve tissue stains. Narcotic effects were noted in the 1500- and 5000ppm animals but only during exposure. There were no treatment-related effects in FOB. Increased motor activity was observed in the 5000-ppm females following Weeks 9 and 13 (57 and 26% above controls, respectively). There were no exposure-related neuropathologic lesions in the central or peripheral nervous systems of IPA-exposed rats. The NOAEL for acute effects in this study is 500 ppm based on the clinical signs of narcosis, and the NOAEL for subchronic neurotoxicity is 1500 ppm based on the increase in motor activity in the female rats. An additional subchronic neurotoxicity was conducted in order to clarify the increased motor activity findings. Female Fischer 344 rats were exposed to 0 or 5000 ppm IPA vapor 6 hr/day, 5 days/week (Gill et al., 1994). Half of the animals in each group were exposed for 9 consecutive weeks and the other half for 13 consecutive weeks. Motor activity was assessed for both subgroups prior to exposure, and following 4, 7, and 9 weeks of exposure. Rats in the 13-week subgroup were also assessed for motor activity after 11 and 13 weeks of exposure. These motor activity measurements were conducted 18–20 hr following the end of exposure for that week. To determine the reversibility of the motor activity effects, measurements were made for rats in the 9- and 13-week subgroups during the 10th and 14th through 21st weeks, respectively. Total motor activity counts were increased following 4, 7, 9, 11, and 13 weeks of exposure. After 9 weeks of exposure, the effect was reversible within 2 days following the last exposure. Subtle differences in the shape of the motor activity versus test session time curve were noted during the recovery period in both the 9-week- and the 13-week-exposed animals, although it was unclear whether these changes were treatment related. Complete reversibility of these subtle changes did not occur until 1 and 6 weeks following the last IPA exposure in the 9- and 13-week exposure groups, respectively.

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Genotoxicity An in vitro Chinese hamster ovary (CHO) cell/hypoxanthine–guanine phosphoribosyl transferase (HGPRT) gene mutation assay and a bone marrow micronucleus assay in mice were conducted to assess genotoxicity (Kapp et al., 1993). In the CHO/HGPRT assay, there was no evidence of a mutagenic response in CHO cells treated with 0.5 to 5.0 mg/ml IPA in the presence or absence of a metabolic activation system. Likewise, micronuclei were not increased in bone marrow polychromatic erythrocytes 24, 48, or 72 hr after mice were injected intraperitoneally with 350 to 2500 mg IPA per kilogram body weight (Kapp et al., 1993). Chronic Toxicity/Carcinogenicity Mice. CD-1 mice were exposed by inhalation to 0, 500, 2500, or 5000 ppm IPA vapor for 6 hr/day, 5 days/ week for 18 months (Burleigh-Flayer et al., 1994b). At 12 months, 10 animals/sex/group were euthanized, and 10 animals/sex/group did not receive any further exposure but were retained until study termination. Mortality rates of both the main and the recovery groups at all dose levels were not statistically different from the controls. Clinical signs noted during exposures in the 5000-ppm group included hypoactivity, lack of a startle reflex, ataxia, prostration, and narcosis. Some of the animals in the 2500-ppm group also showed hypoactivity, lack of startle reflex, and narcosis. No clinical signs were noted in the 500-ppm group during or after exposure. Ataxia was the only exposure-related clinical sign that was noted for the 5000-ppm male and female animals following exposure. Concentration-related increases in body weights and body weight gain were noted in both the male and the female mice in the 2500- and 5000-ppm groups. No exposure-related changes in any hematological parameters were noted in any of the exposure groups at either 12 or 18 months. At 12 months, liver weights were increased in the 5000-ppm males. At study termination (18 months), there was a concentration-related increase in liver weights for female mice, although only the 5000-ppm group was statistically different from controls. Also, there was a decrease in brain weights in the 5000-ppm female mice. Nonneoplastic lesions were limited to the testes (males) and the kidney. In the testes, enlargement of the seminal vesicles occurred in the absence of associated inflammatory or degenerative changes. The kidney effects included tubular proteinosis and/or tubular dilation. The incidence of testicular and kidney effects were not increased in the IPA-exposed recovery animals. There was no increased frequency of neoplastic lesions in any of the IPA exposure groups. The NOAEL for chronic toxicity in this study was 500 ppm with no evidence of an oncogenic effect at any dose level.

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Rats. Fischer 344 rats were exposed by inhalation to 0, 500, 2500, or 5000 ppm IPA vapor for 6 hr/day, 5 days/week for 24 months (Garman et al., 1995). At 18 months, 10 animals/sex/group were euthanized. The mortality rates for all male rats were 82, 83, 91, and 100% for the 0-, 500-, 2500-, and 5000-ppm groups, respectively. The corresponding values for the female rats were 54, 48, 55, and 69%. The main cause of death for the 5000-ppm rats (both sexes) was chronic progressive nephropathy (see below). Chronic progressive nephropathy also accounted for much of the mortality of the 2500-ppm male rats. In contrast, the main cause of death for the control animals was large granular lymphocyte leukemia. Clinical signs noted in the 5000-ppm group included hypoactivity, lack of a startle reflex, and narcosis. Some animals in the 2500-ppm group also showed a lack of a startle reflex, while the 500-ppm group showed no clinical signs. There was initially a decrease in body weights in the 5000-ppm-exposed animals; then, from Week 6 through approximately Week 72, these animals showed increased body weights and body weight gain. The 2500-ppm group males also showed a similar pattern of body weight changes. Urinalysis and urine chemistry findings indicative of impaired kidney function (i.e., decreased osmolality and increased total protein volume and glucose) were noted for the 2500- and 5000-ppm-exposed male rats and the 5000-ppm-exposed female rats. There was a concentration-related increase in testicular weights in male rats at 18 months, but not at study termination. Liver weights were increased in the 2500- and 5000-ppm male rats at 18 months, and in the 2500-ppm male rats and 5000ppm female rats at 24 months. Kidney weights were increased in the 5000-ppm male rats at 18 months and in the 5000-ppm female rats at 24 months. The kidney appeared to be a target organ for nonneoplastic effects in rats. IPA exposure resulted in impaired kidney function, as indicated by various urine chemistry changes in male (2500- and 5000-ppm) and female (5000-ppm) rats. Animals in these groups also exhibited histopathologic effects in the kidneys, which included mineralization, tubular dilation, glomerulosclerosis, interstitial nephritis and fibrosis, hydronephrosis, and transitional cell hyperplasia. The lesions in the kidneys of IPA-exposed rats appear to be an exacerbated form of chronic progressive nephropathy. The only neoplastic lesion noted was increased interstitial (Leydig) cell adenomas in male rats. The frequencies of this tumor for all male rats were 65, 77, 87, and 95% for the 0-, 500-, 2500-, and 5000-ppm exposure groups. The frequencies of this tumor were similar to the mean incidence of 88% reported by Haseman and Arnold (1990) for control male Fischer rats from numerous 2-year National Toxicology Program (NTP) carcinogenicity studies. In addition, the incidences observed for the IPA exposure groups were similar to

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frequencies noted during previously conducted studies at Bushy Run Research Center (BRRC) for male control Fischer 344 rats (86 and 91%; BRRC historical control data). Thus, the incidence observed for the control group in this study (65%) was well below the historical control values of NTP and BRRC for male Fischer 344 rats. The NOAEL for chronic toxicity in the rat was 500 ppm. There is no evidence that IPA has oncogenic activity in the rat at any dose level. Developmental Toxicity A rat developmental toxicity study was conducted using Sprague–Dawley rats dosed by oral gavage with 0, 400, 800, or 1200 mg/kg IPA during Gestational Days (GD) 6 through 15 (Tyl et al., 1994). Two dams (8%) died at 1200 mg/kg and one dam (4%) died at 800 mg/ kg. At 1200 mg/kg, maternal body weights were reduced throughout gestation (GD 0–20; 89.9% of control value), associated with reduced gravid uterine weight at 1200 mg/kg (89.2% of control value). There were no other treatment-related effects on the dams. Fetal body weights per litter were statistically significantly reduced at the 800- and 1200-mg/kg levels, but no teratogenic effects were noted. The NOAEL for maternal and developmental toxicity in this study was 400 mg/kg. In a rabbit developmental toxicity study, New Zealand White rabbits were dosed by oral gavage with 0, 120, 240, or 480 mg/kg IPA during Gestational Days 6 through 18 (Tyl et al., 1994). At 480 mg/kg, IPA was unexpectedly toxic to the pregnant female rabbit, resulting in the deaths of four does (25.7%). Maternal body weights were statistically significantly reduced during treatment (GD 6–18) at 480 mg/kg (45.4% of control value). This effect was associated with reduced maternal food consumption during this period. Profound clinical signs were noted at 480 mg/kg and included flushed and/or warm ears, cyanosis, lethargy, and labored respiration. No evidence of developmental toxicity was noted at any exposure concentration. The NOAELs for maternal and developmental toxicity in this study are 240 and 480 mg/kg, respectively. Reproductive Toxicity A two-generation reproductive toxicity study was conducted with IPA in Sprague–Dawley rats (Bevan et al., 1995). Parental animals (30/sex/group, designated F0 and F1 for their respective generations) were dosed once daily by oral gavage with 0, 100, 500, or 1000 mg/kg IPA. These doses were selected from a range-finding study in which there was excessive mortality in parental rats dosed with 1750 and 2500 mg/ kg IPA. Two treatment-related deaths also occurred in the 1000-mg/kg group. In the definitive study, there were seven parental deaths that were considered treatment-related: two high-dose F0 females, two F1 high-dose females, one

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mid-dose F0 female, and two low-dose F1 males. Lactation body weight gain was increased in the 500- and 1000-mg/kg females in both generations, and liver and kidney weights were increased in the 500- and 1000mg/kg groups of both sexes. Centrilobular hepatocyte hypertrophy was noted in some 1000-mg/kg F1 males. There were some kidney effects in the 500- and 1000mg/kg F0 male rats, and in all treated F1 male rats. The kidney effects were characterized by increased number of hyaline droplets in convoluted proximal tubular cells, epithelial degeneration and hyperplasia, and proteinaceous casts. Increased mortality occurred in the high-dose F1 offspring during the early postnatal period; no other clinical signs of toxicity of the offspring from either generation were observed. Offspring body weight, however, in the 1000-mg/kg group was reduced during the early postnatal period. There was significant mortality in the F1 weanlings (18/70) prior to the selection of the F1 adults. It is likely that these deaths were the result of a diminished metabolic capacity or altered disposition in young groups compared to that in older animals. This hypothesis is supported by Kimura et al. (1971) who noted a lower oral LD50 in 14-day-old rats compared to that in older animals. The F2 offspring body weights were also decreased in the 1000-mg/kg group. A statistically significant reduction was observed in the F1 male mating index of the 1000-mg/kg IPA group (73% versus 97% in the controls). There were no other treatment-related effects on reproduction, including fertility and gestational indices, or histopathology of the reproductive organs. Based on the single finding of reduced F1 mating index, the NOAEL for reproductive toxicity was 500 mg/kg/day. Developmental Neurotoxicity IPA was administered by oral gavage to pregnant Sprague–Dawley rats from GD 6 through Postnatal Day (PND) 21 (Bates et al., 1994). Doses were 0, 200, 700, or 1200 mg/kg. The dams were allowed to deliver; pups were counted, examined, and sexed. Pup weights were obtained and clinical examinations conducted frequently throughout the study. Litters were culled to eight pups (4:4 or 5:3 sex ratio) on PND 4. On PND 22, pups were divided into two groups. One group consisting of two pups from each litter was euthanized. Neuropathological examinations of the central nervous system were conducted on six pups from each group. The weight of each of four regions of the brain was obtained on the remaining pups in this group. The second group, consisting of the remaining pups, was weaned and raised through PND 68. These pups were assessed for the developmental landmarks ‘‘day of testes descent’’ or ‘‘day of vaginal opening.’’ Motor activity was assessed on PNDs 13, 17, 21, and 47 and PNDs 60–64.

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TABLE 3 Summary of NOAEL and LOAEL Values for IPA Test Rule Studies Toxicity test Subchronic toxicity Subchronic toxicity Acute neurotoxicity Subchronic neurotoxicity Developmental toxicity (oral) Developmental toxicity (oral) Reproduction toxicity (oral) Developmental neurotoxicity (oral) Chronic toxicity Chronic toxicity Oncogenicity Oncogenicity

NOAEL (mg/kg)a

LOAEL (mg/kg)a

Rats Mice Rats

ú1820 ú3800 255

NA NA 765

Rats

546

1820

Rats

400

800

Rabbits

480

NA

Rats

500

1000

Rats Rats Mice Rats Mice

1200 182 380 ú1820 ú3800

NA 910 1900 NA NA

Species

a Conversion assumptions: inhalation rates for rats and mice are 0.29 and 0.052 m3/day, respectively; the body weights of rats and mice in kilograms are 0.35 and 0.03 kg, respectively.

One dam in the 1200-mg/kg-dose group died on PND 15. No other exposure-related effects were observed in the dams during the course of the study. Pup body weights and body weight gains were unaffected by exposure to IPA. Furthermore, no exposure-related effects were noted on motor activity, weights of the four regions of the brain, developmental landmarks, or morphological changes to the tissues of the central nervous system. The NOAEL for developmental neurotoxicity in this study was 1200 mg/kg. DISCUSSION

The U.S. EPA issued the Test Rule for health effects testing of IPA because of the substantial quantities of IPA produced and the potential for human exposure. At the time, the U.S. EPA had concluded that the existing toxicity data for IPA were inadequate to assess the human health risks associated with IPA exposure. Now a comprehensive toxicity testing program is complete for IPA that will allow for improved human health hazard assessment. The results of the testing program provide insight into IPA’s capacity to induce systemic (noncancer) injury, effects on the nervous system, effects on developing offspring, deficits in reproduction, genotoxicity, and cancer. Table 3 lists the lowest observed advserse effect levels (LOAELs) and the NOAELs of all of the toxicity endpoints in the IPA Test Rule studies. The exposure concentrations from the inhalation studies (acute neurotoxicity, subchronic, and chronic studies) were converted to a mg/kg/day dose so that the

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LOAELs and NOAELs could be compared to the oral gavage studies. Systemic (noncancer) toxicity assessment for IPA is provided by the subchronic, chronic, and two-generation reproductive toxicity studies. These studies demonstrate that IPA produces systemic toxicity with little relevance to human health. IPA does, however, target the male rat kidney. In the two-generation reproductive toxicity study, kidney effects were observed in the male rats dosed orally with 100, 500, and 1000 mg/kg IPA for at least 10 weeks. Increased hyaline droplets in the convoluted proximal tubular cells, epithelial degeneration and hyperplasia, and proteinaceous casts were observed. These renal changes appear to be similar to that reported for hyaline droplet nephropathy, also known as a2u-globulin nephropathy (Swenberg et al., 1989). This nephropathy is characterized by a malerat-specific nephrotoxicity due to excessive accumulation of a2u-globulin (a low-molecular-weight protein) in the phagolysosomes of renal proximal tubular cells (Swenberg et al., 1989; Borghoff et al., 1990). Because a2u nephropathy is unique to the male rat, it is not considered relevant for assessing human health risk (U.S. EPA, 1991). Although the kidney lesions in the IPA reproductive study appear histopathologically similar to a2u-globulin, confirmation will require further studies. Immunohistochemical studies using a2u-globulin antibody staining are needed to show that the accumulating protein in the hyaline droplets is a2u-globulin. Other studies with IPA have also shown male-ratspecific kidney effects. Increased formation of hyaline casts and content of hyaline droplets in the proximal tubules were reported in male rats given 1–4% IPA in the drinking water for 12 weeks (Pilegaard and Ladefoged, 1993). In the subchronic studies, however, hyaline droplets were increased in size and frequency in the kidneys of male rats, but these changes were not associated with any cytotoxic effects. Thus, although treatment-related, the hyaline droplet accumulation observed in the rat subchronic study may not be considered an adverse effect. It is interesting to note that pathological kidney damage occurred in male rats when exposure was by oral gavage or via drinking water, but not by inhalation exposure. One possible explanation for this difference could be pharmacokinetic differences of IPA or its metabolites, which could be binding to the a2u-globulin to produce the kidney effects. Kidney disease was also evident in rats chronically exposed to IPA. The major cause of death for the highdose rats and for many of the middose male rats in the oncogenicity study was chronic progressive nephropathy. Chronic progressive nephropathy is the single most common spontaneous renal disease in adult and aged rats (Gray, 1977; Goldstein et al., 1988). The pathogenesis of chronic progressive nephropathy is not known; however, consumption of a high-protein diet

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many be a contributing factor (Goldstein et al., 1988). The mechanism by which IPA exacerbates chronic progressive nephropathy is not known. IPA effects on the nervous system are assessed from studies conducted in both adult and developing animals. In general, the findings of the neurotoxicity studies indicate that IPA is a low hazard to the nervous system. The studies in the adult rats demonstrate that acute or repeated exposure to high concentrations of IPA vapor produces a reversible central nervous system depression. This finding is consistent with the narcotic action of other aliphatic alcohols, such as ethanol (Rall, 1990). Repeated exposure to a high concentration of IPA (5000 ppm) for at least 4 weeks resulted in increased motor activity in female rats. Increased motor activity has been previously reported in rats with ethanol in the drinking water over an extended period of time (Cicero et al., 1971; Capaz et al., 1981; Barros et al., 1991). This response is also typically observed with chemicals and drugs that are CNS depressants. The study of the developing rodent also demonstrates a lack of neurotoxic effects for IPA. This finding is consistent with previous studies conducted prior to the IPA Test Rule on other short-chain alcohols. Ethanol, 1-propanol, 1-butanol, and t-butanol have not shown any neurotoxic effects during fetal development from in utero exposure (Nelson et al., 1985, 1988a,b, 1989a,b,c). IPA developmental hazard potential is characterized in rat and rabbit developmental toxicity studies and in the above developmental neurotoxicity study. These studies indicate that IPA is not a selective developmental hazard. IPA produced developmental toxicity in rats, but not in rabbits. In the rat, the developmental toxicity occurred only at maternally toxic doses and consisted of decreased fetal body weights, but no teratogenicity. IPA has also been tested for developmental toxicity in rats by the inhalation route of exposure (Nelson et al., 1988b). Female Sprague–Dawley rats were exposed by inhalation to 0, 3500, 7000, or 10,000 ppm IPA 7 hr/day during Gestational Days 1 to 19. At 7000 and 10,000 ppm, the dams showed unsteady gait and narcotization during the initial exposures, decreased food consumption, and decreased weight gain. Fetal body weights per litter were reduced in all exposure groups. Exposure to 10,000 ppm also resulted in failure of implantation, fully resorbed litters, increased resorptions per litter, and increased incidence of rudimentary cervical ribs. To compare the findings of both the rat developmental toxicity studies, the inhalation exposure was converted to a mg/kg/day dose. Thus, inhalation exposures of 3500, 7000, and 10,000 ppm (8603, 17,207, and 24,581 mg/m3) are converted to 2079, 4158, and 5940 mg/kg/day, respectively3. The corresponding oral 3 mg/kg day Å mg/m3 1 inhalation rate of a rat (0.29 m3/day) 1 conversion from a day or 24-hr exposure to a 7-hr exposure 1 1/body weight of a rat in kilograms (1/0.35 kg).

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doses are 400, 800, and 1200 mg/kg/day, respectively. Reduced fetal body weights were observed in both studies, but the inhalation study reported pre- and postimplantation loss and increased incidence of rudimentary cervical ribs. The differences in the developmental findings are likely due to the higher doses employed in the inhalation study, as well as the rat strain. A two-generation reproductive study depicts the reproductive hazard for IPA. This study found that the only reproductive parameter apparently affected by IPA exposure was a statistically significant decrease in male mating index of the F1 males. It is possible that the change in this reproductive parameter was treatment-related and significant, although the mechanism of this effect could not be discerned from the results of the study. However, the lack of a significant effect of the female mating index in either generation, the absence of any adverse effect on litter size, and the lack of histopathological findings of the testes of the high-dose males suggest that the observed reduction in male mating index may not be biologically meaningful. Additional support for this conclusion is provided by the fact that most of the females became pregnant. Furthermore, male and female fertility and female fecundity indices of rats dosed with IPA were not different from those of controls by statistical analysis and were within, or relatively close to, historical control values. No reproductive effects were noted in other studies conducted prior to the IPA Test Rule in which rats were dosed up to 2% in the drinking water (BIBRA, 1988, 1990; FDRL, 1975; Gallo et al., 1977). Characterization of the genotoxicity hazard for IPA is provided by both in vitro and in vivo mutation/chromosomal studies. These studies demonstrate that IPA is not a hazard for genotoxic effects. IPA was found not to be mutagenic in an in vitro CHO/HGPRT assay and did not increase micronuclei in an in vivo micronuclei assay in mice. Mutagenicity studies conducted prior to the Test Rule also showed that IPA was not mutagenic in the Salmonella reverse mutation plate incorporation assay using strains TA97, TA98, TA100, TA1535, and TA1537 in the presence or absence of an S9 metabolic activation system (Zeiger et al., 1992). IPA was similarly not mutagenic in the Salmonella/microsomal assay using the spot test in strains TA98, TA100, TA1535, and TA1537, both with and without S9 from Arochlor-induced rats (Florin et al., 1980). In vitro sister chromatic exchange assays on IPA using cultured V79 cells both with and without S9 activation were also negative (von der Hude et al., 1987). No effect on meiotic nondisjunction or subsequent aneuploidy was observed in crossed strains of Neurospora crassa following treatment with IPA (Brockman et al., 1984). Finally, IPA did not induce transformation in Syrian hamster embryos infected with Simian SA7 virus (Heidelberger et al., 1983). Two chronic-exposure, rodent studies evaluated IPA

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for cancer potential. These studies demonstrate that IPA does not exhibit carcinogenic potential for relevant cancer effects. The only statistically significant elevated tumors concluded from either study were interstitial (Leydig) cell tumors in the male rats. Interstitial cell tumors of the testis is typically the most frequently observed spontaneous tumor in aged male Fischer 344 rats (Takaki et al., 1989; Haseman and Arnold, 1990). Nearly all male Fischer rats will develop these proliferative tumors if they are allowed to complete their lifespan (Boorman et al., 1990). From numerous 2-year NTP carcinogenicity studies, the mean incidence of these tumors is 88% in control male Fischer rats (Haseman and Arnold, 1990). Interstitial cell adenomas of the testes are believed to represent severe hyperplasia rather than autonomous growth (Boorman et al., 1990). It is often difficult to differentiate between focal hyperplasia and adenomas in the testes of male F344 rats because these interstitial adenomas originate as focal hyperplasia and the transformation from hyperplasia to adenoma represents a continuous spectrum of morphologic change occurring within the testes of these aged male rats. Furthermore, there was no evidence from this study to indicate the development of carcinomas of the testes in the male rat, nor has IPA been found to be genotoxic (Kapp et al., 1993). Thus, the relevance of the testicular tumors in the IPA-exposed male rats is probably of no significance in terms of human cancer risk. CONCLUSIONS

In summary, the TSCA Section 4 studies demonstrate that IPA in general has a low overall hazard potential for human health. IPA has a low order of acute and repeated exposure systemic (noncancer) toxicity, does not produce adverse effects on reproduction, is not a selective developmental toxic substance or a developmental neurotoxicant, is not genotoxic, and is not an animal carcinogen. IPA is, however, a potential hazard for transient central nervous system depression effects at high exposure levels. In addition, IPA produces effects to several rodent toxicity endpoints at high dose levels (including motor activity, male mating index, and exacerbated renal disease), which are of unclear relevance to human health. ACKNOWLEDGMENTS This work was sponsored by the Isopropanol Panel of the Chemical Manufacturers Association. The authors acknowledge the numerous significant contributions and suggestions made during the course of this work by James F. Quance, Heather D. Burleigh-Flayer, Rochelle W. Tyl, Robert H. Garman, Richard H. McKee, Michael W. Gill, Larry S. Andrews, Lawrence W. Masten, Bruce K. Beyer, Dale E. Strother, Carolyn A. Matula, J. Michael Cleverdon, and Richard C. Wise. The authors also thank Cecilia W. Spearing, Isopropanol Panel Manager, for her oversight of the Panel activities.

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