Inhalation Two-Generation Reproductive Toxicity Study of Methyl Isobutyl Ketone in Rats

Abstract

To evaluate whether methyl isobutyl ketone (MIBK) affects reproductive performance, a two-generation reproduction study was conducted. MIBK was administered to 30 Sprague-Dawley rats/sex/group via whole-body inhalation at concentrations of 0, 500, 1000, or 2000 ppm, 6 h daily, for 70 days prior to mating. F₀ and F₁ females were exposed from mating through gestation day 20 and from postnatal day 5; F2 litters were maintained through post-natal day 21. No treatment-related mortality of adult animals occurred. There was a dose-related increase in adult animals with no or a decreased response to a sound stimulus at 1000 and 2000 ppm; however, no adverse clinical signs occurred 1 h after exposure, suggesting this was a transient sedative effect. Clinical signs of central nervous system (CNS) depression in the pups were observed and one F₁ pup died after initial exposure to 2000 ppm on postnatal day 22; subsequently exposure was delayed until postnatal day 28. Decreased body weight gain and slight decreased food consumption were observed during the first 2 weeks of exposure in both generations at 2000 ppm. There were no adverse effects on male and female reproductive function or landmarks of sexual maturation. Increased F₀ and F₁ liver weights with associated centrilobular hypertrophy occurred in rats at 2000 ppm, indicative of an adaptive response. Increased male kidney weights at all exposure concentrations, associated with hyaline droplets, were indicative of male rat-specific nephropathy. Other than acute sedative effects, the no-observed-adverse-effect level (NOAEL) for parental systemic effects (excluding male rat kidney) was 1000 ppm, based on transient decreased body weight and food consumption; for reproductive effects, 2000 ppm, the highest concentration tested; and for neonatal toxicity, 1000 ppm (based on acute CNS depressive effects).

Methyl isobutyl ketone (MIBK: 2-methyl-4-pentanone, CAS RN 108-10-1) is a clear, flammable liquid (United States Environmental Protection Agency [USEPA] 1994) produced for general commercial use as a solvent in lacquers, paints, and varnishes; in vinyl, epoxy, acrylic, and natural resins, and in nitro-cellulose and dyes. To a lesser extent, MIBK is used as a solvent for rare metal extraction, extraction and purification of antibiotics, and pesticide manufacturing (USEPA 1994). The most common route of human exposure to MIBK is expected to be via vapor inhalation; dermal contact is also possible in the work-place (USEPA 1994). The threshold-limit value (TLV) is 50 ppm (American Conference of Industrial Hygienists [ACGIH] 2001), and the Occupational Safety and Health Administration (OSHA) permissible exposure level (PEL) is 100 ppm as an 8-h time-weighted average (TWA) (Federal Register 58:35338–35351, June 30, 1993: corrected Federal Register 58:40191, July 27, 1993).

The toxicity of MIBK in experimental animals has been extensively investigated. Exposure of female and male rats and mice to MIBK at levels up to 1000 ppm, 6 h/day, for 14 weeks showed no effects, except for kidney damage in male rats characterized by hyaline droplet formation (Phillips et al. 1987). In a more recent study designed to evaluate MIBK’s potential to produce neurobehavioral effects, male Sprague-Dawley rats were exposed to 0, 250, 750, or 1500 ppm MIBK for 6 h/day, 5 days/week, for 13 weeks, using schedule-controlled operant behavior (SCOB) paradigm. Although acute clinical signs of transient, reduced activity were reported at 750 and 1500 ppm during the first 10 weeks of exposure, the authors concluded that 13 weeks of repetitive exposures to high concentrations of MIBK did not alter operant behavior in the rat (David et al. 1999).

Developmental toxicity studies on MIBK have been conducted in two species (Tyl et al. 1987), where MIBK was administered by whole-body inhalation to Fischer 344 rats and CD-1 mice on gestation days (GDs) 6 to 15 at 0, 300, 1000, or 3000 ppm. Clinical signs of central nervous system (CNS) depression were reported in both species from the 3000-ppm exposure. The authors concluded that, under maternally toxic conditions of exposure, the top concentration was associated with fetal toxicity described as reduced fetal body weight and delayed skeletal ossification in both the rat and mouse. Inhalation exposure to 300 or 1000 ppm showed neither maternal nor developmental toxicity.

The present MIBK whole-body inhalation two-generation reproduction study was conducted in rats to assess reproductive performance. Based on results from the studies described above, rats were exposed to MIBK at 0, 500, 1000, or 2000 ppm, 7 days/week for at least 70 days prior to mating and throughout mating, gestation, and lactation. This study was conducted as part of the Toxic Substance Control Act (TSCA) enforceable consent agreement with the USEPA (Federal Register 64:20298–20300, April 26, 1999) and was performed according to the most recent regulatory guidelines (USEPA 1998) and principles for Good Laboratory Practices (USEPA 1989a, 1989b).

MATERIALS AND METHODS

Test Material

MIBK was supplied as a clear, colorless liquid from TR Metro Chemicals, Avenel, NJ. The purity and stability of MIBK were verified by gas chromatography and infrared analysis. Results obtained indicated the MIBK was 99.79% to 99.93% pure. Although the identity and composition of impurities in the MIBK were not characterized, their trace presence in the test sample were not considered to affect the outcome of the study.

Animals and Husbandry

Virgin female and male Crl:CD (SD)IGS BR Sprague-Dawley rats (approximately 29 days old upon arrival) were obtained from Charles River Breeding Laboratories, Portage, MI. During the 23-day acclimation period, animals were observed twice daily for mortality and moribundity. All rats were individually housed, except during mating, in suspended wire-mesh-bottomed, stainless steel cages. Basal diet (PMI Nutrition International, Certified Rodent Lab Diet 5002) and reverse osmosis–treated water were available ad libitum throughout the study, except during exposure. Prior to and after the exposure period, all animals were kept on a 12-h photoperiod, at 72°F ±4°F and 30% to 70% humidity. Animals found to be in good general health were allocated to groups based upon body weight stratification and randomized in a block design by a computer-generated program (WIL Toxicology Data Management System). This study was conducted in accordance with the Animal Welfare Act (9 CFR Parts 1, 2, and 3).

Exposure Conditions

Each group of animals was exposed in a 2.0-m³ stainless steel and glass whole-body Hazleton 2000 inhalation chamber operated under dynamic conditions. MIBK was generated as a vapor independently for each exposure chamber. Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 min) during each daily exposure period via on-line gas chromatography, via sensors placed in approximately the center of the chamber, within the general breathing zone of the animals. At least one standard was analyzed each day prior to exposure to confirm gas chromatographic (GC) calibration. There were no interferences with GC chamber concentration determinations. Chamber temperature (18°C to 26°C), relative humidity (30% to 60%), ventilation rate (12 to 15 air changes per hour), and negative pressure within the chambers were monitored. Each chamber was dedicated to one exposure group. In order to minimize any potential variation occurring due to positioning within the chamber, the cages were sequentially rotated around the available rack positions within the chamber on a daily basis throughout the study.

Experimental Design

Four groups of 30 F₀ males and 30 F₀ females were randomly bred to produce an F₁ generation. A replication of the breeding procedure (avoiding sibling mating) was conducted in the second generation (30 animals/sex/group) to produce an F₂ generation. The F₀ and F₁ generations were approximately 7 weeks old and 4 weeks old at initiation of their respective exposures. The animals were exposed 6 h/day, 7 days/week, to MIBK at concentrations of 500, 1000, or 2000 ppm (Figure 1). The control group was exposed to clean, filtered air under identical conditions as the MIBK-exposed groups. F₀ and F₁ males were exposed for 10 weeks prior to mating and throughout mating until one day prior to euthanasia. F₀ and F₁ females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until 1 day prior to euthanasia. During the mating period, each female was housed overnight in the home cage of a specific, nonsibling, male (1:1) throughout the mating period until evidence of mating was detected. The observation of a copulatory plug in the vagina or the presence of sperm in a vaginal smear confirmed positive evidence of mating and that day was termed GD 0 and the animals were separated. Exposure of the F₀ and F₁ dams was suspended for 5 days following parturition (lactation/postnatal days 0 to 4), to avoid confounding nesting and nursing behavior and neonatal survival during early post-natal development; exposure resumed on postnatal day (PND) 5. The dams were removed from the litters in the home room for the daily 6-h exposure period during lactation. The animals were exposed to MIBK at approximately the same time each day.

Each dam and litter remained housed together until weaning on PND 21. Male and female F₁ pups (30/sex/group) were randomly selected prior to weaning (PND 21) to comprise the F₁ generation. The offspring of the F₀ and F₁ generations (F₁ and F₂ pups, respectively) were potentially exposed to MIBK in utero, via milk through nursing during PNDs 0 to 21 and for F₁ pups, via direct exposure following weaning. These F₁ weanlings were first exposed to MIBK for 6 h beginning on PND 22; however, exposures were suspended on PND 22 due to the death of one male pup in the 2000-ppm group and clinical signs of CNS depression indicative of a sedative effect. Exposures for all subsequent groups of F₁ weanlings were also suspended but daily exposures were reinitiated for all surviving animals on PND 28.

Observations and Measurements

Detailed physical examinations were recorded weekly for all parental animals (F₀ and F₁). All animals were observed twice daily for appearance, behavior, moribundity, mortality, and pharmacotoxic signs within 1 h after completion of exposure. Additionally, at the approximate midpoint of each day’s exposure, a striker device, consisting of schedule 80 PVC ¾-inch pipe (∼50 g) attached to a 70-cm cotton rope with a 60-cm fulcrum point, was used to produce a loud noise/novel stimuli on the front glass of the exposure chamber. The animals in cages accessible to viewing (8 to 17 animals/group) were observed qualitatively for a reaction to the stimulus and their responses were classified as either no reaction; ear flick or some evidence the stimulus was heard; or more energetic response (e.g., jump, flinch, vocalization). Females were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

Individual F₀ and F₁ male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual F₀ and F₁ female body weights were recorded weekly until evidence of copulation was observed and on GDs 0, 4, 7, 11, 14, and 20 and PNDs 1, 4, 7, 14, and 21. Parental food consumption was determined at the same time as the body weight measurements, except during the mating period when measurement of food consumption was suspended due to cohabitation.

To assess estrous cyclicity, vaginal smears from each F₀ and F₁ female was evaluated daily beginning 3 weeks prior to mating and continuing until mating was observed. Females were allowed to deliver naturally and nurture their young to weaning (PND 21). On the day parturition was judged complete (PND 0), pups were sexed and examined for external malformations, and the numbers of stillborn and live pups were recorded. Intact offspring dying from PNDs 0 to 4 were necropsied using a fresh dissection technique (Stuckhardt and Poppe 1984). Pups observed with external abnormalities that warranted further skeletal examination were eviscerated and stained (Dawson 1926) for sub-sequent skeletal evaluation (F₁ pups were stained accordingly, i.e., only if warranted).

To reduce variability among the litters, eight pups/litter (four/sex when possible) were randomly selected on PND 4 using a computer-generated selection procedure, except for litters with fewer than eight pups. Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup was individually weighed and received a detailed physical examination on PNDs 1, 4, 7, 14, and 21. Pups were also individually sexed on PNDs 0, 4, 7, 14, and 21.

Each male pup was examined for balanopreputial separation beginning on PND 35 (Korenbrot, Huhtaniemi, and Weiner 1977) and each female for vaginal perforation beginning on PND 25 (Adams et al. 1985). These observations continued until all animals reached these criteria (i.e., balanopreputial separation and vaginal perforation). Pup body weights were recorded on the day of acquisition of these landmarks.

Samples of sperm from the right epididymis were collected from each adult F₀ and F₁ male and evaluated for the percentage of progressively motile sperm. Motile sperm were evaluated using the Hamilton-Thorne HTM-IVOS (Integrated Visual Optical System) Version 10 computer-assisted sperm analysis (CASA) system. Sperm morphology was evaluated by light microscopy via a modification of the wet-mount evaluation technique (Linder et al. 1992). Also, the left testis and epididymis from all F₀ and F₁ males in all dose groups were evaluated for homogenization-resistant spermatid counts and sperm production rate (Blazak, Ernst, and Stewart 1985) using the HTM-IVOS system.

Necropsy and Histopathologic Examination

Surviving F₀ adults were euthanized and necropsied following the selection of the F₁ generation and surviving F₁ adults were euthanized and necropsied following weaning of the F₂ pups. The stage of estrus on the day of necropsy was determined for all F₀ and F₁ females and selected F₀ and F₁ parental tissues and organs were fixed in 10% neutral-buffered formalin for possible histopathological examination. Microscopic evaluations were performed on the following tissues for F₀ and F₁ parental animals (10/sex/group) from the control and high-dose groups and for all adult animals found dead or euthanized in extremis: adrenal glands, prostate, brain, spleen, thymus, liver (all F₀ animals examined and all F₁ males examined), kidneys (all F₀ animals examined and all F₁ males examined), lung, pituitary, seminal vesicles, the right epididymis (caput, corpus and cauda), the right testis, vas deferens, vagina, cervix, coagulating gland, uterus, oviducts, ovaries (one section from each ovary from F₀ females was examined). Periodic Acid Shift (PAS) and hematoxylin staining were used for the right testis and epididymis and hematoxylineosin staining was used for all other tissues. Quantitative histopathologic evaluation of 10 sections of the inner third of the ovary (including enumeration of primordial follicles) was conducted on 10 F₁ females from the 0-and 2000-ppm groups (Bolon et al. 1997; Bucci et al. 1997). A qualitative assessment for the presence or absence of growing follicles, astral follicles, and corpora lutea was also performed.

Organs weighed from all F₀ and F₁ parental animals included adrenals, prostate, brain, seminal vesicles with coagulating glands (with accessory fluids), epididymides (weighed separately: total and cauda), kidneys, spleen, liver, testes (weighed separately), ovaries thymus, pituitary, and uterus with oviducts and cervix.

On PND 21, a complete necropsy similar to that performed on parental animals was conducted on F₁ pups not selected for MIBK exposure and on F₂ pups. Brain, spleen, and thymus gland weights were also recorded from these pups.

Statistical Analyses

All analyses were conducted using two-tailed tests (except as noted below) for a minimum significance level of 5% comparing each treated group to the control group. Data obtained from nongravid animals were excluded from statistical analyses following the mating period.

Parental mating and fertility indices were evaluated by the χ² test (Dixon and Brown 1979) with Yates’ correction factor. Parental weekly body weights and weight changes, gestation and lactation body weights and body weight changes, parental food consumption, food efficiency, mean gestation length, pre-coital interval, implantation sites, unaccounted implantation sites, pup body weights, mean litter weights, absolute and relative organ weights, live litter size, sperm production rate, sperm numbers, ovarian primordial follicle counts, number of pups born, balanopreputial separation (mean day of acquisition and body weight), and vaginal patency (mean day of acquisition and body weight) were subjected to a one-way analysis of variance (Dixon and Brown 1979). If the analysis of variance (ANOVA) was significant, Dunnett’s test (Dunnett 1964) was used to determine which treated groups differed from the control.

Sperm motility and morphology, and proportional postnatal offspring survival were analyzed by the Kruskal-Wallis test (Dixon and Brown 1979) to assess differences in group means. The Mann-Whitney U test (Dixon and Brown 1979) was used to determine which treatment groups differed significantly from control. Histopathologic findings were evaluated using a one-tailed Kolmogorov-Smirnov test (IBM 1971). Pup organ weights by litter were analyzed by using an analysis of covariance (with the litter size as the covariant) and Student’s t test (SAS Institute 1997).

RESULTS

Chamber Exposure Concentrations

Figure 1 indicates that the targeted exposure concentrations of 0, 500, 1000, and 2000 ppm were achieved and remained consistent throughout the study.

F₀ Observations and Reproduction Data

F₀ Adults

There were no MIBK-related mortalities or clinical signs of toxicity noted during the study. However, an increase in the number of observations of F₀ rats having an absent or decreased response to a novel sound stimulus (a single loud noise at the midpoint of exposure) was observed in the 1000- and 2000-ppm groups (Table 1); however, the animals appeared normal at the 1 h postexposure observation (data not shown). No other MIBK-related clinical findings were observed at any exposure level evaluated (data not shown).

The regularity and duration of estrus were not affected by exposure. The mean lengths of estrous cycles were 4.2, 4.1, 5.0, and 4.2 days in the 0-, 500-, 1000-, and 2000-ppm groups, respectively (data not shown). Furthermore, no adverse exposure-related effects were observed on the number of days between pairing and coitus, gestation lengths or reproductive performance, e.g., fertility and mating indices (Table 2). Exposure to MIBK also had no effect on F₀ spermatogenic end points, e.g., mean testicular and epididymal sperm numbers, sperm production rate, and sperm motility and morphology (Table 3).

Weekly body weights and body weight gains were unaffected by MIBK exposure in the F₀ female rats in the 500- and 1000-ppm groups and in F₀ male rats at all exposure levels evaluated (Figures 2 and 3). Statistically significant reductions in body weight gains in the 2000-ppm group F₀ females were observed during weeks 0 to 1 and 1 to 2 (data not shown). Although these slight, transient reductions were considered to be exposure related, weekly body weights were unaffected at these intervals. Female body weights and body weight gains in the 500-, 1000-, and 2000-ppm groups were comparable to the control group throughout gestation and lactation (Figure 3).

Statistically significant reductions in food consumption for both sexes were observed during weeks 0 to 1 at 2000 ppm (data not shown). These transient reductions, although slight, were considered to be exposure related. No other exposure-related trends were apparent. Weekly food consumption and food efficiency in the males and females were unaffected by 500- and 1000-ppm MIBK exposures. These parameters were unaffected in females during the gestation and lactation period exposures at 500, 1000, and 2000 ppm (data not shown).

Exposure-related increases in liver weights (absolute and relative to final body weights) occurred in the 2000 ppm group males and females (Table 4, absolute weights). Also, MIBK-related centrilobular hepatocellular hypertrophy was noted in 0, 3, 15, and 26 F₀ males in the 0-, 500-, 1000-, and 2000-ppm groups, respectively. This hepatic hypertrophy was characterized by centrilobular random disruption of normal plate structure/architecture by enlarged, rounded hepatocytes with variable increases in acidophilic cytoplasm containing basophilic clumping of organelles (Figure 4). Increased male absolute and relative kidney weights occurred in all exposure groups (Table 4) and correlated with an increased occurrence of nephropathy characterized by basophilic tubules with variable inflammation and thickening of the tubular basement membrane in the 1000 and 2000 ppm groups (Figure 5). However, kidney weights in the females were unaffected by exposure at all MIBK concentrations (Table 4).

Increased absolute and relative (to final body weight) seminal vesicle/coagulating gland weight were observed for males at 2000 ppm and increased absolute and relative ovary and adrenal gland weights were noted for females at 2000 ppm (Table 4). As these responses (ovarian and adrenal gland weight changes) were not reproduced in the F₁ parental animals and no correlating histopathologic findings were observed in the aforementioned tissues, the effects were considered spurious and not related to MIBK exposure.

F₁ Observations and Reproduction Data

F₁ Offspring

The number of pups born, live litter size, sex ratio at birth, pup survival at various intervals and pup body weights were unaffected by parental exposure (Table 5, Figure 6). However, on PND 14, transient decreases in mean pup body weights were observed in all MIBK-exposed groups. Although similar a reduction was observed on PND 14 in the 2000-ppm F₂ offspring, this reduction was not attributed to MIBK exposure because it was small [about 5%], not dose related, and occurred only on day 14 and not days 7 or 21 [Figure 6]. The day of acquisition of balanopreputial separation and vaginal patency was also un-affected by parental MIBK exposure (Table 6). Balanopreputial separation occurred in all male pups by PND 55 and all female pups had vaginal openings by PND 40.

No internal findings that could be attributed to parental MIBK exposure were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the presence or absence of milk in the stomach, no internal findings were noted. No exposure-related effects on absolute or relative brain, spleen, or thymus weights were observed in any of the MIBK exposure groups (Table 7).

F₁ Adults

In the second parental generation, one test article-related mortality occurred in the 2000-ppm male group on PND 22. How-ever, approximately 1 h post exposure on PND 22 (the first day of exposure of F₁ pups), 7 males and 11 females in the 2000-ppm group exhibited clinical signs of neuro- or neuromus-cular toxicity, i.e., rocking, lurching, or swaying while ambulating; 5 males and 2 females were prostrate, and 3 males had half-closed eyelids. Of these animals, 9 males and 10 females also exhibited bilateral lacrimation. Because of the single mortality of the 2000-ppm F₁ male and the CNS depression or sedation noted in these animals, exposures for all groups of F₁ weanlings were suspended through PND 27. After exposures were reinitiated on PND 28, only 6 males (out of 30) in the 2000-ppm group exhibited the previously observed clinical signs of sedation 1 h post exposure. CNS depression proved to be transient as it was not observed on subsequent days. Furthermore, no recurrence was observed in females upon resumption of exposure. Similar to findings in the F₀ rats, an increased number of observations of rats having absent or diminished response to a novel sound stimulus was observed in the 1000-ppm group males and the 2000-ppm group males and females during the exposure period (Table 1).

The regularity and duration of estrus were not affected by exposure. The mean lengths of estrous cycles were 5.1, 4.5, 4.7, and 4.3 days in the 0-, 500-, 1000-, and 2000-ppm groups, respectively (data not shown). Furthermore, no adverse exposure-related effects were observed on the number of days between pairing and coitus, gestation lengths or reproductive performance, e.g., fertility and mating indices (Table 2). Exposure to MIBK also had no effects on F₁ spermatogenic endpoints, e.g., mean testicular and epididymal sperm numbers, sperm production rate, and sperm motility and morphology (Table 3).

Weekly body weights were slightly reduced throughout the study in the 2000-ppm group males and throughout the prebreeding and postlactational phases in the 2000-ppm group females (Figures 7 and 8). Although these body weight differences from the control group were statistically significant throughout the study, weekly body weight gains were comparable among all exposure groups after weeks 17 to 18 for F₁ males and weeks 18 to 19 for F₁ females. The differences in body weight noted throughout the remainder of the F₁ generation exposures were due primarily to these initial reductions in weight gain, and not to any differences in weight gain induced by exposure to the test substance.

Reductions in body weight were observed in the 1000-ppm group males and a transient reduction in body weights was noted for males in the 500-ppm group only during weeks 17 to 19 (Figure 7). Mean body weight gain in the 1000 ppm group was reduced only during the first week of the generation (data not presented). The value for this group, however, was only 8.3% (or 4 g) lower than the control group. A statistically significant reduction in body weight for the 1000-ppm group females was observed during week 17 (Figure 8). With the exception of weeks 17 to 18 for the males, no correlating reductions in mean body weight gain or food consumption were observed in the 500- or 1000-ppm groups. Therefore, the transient reductions in mean body weight observed in these groups were not considered adverse effects of exposure. Slight increases in body weight gains for the 2000-ppm group during gestation and lactation had no effect on mean body weight during those periods (Figure 8).

Statistically significant reductions in food consumption were observed for both sexes in the 2000-ppm group during the first week that food consumption was measured for the F₁ generation (data not shown). No other exposure-related trends were apparent. Weekly food consumption and food efficiency in the males and females were unaffected by 500- and 1000-ppm MIBK exposures. Additionally, these parameters were unaffected in females during the gestation and lactation period exposures at 500, 1000, and 2000 ppm (data not shown).

There were no unusual or MIBK-related postmortem findings noted at necropsy (data not shown). However, absolute and relative (to final body weight) liver weights were significantly increased for males and females in the 2000-ppm group (Table 4, absolute weights). As with the F₀ animals, exposure-related increases in centrilobular hepatocellular hypertrophy were noted in 18/30 F₁ males in the 1000-ppm group and in 20/30 F₁ males in the 2000-ppm group, with increased severity in the 2000-ppm group males (Figure 4). Other slight changes, e.g., brain, spleen, prostate weights and seminal vesicles/coagulating gland weight, were not considered MIBK-exposure related, as there were no corresponding histopathologic alterations and/or effects on spermatogenic endpoints. Ovarian primordial follicle counts (Table 4) and the presence or absence of corpora lutea (data not shown) were unaffected by MIBK exposure in the 2000 ppm group.

Nephropathy and homogeneous, developed acidophilic and spherical inclusions/droplets (up to 4 to 5 μgn diameter) in the renal cortical tubular epithelium were observed in the MIBK-exposed males (Figure 5). Correlating increases in absolute and relative (to final body weight) kidney weights were also observed (Table 4, absolute weights). However, absolute kidney weights were unaffected by MIBK exposure and nephropathy was not observed in females. Relative kidney weights were statistically increased in the female 2000-ppm group, but no correlating histopathologic findings were observed.

F₂ Observations

F₂ Offspring

The number of pups born, live litter size, sex ratio at birth, pup survival at various intervals, and pup body weights were unaffected by parental exposure to MIBK (Table 5). A transient reduction was observed in pup body weights (males, females, and both sexes combined) on PND 14 in the 2000 ppm F₂ off-spring. This reduction was not attributed to MIBK exposure because it was small (about 5%), not dose related, and occurred only on day 14 and not day 7 or 21 (Figure 9). There were no unusual postmortem observations that were considered to be exposure related (data not shown). No exposure related internal findings were observed at necropsy of the F₂ weanlings selected for organ weights. Furthermore, no exposure-related effects on absolute or relative (to final body weight) brain, spleen, or thymus weights were observed in any of the MIBK exposure groups (Table 7).

DISCUSSION

The objective of this study was to evaluate the potential adverse effects of MIBK inhalation exposure on the reproductive capacity in two generations of Crl:CD (SD)IGS BR male and female Sprague-Dawley rats. The current study confirmed earlier findings of MIBK-induced CNS depression; a dose-related increase in the number of F₀ and F₁ parental animals with absent or diminished response to a novel sound stimulus was noted during exposure at the 1000- and 2000-ppm concentrations. The response rate was unaffected at 500 ppm, suggesting a sedative effect during exposure at the higher concentrations. A previous MIBK inhalation study has reported CNS depression at concentrations of 1500 ppm and above (David et al. 1999). Because weanling animals (2000 ppm) proved to be more susceptible to the acute sedative effects of MIBK, exposures for all groups of F₁ weanlings were suspended through PND 27. Rats at 21 days old were very sensitive to CNS depression of MIBK, as for other solvents, which is considered an acute pharmacological effect. Effects were much less pronounced when exposures were restarted at PND 28, a more usual age to begin perinatal exposures. These clinical signs of CNS depression lessened once weanling exposures were reinitiated.

Parental effects of MIBK exposure were expressed at the 2000-ppm exposure level in both F₀ and F₁ generation males and females. Initial, transient reductions in body weight gain were paralleled by and reflective of transient reductions in food consumption. However, food efficiency was unaffected at all exposure levels evaluated in both the F₀ and F₁ generations. Therefore, these slight decreases in body weight gain and food consumption may be related to transient sedative effects of MIBK at high exposure concentrations.

Parental effects of MIBK were expressed by changes in absolute and relative organ weights, i.e., increased absolute and relative liver weights for F₀ and F₁ males and females at 2000 ppm, and increased relative liver weights for F₁ males at 1000 ppm. Upon histopathologic examination, a correlating MIBK exposure–related centrilobular hepatocellular hypertrophy was noted with increased severity at the 2000-ppm level. Although similar histopathologic effects were reported in a rat subchronic inhalation study (Phillips et al. 1987), they are considered an adaptive physiological response to an intensified metabolic liver burden (Schulte-Hermann 1974). This adaptive effect has been shown to regress after termination of exposure to xenobiotics, and generally is deemed a nontoxic effect (Schulte-Hermann 1974; Farber 1980; Popp and Cattley 1991). Parental toxicity was also expressed in F₀ and F₁ males, at all exposure levels, as increased kidney weights and nephropathy. Both generations exhibited basophilic tubules with inflammation and thickening of the tubular basement membrane but the F₁ males also exhibited acidophilic inclusions/droplets. Tubular degeneration, cell loss and regeneration, and large hyaline droplets typical of fully developed lesions of α2μ-globulin nephropathy have previously been reported to be induced in male rats by MIBK (Phillips et al. 1987). Because these findings were only observed in male rats and the α2μ-globulin–mediated nephropathy is considered a male rat-specific effect (Haschek and Rousseaux, 1998), this response was not considered a relevant endpoint for human hazard identification (Baetcke et al. 1991) and is not considered in the determination of the no-observed-adverse-effect level (NOAEL).

In conclusion, MIBK, at all exposure levels, did not affect any reproductive parameters nor offspring growth or development. The NOAEL for parental systemic toxicity (apart from male nephropathy) was considered to be 1000 ppm, based on transient reduced body weight gain and food consumption. The NOAEL for neonatal toxicity (based on acute CNS depressive effects, and one death on PND 22) was also considered to be 1000 ppm. The NOAEL for reproductive toxicity was considered to be 2000 ppm, the highest concentration tested.