Two-Generation Reproductive Toxicity Study of Inhaled Acrylonitrile Vapors in Crl:CD(SD) Rats

Abstract

To assess the effects of acrylonitrile (AN) exposure on reproduction, Sprague-Dawley rats (25/sex/group) were exposed to vapor atmospheres of AN via whole-body inhalation at concentrations of 0, 5, 15, 45 (two offspring generations) and 90 ppm (one offspring generation), 6 h daily, 1 litter/generation, through F2 weanlings on postnatal day 28. After approximately 3 weeks of direct exposure following weaning, exposure of the F1 animals at 90 ppm was terminated due to excessive systemic toxicity in the males. There were no exposure-related mortalities in adult animals, no functional effects on reproduction or effects on reproductive organs, and no evidence of cumulative toxicity or of enhanced toxicity in pregnant and lactating dams or in developing animals. Adult systemic toxicity was limited to body weight and/or food consumption deficits in both sexes and generations (greater in males) at 45 and 90 ppm and increased liver weights in the 90 ppm F0 males and females and 45 ppm F1 males. Neonatal toxicity was expressed by F1 offspring weight decrements at 90 ppm. Clinical signs of local irritation during and immediately following exposure were observed at 90 ppm. Microscopic lesions of the rostral nasal epithelium, representing local site-of-contact irritation, were observed in some animals at 5 to 45 ppm. The no-observed-adverse-effect level (NOAEL) for reproductive toxicity over two generations and neonatal toxicity of AN administered to rats via whole-body inhalation was 45 ppm. The NOAEL for reproduction was 90 ppm for the first generation. The NOAEL for parental systemic toxicity was 15 ppm.

Acrylonitrile (CH₂=CH-CN) (AN) is a building-block chemical used to make acrylic fiber for clothing, blankets, carpeting and other fabrics, rugged plastics for computer and TV housings, and nitrile rubber for oil-resistant hoses at gasoline stations and under car hoods. Each year, more than 10 billion pounds of AN is produced worldwide at more than 50 manufacturing sites, including six in the United States.

AN is a strong contact irritant (EU Risk Assessment 2004). If inhaled at high concentrations, AN can produce serious short-term toxicity, including loss of consciousness or death. AN is carcinogenic in rats and mice (Kirman et al. 2005). A recent peer-review panel (Haber et al. 2005) described the occupational epidemiology data on AN as unusually extensive, with three striking features: (1) the size and completeness of the database, (2) the lack of consistently positive findings across studies, and (3) the lack of a clear dose-response relationship for human cancer. The International Agency for Research on Cancer (IARC 1999) has classified AN in Group 2B (possible human carcinogen). The American Conference of Governmental Industrial Hygienists (ACGIH) threshold-limit value (TLV) and the Occupational Safety and Health Administration (OSHA) permissible exposure level (PEL) for AN are both 2 ppm (4.3 mg/m²) as an 8-h time-weighted average. OSHA has also established a 15-min ceiling limit value of 10 ppm.

Reports from China of adverse reproductive effects among chemical workers exposed to AN have prompted renewed attention to the potential of AN to affect human reproduction (Li 1996; Wu et al. 1994, 1995). These studies reported findings of increased incidence of stillbirths, shortened duration of gestation, and increased incidence of developmental malformations (Collins et al. 2003; Neal et al. 2006). In addition, the Chinese studies reported evidence of male-mediated toxicity, impotence, male infertility, female sterility, increased incidence of spontaneous abortion and increased menstrual irregularities, and some evidence of concurrent systemic toxicity (primarily anemia and clinical signs). However, several issues were noted with the Chinese studies that reduced confidence in the reported conclusions, including lack of individual exposure monitoring, lack of information on concurrent chemical exposures, and a high background incidence of neural tube defects in some areas of China (Neal et al. 2006). The Neal review concluded that sufficient data exist to postulate an association between human AN exposure and adverse reproductive outcome, but the data were deemed insufficient to establish causation.

Numerous animal toxicity studies of AN exposure have been conducted, including assessments of developmental and reproductive toxicity (EU Risk Assessment 2004). The previously mentioned review by Neal found that the existing data were robust enough to expect that developmental toxicity was unlikely to occur in the absence of biologically significant and clinically noticeable maternal toxicity. The potential developmental toxicity of AN has been thoroughly assessed in the rat; this assessment includes three well-conducted studies with concordant NOAELs for maternal and developmental toxicity (Murray et al. 1978 [gavage and inhalation]; Saillenfait et al. 1993 [inhalation]). Decreased fetal body weights were noted in two of these studies (at 65 mg/kg/day [gavage] and 25 ppm and higher [inhalation], respectively) in the presence of maternal toxicity. Although not seen consistently across these studies, Murray et al. also reported adverse developmental outcomes at the highest doses of their studies.

Two previous reproductive toxicity studies of AN were conducted using the drinking water route. The first study was a one-generation study performed by Schwetz et al. (1975) and the second was a three-generation assessment conducted by Beliles et al. (1980) and subsequently published by Friedman and Beliles (2002). Based on the poor palatability of AN in water, the effects on pups during lactation in these studies were likely the result of decreased milk production stemming from inadequate water intake. Although there was no evidence of an effect on fertility in either study, assessment of this end point was compromised by poor control fertility in most generations of both studies. In addition, review of these studies revealed inadequate data on spermatogenesis end points, regularity and duration of estrus, reproductive organ histopathology in adult animals, and potential postnatal toxicity following prenatal exposure (relevant to an occupational exposure scenario).

The present two-generation reproduction study of AN in rats was conducted to provide information on potential reproductive effects via inhalation, the most likely route of occupational exposure, and to fully characterize effects on reproductive performance following AN exposure, including assessment of andrology, estrous cyclicity, and testicular and ovarian histopathology. Based on results from the studies described above, rats were exposed to AN at 0, 5, 15, 45, and 90 ppm, 7 days/week, for at least 70 days prior to mating and throughout mating, gestation, and lactation (Figure 1). Histopathological evaluation of nasal tissue was included in the study design to allow dose-response comparisons of the local irritant effects of AN to systemic responses. This study was performed in general accordance with the most recent regulatory toxicity test guidelines (US EPA 1998; OECD 2001) and in compliance with the principles for Good Laboratory Practices (US EPA 1989; OECD 1997).

MATERIALS AND METHODS

Test Material

AN was supplied as a clear, colorless liquid from BP Chemicals America, Port Lavaca, TX. The purity and stability of AN were verified by a gas chromatography method with flame ionization detection (GC/FIC). Results obtained indicated that AN was 99.94% to 99.98% pure.

Animals and Husbandry

Virgin male and female Crl:CD(SD) rats (38 days old upon arrival) were obtained from Charles River Laboratories, Raleigh, NC. During the 14-day acclimation period, animals were observed twice daily for mortality and moribundity. Rats were gang-housed by sex for 3 days, then individually housed, except during mating, in suspended wire-mesh, stainless steel cages. Following mating, the females were transferred to plastic maternity cages with nesting material. Basal diet (PMI Nutrition International, Certified Rodent LabDiet 5002) and reverse osmosis–treated water were available ad libitum throughout the study, except during exposure. Animals were kept on a 12-h photoperiod, at 71°F ± 5°F and 30% to 70% humidity. Animals found to be in good general health were allocated to groups based on body weight stratification and randomized in a block design by a computer-generated program (WIL Toxicology Data Management System). Animals in this study were maintained in accordance with the Animal Welfare Act (AWA 1966, as amended) and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources et al. 1996). The animal facilities at WIL Research Laboratories, LLC are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

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 (Figure 2 depicts the test atmosphere exposure generation system). AN 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 gas chromatography (GC), using sensors placed approximately in the center of the chamber, within the general breathing zone of the animals. One standard was analyzed each day prior to exposure to confirm GC calibration. Chamber temperature (20°C to 25°C), relative humidity (30% to 70%), ventilation (12 to 15 air changes per hour), and negative pressure within the chambers were monitored. Each chamber was dedicated to one exposure group. The control group was exposed to clean, filtered air under identical conditions to those used for the AN-exposed groups. 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

Twenty-five males and 25 females in each of five groups (F0 generation) were exposed 6 h/day, 7 days/week for 10 weeks to AN vapor concentrations of 0, 5, 15, 45, or 90 ppm; these animals were randomly bred to produce an F1 generation. Following weaning on postnatal day (PND) 28, animals selected to be parents from the F1 generation were similarly exposed. Due to excessive toxicity at the 90 ppm exposure level, exposure of the F1 parental animals at 90 ppm was terminated after 16 to 29 exposures. A replication of the breeding procedure (avoiding sibling matings) was conducted with the remaining four groups in the second generation (25 animals/sex/group). The F0 and F1 generations were approximately 8 weeks old and 4 weeks old at initiation of their respective exposures. The F0 and F1 males were exposed for 10 weeks prior to mating and throughout mating until one day prior to euthanasia, and the F0 and F1 females were exposed for 10 weeks prior to mating and throughout mating, gestation, and lactation until one day prior to euthanasia. Throughout the mating period, each female was housed overnight in the home cage of a specific, nonsibling male (1:1) until evidence of mating was detected. Observation of a copulatory plug in the vagina or the presence of sperm in a vaginal lavage confirmed positive evidence of mating; that day was termed gestation day (GD) 0, and the animals were separated. Exposure of the F0 and F1 dams was suspended for five days following parturition (lactation days [LDs] 0 to 4), to avoid confounding nesting and nursing behavior and neonatal survival during early postnatal development. Exposure of the dams resumed on LD 5; they were removed from the litters in the home room for the daily 6-h exposure period at approximately the same time each day.

Each dam and litter remained housed together until weaning on PND 28. Weaning occurred on PND 28 because it has been the experience of the performing laboratory that removal of dams from their pups for inhalation exposure during the lactation period frequently results in a reduced growth curve in F1 progeny if they are weaned at PND 21, thereby potentially compromising long-term survival. Male and female F1 pups (25/sex/group) were randomly selected prior to weaning (PND 28) to compose the F1 generation. The offspring of the F0 and F1 generations (F1 and F2 pups, respectively) were potentially exposed to AN in utero, via milk through nursing during PNDs 0 to 28 and for F1 pups, via direct exposure following weaning. These F1 weanlings were first directly exposed to AN for 6 h beginning on PND 28; however, exposures of the 90 ppm group were suspended after 16 to 29 days due to excessive systemic toxicity. These animals were maintained without exposure for 4 days (through PNDs 48 to 61) prior to macroscopic examination. No further post-mortem evaluations were made on the 90 ppm group animals.

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 prior to exposure and within 1 h after completion of exposure. Females were also observed twice daily during the period of expected parturition and at the time of parturition for dystocia (prolonged or 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 on LDs 1, 4, 7, 14, 21 and 28. Parental food consumption was determined on the same days 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 lavages were performed daily, and the slides from each F0 and F1 female were evaluated daily beginning three weeks prior to pairing and continuing until mating was observed. Females were allowed to deliver naturally and nurture their young to weaning (PND 28). On the day of parturition (PND 0), pups were sexed and examined for external malformations, and the numbers of stillborn and live pups were recorded. Stillborn and intact offspring dying from PND 0 to 4 were necropsied using a fresh dissection technique (Stuckhardt and Poppe 1984). A detailed gross necropsy was performed on any pup dying after PND 4 and prior to weaning.

To reduce variability among the litters, large litters were reduced to 10 pups/litter (5/sex when possible) on PND 4 using a computer-generated random selection procedure. 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, 21, and 28. Pups were also individually sexed on PNDs 0, 4, 7, 14, 21, and 28.

Each male pup selected as a parent for the F1 generation was examined for balanopreputial separation beginning on PND 35 (Korenbrot, Huhtaniemi, and Weiner 1977), and each selected F1 female pup was examined for vaginal perforation beginning on PND 25 (Adams et al. 1985). These observations continued until all animals attained these criteria (i.e., balanopreputial separation or vaginal perforation). Pup body weights were recorded on the day of acquisition of these landmarks.

Plasma and red blood cell (RBC) cholinesterase determinations were conducted on 10 rats/sex of the F0 parental generation from the control and 90 ppm groups and from 10 rats/sex of the F1 parental generation from the control, 5, 15, and 45 ppm groups. Blood samples were collected from the tail vein following the daily 6-h exposure 2 days prior to scheduled euthanasia. EDTA was used as the anticoagulant. Plasma and RBC cholinesterase activities were determined using an assay based on a modification (Hunter, Marshall, and Padilla 1997) of the Ellman reaction (Ellman et al. 1961).

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 12.1 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). The left testis and/or epididymis from all F0 and F1 males in all dose groups were evaluated for homogenization-resistant spermatid counts (testis and epididymis) and sperm production rate (testis only; Blazak, Ernst, and Stewart 1985) using the HTM-IVOS system.

Euthanasia, Necropsy, and Histopathologic Examination

Surviving F0 and F1 adults were euthanized and necropsied following completion of weaning of their offspring (F1 and F2 pups, respectively). 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 by immersion in 10% neutral-buffered formalin for possible histopathological examination. Microscopic evaluations were performed on the following tissues for 10 randomly selected F0 and F1 parental animals per sex (with confirmed sire or pregnancy) from the control and high-exposure groups: adrenal glands, prostate, brain, pituitary, seminal vesicles, right epididymis (caput, corpus and cauda), right testis, vagina, cervix, coagulating gland, uterus, oviducts, and ovaries (one section from each ovary from F0 females was examined). Nasal cavities (levels I, II, III and IV; Young 1981), lungs, and gross lesions from all F0 and F1 animals in the control, 5, 15, and 45 ppm groups were examined microscopically. The lungs and nasal cavities of the 90 ppm animals were not examined histologically because the grossly observable, severe irritation indicated that histopathology would not add useful information to the study. Periodic acid–Schiff (PAS) and hematoxylin staining were used for the right testis and epididymis and hematoxylin-eosin 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 control and 45 ppm groups (Bolon et al. 1997; Bucci et al. 1997). A qualitative assessment for the presence or absence of growing follicles, antral follicles, and corpora lutea was also performed. In addition, the following tissues were examined for F0 animals in the 5, 15, and 45 ppm groups and F1 animals in the 5 and 15 ppm groups that failed to mate or produce offspring or otherwise exhibited potential reproductive dysfunction (e.g., abnormal estrous cyclicity or andrological changes): pituitary, cervix, ovaries, oviducts, uterus, vagina, coagulating gland, right epididymis, right testis, prostate, and seminal vesicles.

Organs weighed from all F0 and F1 parental animals included adrenals, brain, total and cauda epididymides (weighed separately), kidneys, liver, lungs (prior to inflation with 10% neutral-buffered formalin), ovaries, pituitary, prostate, seminal vesicles with coagulating glands and accessory fluids, spleen, testes (weighed separately), thyroid, and uterus with oviducts and cervix.

On PND 28, a complete necropsy similar to that performed on parental animals (with emphasis on developmental and reproductive system morphology) was conducted on F1 pups not selected for AN exposure and on F2 pups. Brain, spleen, thymus gland, epididymis, ovary, pituitary gland, seminal vesicle, testis, and uterus (with oviduct/cervix) weights were also recorded from these pups.

Statistical Analyses

All statistical analyses were conducted using two-tailed tests (except as noted below) for minimum significance levels of 5% and 1%, comparing each AN-exposed group to the control group. Data obtained from nongravid animals were excluded from statistical analyses following the mating period. Parental mating, fertility, copulation, and conception indices were evaluated by the chi-square test with Yates’ correction factor (Hollander and Wolfe 1999). Parental body weight and food consumption data, estrous cycle and gestation lengths, precoital intervals, implantation sites, unaccounted-for implantation sites, numbers of pups born, live litter sizes, pup body weights and weight changes, balanopreputial separation and vaginal patency data (day of acquisition and body weight), anogenital distances, absolute and relative organ weights, sperm production rate, sperm numbers, ovarian primordial follicle counts, and red blood cell and plasma cholinesterase data were subjected to a one-way analysis of variance (ANOVA; Snedecor and Cochran 1980) among all groups. If the ANOVA was significant, Dunnett’s test (Dunnett 1964) was used for the pairwise comparisons to the control group. Sperm motility and morphology and proportional postnatal offspring survival and sex at birth were statistically analyzed by the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis 1952) followed by the Mann-Whitney U test (Kruskal and Wallis 1952), when appropriate. Histopathologic findings in protocol-specified tissues were evaluated using a two-tailed Fisher’s Exact test (Steel and Torrie 1980).

RESULTS

Chamber Exposure Concentrations

The periodic analyses of the chamber atmospheres indicated that the mean analytical values of AN ± SD for the 5, 15, 45, and 90 ppm groups were 5.0 ± 0.30, 15.1 ± 0.69, 45.3 ± 1.51, and 89.4 ± 3.58 ppm, respectively, for the F0 generation, and 5.0 ± 0.25, 15.2 ± 0.59, 45.4 ± 1.57, and 86.5 ± 2.45 ppm, respectively, for the F1 generation. No test chemical was detected in the control atmospheres. These data confirm that the targeted exposure concentrations were achieved and remained consistent throughout the study (Figure 3).

Parental Systemic End Points

There were no AN-related mortalities at any exposure level evaluated. There were no effects on body weights, weight gains, or food consumption at exposure levels of 5 and 15 ppm.

Clinical findings consistent with the known irritant properties of AN (clear/red material around the nose, eyes, and mouth and on the forelimbs) were observed for the F0 males and females exposed to 90 ppm throughout the exposure period within 1 h following completion of daily exposures, but generally did not persist to the following day. Wet, cool tails were also noted for these animals, to a greater extent in the males, within 1 h following exposure. Body weight gains for the 45 and 90 ppm F0 males were statistically reduced relative to controls during the first three weeks of exposure, resulting in persistent and generally statistically significant body weight depressions (up to 11.8%) throughout the F0 generation (Figure 4). Food consumption for these males was also decreased (data not shown), generally in parallel with the body weight effects. Decreased food consumption and body weight gains were also noted for F0 females exposed to 45 and 90 ppm during the first 2 weeks of treatment and throughout gestation, resulting in decreased body weights (generally statistically significant) for 45 ppm females at study week 2 (–4.5%) and 90 ppm females throughout the 10-week premating period and gestation (7.5% to 9.1%) (Figure 5). Body weights in the 90 ppm F0 females were also depressed during lactation (5.8% to 11.5%), but did not achieve statistical significance and were not accompanied by food consumption deficits. Overall, body weight effects in the F0 generation were more pronounced in the males than in the females.

Marked clinical signs, including sensitivity to touch, vocalization upon handling, and evidence of local irritation (red and/or clear material on various body surfaces), an approximate 10% to 15% decrease in food consumption for both sexes, and body weight decrements in excess of 20% for males and approximately 12% for females were noted for F1 animals during the first 3 to 4 weeks of direct exposure to 90 ppm AN following weaning (Figures 6 and 7). As a result of these findings, exposure of the 90 ppm F1 weanlings was discontinued following a total of 16 to 29 exposures, precluding mating and production of an F2 offspring generation at this exposure level. There were no other AN-related clinical findings observed at any F1 exposure level (data not shown). Body weight gains in the 45 ppm F1 males were slightly reduced (generally statistically significant) during the first 3 weeks of AN exposure, but the effects were less pronounced than in the F0 males. Body weights for the 45 ppm F1 males were decreased by up to 9.4% during study weeks 18 to 26 (Figure 6). Decreased food consumption in these animals generally paralleled the observed body weight deficits (data not shown). There were no other effects on F1 female body weights or food consumption.

Reproductive Function

The regularity and duration of estrus were not affected by exposure to AN during either generation. Mean estrous cycle lengths in all groups evaluated were similar to controls, with the exception of slightly increased values in the 45 ppm F0 and F1 females (Table 1). However, a similar increase was not observed in the 90 ppm F0 females, and the increase in the 45 ppm F1 females was due to a single female with an atypically long estrous cycle (16 days). Therefore, the increased mean estrous cycle lengths in the 45 ppm F0 and F1 females were not attributed to AN exposure. Furthermore, no adverse exposure-related effects were observed on the number of days between pairing and coitus, gestation length, or reproductive performance (fertility, mating, copulation, and conception indices) in either generation (Table 1).

The process of spermatogenesis (mean testicular and epididymal sperm numbers, sperm production rate, and sperm motility and morphology) was unaffected by AN exposure in both generations (Table 2). A slight statistical decrease in sperm motility (including progressive motility) was noted for the F0 males exposed to 90 ppm AN when compared to controls (Table 2). However, there were no other andrological changes in this group, nor were there effects on fertility or on reproductive organ weights and histopathology. The sperm motility values in this group were similar to those in the F1 generation control group, which illustrates the variability of these data (Table 2). In addition, the sperm motility in the 90 ppm group F0 males was within the range of the historical control data for inhalation reproductive toxicity studies conducted in the performing laboratory. Therefore, the slightly decreased sperm motility in the 90 ppm F0 males was considered unrelated to AN exposure.

Offspring Survival, Growth, and Sexual Development

The numbers of F1 and F2 pups born, live litter sizes, sex ratios at birth and postnatal survival were unaffected by parental AN exposure (Table 1). Anogenital distances (absolute and relative to the cube root of pup body weight) on PND 1 were also unaffected by parental AN exposure (Table 3). Slight, generally statistically significant increases in absolute and relative anogenital distance were noted for the F1 males exposed to 45 and 90 ppm AN. However, there is no known mechanism for increasing male anogenital distance, and there were no effects on anogenital distance in the 45 ppm F2 pups; therefore, the slight increases in anogenital distance in the 45 and 90 ppm group F1 males were not considered related to AN toxicity.

F1 pup body weights in the 90 ppm group during the last 2 weeks of lactation (PNDs 14 to 28) were decreased 6.6% to 12.2% for males and 5.8% to 10.7% for females (Figure 8) as a result of decreased body weight gains in these animals from PNDs 7 to 14 through 14 to 21 (following the reinitiation of maternal exposures on PND 5). Slight delays in the acquisition of sexual developmental landmarks (balanopreputial separation and vaginal patency) and lower body weights on the day of acquisition (relative to control group values) were noted for F1 males in the 45 and 90 ppm groups and females in the 90 ppm group (Table 3). Body weight has been shown to affect the time of acquisition of balanopreputial separation and vaginal patency (Ashby and LeFevre 2000; Clark 1998). Therefore, because the slight delays in acquisition of these sexual developmental landmarks were observed in parallel and/or in the presence of reduced body weights, the delays were not considered to be direct effects of AN exposure. Balanopreputial separation occurred in all F1 male pups by PND 53, and all F1 female pups had vaginal opening by PND 42.

F2 pup weights and weight gains were unaffected by parental exposure to AN throughout the postnatal period. Slightly decreased male pup weights in the 5, 15, and 45 ppm groups on PND 28 achieved statistical significance relative to controls (Figure 9), resulting in lower overall weight gain during PNDs 1 to 28. However, the differences did not display an exposure-related pattern and the mean values in these groups were similar to the performing laboratory’s historical control data for inhalation reproductive toxicity studies; therefore, these slight weight differences were not attributed to parental AN exposure.

Parental Cholinesterase Evaluations

RBC cholinesterase activity was unaffected in males and females at AN exposure levels of 90 ppm in the F0 generation and 5, 15, and 45 ppm in the F1 generation. Plasma cholinesterase activity in the F0 females exposed to 90 ppm AN was 40% lower than controls (statistically significant; Table 4) and was also lower than the mean value in the performing laboratory’s historical control database for approximately age-matched animals. However, there were no corresponding clinically observed functional deficits or inhibition of RBC cholinesterase activity in these females, and no effects on plasma or RBC cholinesterase activity were noted for F0 males, F1 males, and F1 females at any exposure level evaluated (Table 4). Because plasma cholinesterase activity was not evaluated at 90 ppm in the F1 generation (due to early group termination because of excessive systemic toxicity), the relationship of the decreased mean plasma cholinesterase activity in the 90 ppm F0 females to AN exposure could not be conclusively determined, but is not considered to be of toxicological significance in the absence of corresponding changes in RBC cholinesterase levels or associated clinical observations.

Necropsy and Histopathology

F0 and F1 Adults

Spontaneous deaths occurred in parental animals of both generations in this study. One F0 female each in the 5 and 45 ppm groups were found dead. There were no signs of toxicity for these females, although there was evidence of dystocia for the 45 ppm female and the 5 ppm female failed to initiate parturition. However, there were no occurrences of dystocia or mortality in the 90 ppm group. Therefore, these findings at the lower exposure levels were not considered AN-related. One F1 female in the control group was found dead and one F1 male in the 45 ppm group was euthanized in extremis prior to the breeding period. Each of these animals was found to have malignant lymphoma. Because the condition appeared in both control and treated animals and did not exhibit a dose-related response, the moribund condition of the 45 ppm F1 male was not attributed to AN exposure.

Increased liver weights occurred in the 90 ppm F0 males (statistically significant) and females and the 45 ppm F1 males (Tables 5 and 6; absolute weights presented). The relative (to final body weight and brain weight) increases were generally statistically significant (data not shown) and occurred to a greater degree than could be explained by decreased body weight alone. Gross liver changes were not observed, so the livers of these animals were not examined microscopically. No other AN-related changes in parental organ weights were noted for either sex in either generation (selected absolute organ weights are presented in Tables 5 and 6).

There were no morphologic differences noted during the microscopic examination of male or female reproductive organs in the animals that were suspected of reduced fertility (those that failed to mate, conceive, or sire a litter). Ovarian primordial follicle counts (Table 1) were unaffected by AN exposure in the 45 ppm F1 females.

Histopathologic alterations attributed to AN exposure were limited to morphologically similar lesions in the nasal tissues of F0 males and females at 45 ppm, F1 males at 5, 15, and 45 ppm, and F1 females at 15 and 45 ppm (Table 7). The lesions showed a clear exposure-related response in incidence and severity, and included respiratory/transitional epithelial hyperplasia, subacute inflammation, squamous metaplasia, and/or degeneration of the olfactory epithelium. The majority of the lesions were present in the most rostral section (level I) of the nasal tissues examined and were consistent with site-of-contact irritation from an irritant chemical such as AN (Monticello, Morgan, and Uraih 1990; Dungworth, Hahn, and Nikula 1995). All of the nasal lesions noted in this study are common findings in the nasal epithelium of the rat following subchronic to chronic inhalation exposure with an irritating compound and represent the effects of local irritation, rather than a systemic effect. No other AN-related histopathologic alterations were noted at any exposure level.

F1 and F2 Offspring

No internal findings that could be attributed to AN exposure were noted at the necropsies of F1 and F2 pups that were found dead or examined at the scheduled PND 28 necropsies. No AN-related effects on F1 and F2 offspring organ weights were noted in any group (data not shown).

DISCUSSION

The present study was conducted, in part, in response to Chinese reports of adverse reproductive effects in chemical workers exposed to AN. An independent review of these epidemiological studies concluded that there were sufficient data to hypothesize that AN exposure may be associated with adverse reproductive outcomes (Neal et al. 2006). The same review concluded that the existing animal data were insufficient for a definitive evaluation of reproductive toxicity. The objective of the present study was to improve the experimental data set by characterizing the potential adverse effects of AN inhalation exposure on the reproductive capacity in two generations of Crl:CD(SD) male and female rats.

When evaluated across two generations, there were no AN-related effects on estrous cyclicity, reproductive performance, parturition, numbers of implantation sites and unaccounted-for sites, the process of spermatogenesis, and reproductive organ weights and histopathology in the F0 and F1 animals. The mean number of ovarian primordial follicles was unaffected in the 45 ppm group F1 females. No AN-related effects were observed on the mean numbers of F1 or F2 pups born, the pup sex ratio, pup survival, or the clinical condition of the pups during the preweaning period. No AN-related macroscopic findings or effects on organ weights were observed in F1 or F2 pups at the scheduled necropsies; no AN-related findings were noted upon macroscopic examination of F1 or F2 pups that died during the postnatal period. No direct effects of parental AN exposure were observed on F1 or F2 pup anogenital distance on PND 1 or on acquisition of balanopreputial separation or vaginal patency by F1 weanlings. Evaluation of RBC and plasma cholinesterase activities in the parental animals of both generations revealed no changes that were unequivocally of toxicological significance.

There were no AN-related mortalities in parental animals in this study. Evidence of F0 and F1 parental systemic toxicity (decrements in body weights and/or food consumption) was noted for both sexes at exposure levels of 45 and/or 90 ppm. In addition, increased absolute and/or relative (to final body weight) liver weights occurred for the 90 ppm group F0 males and females and the 45 ppm group F1 males. Exposure to xenobiotics commonly induces hepatic metabolic enzymes in laboratory rats. However, the livers in this study were not evaluated histologically (by study design) because no grossly observable liver effects were noted; therefore, the relationship of hepatic enzyme induction to the liver weight increases was not determined. AN-related decreases in F1 offspring body weights in the 90 ppm group were observed during PNDs 14 to 28. These decreases occurred in the presence of maternal toxicity and evidence of significant nasal irritation in dams at the 90 ppm exposure level. The mean weights for these pups were also lower than the performing laboratory’s minimum historical control value for inhalation studies and corresponded to reduced mean maternal body weight gains. The lower body weights for the F1 offspring in this group may have resulted in a higher exposure to AN per gram of body weight for the adult F1 animals. Marked clinical findings and severe decrements in body weight and food consumption were noted for the 90 ppm F1 adults following 3 to 4 weeks of direct test article exposure. Further exposure of this group was terminated, and the animals were euthanized. For the remaining groups (5, 15, and 45 ppm), there was no evidence of cumulative toxicity in pregnant or lactating dams. No evidence of parental systemic toxicity was found in this study at AN exposure levels of 5 and 15 ppm, and no offspring systemic toxicity was noted at exposure levels of 5, 15, and 45 ppm.

AN-related effects that were not indicative of systemic toxicity consisted of clinical signs of irritation 1 h following exposure at 90 ppm and microscopic findings in the nasal tissues. Increases in the incidences of red, clear, and/or yellow material on various body surfaces were noted throughout the exposure period for the 90 ppm F0 and F1 parental males and females. There was also a higher incidence of F1 females at 90 ppm that were sensitive to touch and that vocalized upon handling 1 h following exposure from study week 19 (after 12 days of exposure) through the last day of exposure prior to early termination. Clinical findings noted in the 90 ppm group F0 and F1 males and females, which were attributed to the irritant properties of the test article, generally did not persist to the following day and were not considered evidence of systemic toxicity. There were no clinical signs of irritation 1 h following exposure in the 5, 15, and 45 ppm groups.

Acrylonitrile-related microscopic alterations noted in this study were limited to morphologically similar nasal lesions in the F0 males and females at 45 ppm, the F1 males at 5, 15, and 45 ppm, and the F1 females at 15 and 45 ppm. Four levels of the nasal cavity were examined microscopically for the 5, 15, and 45 ppm groups; the majority of the nasal lesions were present in the most rostral section (level I) examined. The lesions showed a clear exposure-response relationship in incidence and severity, and included respiratory/transitional epithelial hyperplasia, subacute inflammation, squamous metaplasia, and/or degeneration of the olfactory epithelium. The nonspecific changes in the nasal epithelium that were observed are typically reversible (Feron, Woutersen, and Spit 1986, Dungworth, Hahn, and Nikula 1995). Because the majority of the nasal epithelium was unchanged following repeated AN exposure, the lesions would have limited toxicological significance for the rat.

An evaluation of the relevance of these findings to the risk assessment of potential AN exposure to humans must consider the comparative nasal anatomy of rats and humans. Because the rat is an obligatory nose-breather, in contrast to humans and other primates, an understanding of airflow through the nasal passages of a rat is critical to relate measured exposure to an inhaled vapor to actual delivered dose (DeSesso 1993). Experimental attempts to characterize airflow through the nasal passages using nasal molds and computerized simulation have found clear species-specific differences, particularly between rats and humans, suggesting that rats are often much more sensitive to localized nasal insult from inhaled toxicants (Morgan and Monticello 1990; Kimbell et al. 1997; Frederick et al. 2002).

Based on the lack of functional effects on reproduction (estrous cycles, mating and fertility indices, number of days between pairing and coitus, gestation length, or spermatogenesis) at a concentration of 90 ppm when evaluated for one generation, or at 45 ppm when evaluated for two generations, and the expression of neonatal toxicity in the presence of maternal toxicity at 90 ppm in the first generation, the NOAEL (no-observed-adverse-effect level) for parental reproductive toxicity and neonatal toxicity of AN when administered via whole-body inhalation exposure to rats over two generations was 45 ppm. The NOAEL for reproduction was 90 ppm in the first generation. There was no evidence of cumulative toxicity over two generations or of enhanced toxicity in developing animals or in pregnant and lactating dams. The NOAEL for parental systemic toxicity was considered to be 15 ppm.

In conclusion, the results of this study do not indicate concern for reproductive toxicity as an effect of AN exposure, and provide a context for further consideration of the practical restrictions on toxicity testing imposed by the nasal irritant properties of AN vapor.