& 2008 Wiley-Liss, Inc.

Birth Defects Research (Part B) 83:459476 (2008)

Original Article

Review of Reproductive and Developmental Toxicity Studies with Isopropanol

Willem D. Faber,1 Kenneth L. Pavkov,2 and Ralph Gingell3
2 1 WFTC, LLC, Victor, New York ExxonMobil Biomedical Sciences, Inc., Annandale, New Jersey 3 Shell Oil Company, Houston, Texas

Published studies for reproductive and developmental toxicity conducted with isopropanol have been conducted by the inhalation and oral gavage routes of administration. Interpretation of the data from these studies has resulted in discussions regarding NOAELs and additional benchmark dose modeling publications. Unpublished reproductive and developmental toxicity studies administered in the drinking water were also conducted by BIBRA, and the results of those studies are presented here. In addition, all of the reproductive and developmental toxicity studies conducted with isopropanol are summarized and evaluated for concordance of effects and NOAELs. Endpoints of concern for regulatory agencies were decreases in male mating index and reductions in postnatal pup survival. Original study reports were evaluated and data collated to address these two endpoints, and the data summarized. Data are presented suggesting that there were technical problems in the study that implied a decrease in male mating index, and based on the results from the drinking water studies, the weight of evidence suggests that isopropanol does not affect male mating or fertility at dose levels of up to 1000 mg/kg/day. The weight of evidence suggests that isopropanol can cause decreases in postnatal pup survival following oral gavage administration of 10001200 mg/kg/day to the dams. The NOAEL for this endpoint with oral gavage administration was 700 mg/kg/day. Indications of maternal toxicity were also an important predictor for decreased postnatal survival. Decreased postnatal pup survival was also noted in the drinking water studies with isopropanol with a LOAEL of 2278 mg/kg/day and a NOAEL of 1947 mg/kg/day. Birth Defects Res (Part B) 83:459476, 2008. r 2008 Wiley-Liss, Inc.

Key words: isopropyl alcohol; reproductive; developmental toxicity

INTRODUCTION
A two-generation reproductive toxicity study previously conducted (Bevan et al., 1995) has raised questions and concerns regarding the ability of isopropanol to affect male fertility endpoints and postnatal pup survival. Groups of male and female rats (30/sex/group) were administered 0, 100, 500, or 1000 mg/kg bw/day isopropyl alcohol (IPA) by oral gavage, resulting in several deaths in the parental generations prior to study termination (2 high-dose P1 females, 2 high-dose P2 females, 1 mid-dose P2 female, and 2 low-dose P2 males). A decrease in the male mating index was reported in the 1000 mg/kg bw/day P2 group. In the F1 generation, there was a decrease in the live birth index and the day 1 and day 4 survival indices in the 1000 mg/kg bw/day level and a decrease in the day 4 survival index at the 500 mg/kg bw/day dose level. Decreases in day 1 and day 7 survival indices and a decrease in the lactation index were observed in the F2 500 and 1000 mg/kg bw/ day pups. In addition, there was a decrease in the day 4 survival index in 1000 mg/kg bw/day pups. In the 1000 mg/kg bw/day F1 weanling group, 18/70 animals died or were euthanized prior to selection of the P2

generation. Body weights in male F1 1000 mg/kg bw/day pups were lower than controls on PNDs (postnatal days) 0 and 1, whereas body weights in male and female F2 1000 mg/kg bw/day pups were lower on PNDs 0, 1, and 4. The NOEL for parental toxicity (based on lethality and kidney effects) waso100 mg/kg bw/day and 100 mg/kg bw/day in male and female rats, respectively. The NOEL for developmental effects (based on decreases in pup weights and survival indices) was 100 mg/kg bw/day. The data from the two-generation study were the subject of several additional interpretations and modeling exercises. Allen et al. (1998) used data from the Bevan et al. (1995) study to calculate a benchmark dose level of 420 mg/kg/bw/day (lower bound on dose associated with a 5% response rate) for the decrease in the male mating index. The U.S. EPA (1992) and Tyl (1996) interpreted the reductions in postnatal survival as related

*Correspondence to: Willem D. Faber, WFTC, LLC, 1338 Strong Rd., Victor, NY 14564. E-mail: wfaber@msn.com Received 23 April 2008; Accepted 15 August 2008 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bdrb.20167

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water ad libitum, as per the study design, with or without test material. The designated test rooms were used solely for the IPA studies.

to treatment and proposed a NOAEL of 100 mg/kg bw/ day. Harris (1995) and Bevan et al. (1995) disagreed and proposed that 500 mg/kg bw/day was the NOAEL for reproductive effects. Gentry et al. (2002) used a physiologically based pharmacokinetic (PBPK) model to develop an RfD and RfC of 10 mg/kg bw/day and 40 ppm, respectively, for the decrease in fetal body weights, using data from the developmental toxicity studies and the two-generation study. Questions continue to arise regarding the interpretation of the data from the Bevan et al. (1995) study particularly with regards to classification criteria for labeling. The repeated exposure studies with IPA do not describe any effects to the male reproductive organs (Burleigh-Flayer et al., 1994, 1997, 1998), and therefore a plausible explanation for the decrease in male mating index described by Bevan et al. (1995) has not been provided. In an attempt to better understand the decrease in male mating index and decreased postnatal survival reported by Bevan et al. (1995), the original study report (including appendices) from the testing laboratory was reviewed. In addition, earlier studies conducted by the British Industrial Biological Research Association (BIBRA, Surrey, UK) for the Feed and Drink Federation IPA Steering Group (London, UK) were obtained and reviewed. These studies, not previously reported in the literature, included a one-generation pilot study, a developmental toxicity study, and a onegeneration/embryotoxicity study conducted with IPA in drinking water (BIBRA, 1986, 1987, 1988, 1990). Although the studies do not document that a specific guideline was followed, the studies were in compliance with Good Laboratory Practices regulations, and the protocols were similar to developmental toxicity guidelines current at the time (1986). In an effort to broaden the available database available to the public for risk assessment purposes, the data from the developmental toxicity and one-generation/embryotoxicity studies are presented here. A review of the available scientific literature is also provided in an attempt to address the recurring questions raised by the Bevan et al. (1995) paper; namely, whether the decrease in male mating index and the decrease in postnatal pup survival were treatment related and toxicologically relevant.

Developmental Toxicity Study

Females were paired overnight with untreated males (1M:1F) following confirmation of proestrus. The presence of sperm in the vagina or a vaginal plug the morning after pairing defined gestation day 0 (GD 0). Mated females were uniquely identified, housed individually in the same type of cage, and randomly assigned to one of four dose groups containing 20 females.

Reproduction/Embryotoxicity Study
The male rats were obtained approximately 7 weeks earlier than the female rats. On receipt, animals were randomly assigned to one of four dose groups and assigned a unique identification number. Each dose group consisted of 10 males and 30 females, of which 10 females were used for the embryotoxicity determinations and the remaining 20 females for the singlegeneration (littering) phase of the study. Seventy days (male) and 21 days (female) after the start of treatment, two females from those assigned to the littering phase of the study and one female from those assigned to the embryotoxicity phase of the study were housed with one male from the same treatment group for up to 15 days. Each morning during the mating period, vaginal smears were examined from each female still housed with the male. The presence of sperm or a vaginal plug was designated as GD 0. After mating was confirmed, pregnant females were housed individually in similar cages. Those assigned to the littering phase of the study were given cages with solid floors covered with sawdust and provided with paper nesting material, as necessary.

Test Chemical
The test chemical was provided by Shell Chemicals UK Ltd. Isopropanol (IPA; isopropyl alcohol; 2-propanol; CAS No. 67-73-0; CH3CHOHCH3) was from batch 1A1/ 41.3/84 GB1/260 and shown to be 99.89% pure by gas liquid chromatography (GLC). For the developmental toxicity and reproductive toxicity/embryotoxicity studies, drinking water formulations were prepared with domestic mains tap water at 2-week intervals for the duration of treatment of the animals. Due to greater water consumption during pregnancy and lactation, drinking water solutions were prepared more frequently during some stages of the reproduction/embryotoxicity study. Prior to use, formulations were analyzed by GLC to confirm concentration of IPA and stability. All formulations were within 710% of nominal concentrations and stable for at least 28 days.

MATERIALS AND METHODS Animals and Husbandry

Virgin male and female Wistar-derived rats, obtained from a Specified-Pathogen-Free colony (Olac 1976 Ltd., Bicester, Oxon), were acclimated to the lab environment for 79 days. The ages of the rats at receipt were 1112 weeks for the developmental toxicity study and 78 weeks (males) or 1011 weeks (females) for the onegeneration reproductive toxicity study. All animals were examined for health status, kept on a 12:12 hr light/dark cycle with nominal environmental temperature of 20241C and relative humidity of 45%65%. During acclimatization and prior to mating, groups of 5 (females) or 2 (males) animals of the same sex were housed in polypropylene cages with stainless steel tops and grid floors; were given free access to Certified Rat and Mouse No. 3 expanded fine ground laboratory feed (Special Diet Services, Witham, Essex, UK) and domestic mains tap

Dose Selection and Treatment

Dose level selection for both the teratology and reproductive/embryotoxicity studies were chosen based on the preliminary range-finding study previously described. For the teratology study, IPA was given at concentrations of 0.5%, 1.25%, and 2.5% in the drinking water to groups of 20 female rats on GD 616. For the reproductive/embryotoxicity study, IPA was given at concentrations of 0.5%, 1.0%, and 2.0% in the drinking
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water to male and female rats as follows: malesfor 70 days prior to mating, during mating, and continued until animals were euthanized; femalesfor 21 days prior to mating, during gestation, during lactation, and continued until animals were euthanized; offspring during rearing until animals were euthanized. In both studies, controls received tap water vehicle only. Actual intake of IPA was calculated from data on body weights, water intake, and the analyzed (teratology study) or nominal (reproductive/embryotoxicity study) concentrations.

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EVALUATIONS Developmental Toxicity Study

General observations were made each day, with a more thorough examination done once per week. Maternal body weights and feed and water consumption were determined daily from GD 0-20. Dams were euthanized on GD 20. The abdominal and thoracic contents were examined for abnormalities. Ovaries were examined and the number of corpora lutea recorded. The uterus was examined and the numbers and locations of viable and nonviable fetuses, early and late resorptions, total implantations, and pre- and postimplantation losses were recorded. Each live fetus was weighed and examined for gross, externally visible abnormalities. Approximately 50% of all of the fetuses (including all with gross abnormalities) were preserved in ethanol, eviscerated, and processed for skeletal exam after staining with Alizarin Red S (Staples and Schnell, 1964). The stained preparations were examined for skeletal abnormalities, variants, and variations in the degree of ossification. The remaining fetuses were preserved in Bouins solution, and those from the highdose and control groups were examined by freehand sectioning of the head and palate and dissection of the abdomen and thorax (Wilson, 1973). Sex was recorded as part of the evisceration or abdominal dissection procedures.

examined and the number of corpora lutea recorded. The uteri were examined and the numbers and locations of viable fetuses, early and late resorptions, total implantations, and pre- and postimplantation losses were recorded. Each live fetus was weighed and examined for gross, externally visible abnormalities. All fetuses from the embryotoxicity phase were preserved in ethanol. The viscera of fetuses that showed evidence of edema in the embryotoxicity study, along with their littermates, were examined by evisceration under a dissecting microscope, and the sex of each fetus was recorded. The remaining females were allowed to litter. Litters were examined on PND 1 for stillborn or abnormal young and then daily for any subsequent deaths. On PND 4, 7, 10, 14, 17, and 21, numbers of survivors and any abnormalities were recorded. On PND 21, pups were weaned and removed from the dams. Within 21 days of weaning the last litter, each adult animal and one animal of each sex from each litter were fasted overnight (with access to water) and euthanized by exsanguination under barbiturate anesthesia. During the anesthesia phase, blood was collected from the aorta of each adult rat and analyzed for total erythrocyte and leukocyte counts, hemoglobin concentration, and mean cell volume. Twelve females (5 controls, 3 given 1.0% IPA, 4 given 2.0% IPA) that failed to litter were euthanized 24 days after the last day of pairing. Each rat was examined for general condition and gross abnormalities. Weights of the following organs were recorded: adrenal glands, brain, cecum, gonads, heart, liver, kidney, and spleen. Selective tissues were preserved in 10% neutral buffered formalin. Histopathologic examination was carried out on the following tissues from control and high-dose adult animals: bladder, cervix and uteri, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes, uterine horns, and vagina. Approximately 10 days later, the remaining pups (F1 generation) were euthanized by carbon dioxide inhalation and examined for gross, external abnormalities. The liver and kidneys of each animal were weighed and tissue samples preserved in formalin.

Reproduction/Embryotoxicity Study
General observations were made each day, with a more thorough examination done once per week. Male body weights were recorded for three days prior to and four days after the test solutions were first given and then twice weekly throughout the study. Females were weighed daily for three days prior to and four days after the test solutions were first given and then twice weekly for 3 weeks. During gestation, females were weighed on GD 0 and every day until they littered or were euthanized. During lactation, the weight of the female and the total litter weight were recorded on postnatal days (PNDs) 1, 4, 7, and 14. On PND 21, the female and each of the pups were weighed individually. During the mating period, only male body weights were recorded. Consumption of feed and water was determined at the same intervals as the body weight measurements except for the females during the postpartum period when intake of feed and water was measured twice weekly. Males were euthanized on day 126 of the study. Dams assigned to the embryotoxicity phase of the study were euthanized on GD 19. The abdominal and thoracic contents were examined for abnormalities. Ovaries were
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STATISTICAL ANALYSES Developmental Toxicity Study

Body weights, feed intakes, water intakes, number of preimplantation losses, number of early and late resorptions, total number of postimplantation losses, number of live fetuses, and weight of fetuses from the treated and control groups were compared using analysis of variance and the procedure of Least Significant Difference and Students t-test. The incidence of abnormalities in adults at necropsy, the incidence of findings in the skeletal examination of fetuses, and the incidence of findings in visceral examination of the fetuses from the treated and control groups were compared using Chi Square or Fishers Exact tests. Analyses were performed taking into account both between and within litter variation and, in all cases, a probability level of po0.05 was accepted as indication of statistical significance.

Reproduction/Embryotoxicity Study
Body weights, organ weights, hematological results, feed intakes, water intakes, pup numbers, pup weights, number of preimplantation losses, number of early and

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each in the 0.5% and 1.25% IPA groups, and one in the 2.5% IPA group. No abnormalities were observed in the dams at necropsy. All animals given 1.25% or 2.5% IPA showed reduced feed (Fig. 1) and water (Fig. 2) consumption. Administration of 2.5% IPA in drinking water resulted in an immediate reduction in water intake, and this was statistically significant throughout the treatment period. A statistically significant increase occurred on the first day (GD 1617) following the end of treatment for all dose levels. This pattern suggests decreased palatability of the drinking water containing 2.5% IPA, and similar decreases in water consumption were observed in the range-finding study. Although the mid-dose group also showed a similar but less marked reduction in water intake, the values were only statistically significant during GD 69. Except for a slight decrease on the first day of treatment, water consumption in the low-dose group was similar to the control group values. Feed consumption patterns paralleled the

late resorptions, total number of postimplantation losses, number of live fetuses, weight of fetuses, and litter weights from the treated and control groups were compared using analysis of variance, the procedure of Least Significant Difference and Students t-test. The incidence of abnormalities in adults at necropsy and the incidence of histopathological findings in the treated and control groups were compared using Fishers Exact test. In all cases, the treated groups were compared with the control, and a probability level of po0.05 was accepted as indication of statistical significance.

RESULTSDevelopmental Toxicity Study Maternal Effects

There were no deaths, abortions, early deliveries, or dams removed from the study. The numbers of nonpregnant females were three in the control group, two

30

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Food Consumed (g/rat/day)

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10 Control 0.50% 1.25% 2.50%

0 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 17-18 18-19 Gestation Day (GD)

Fig. 1. Developmental toxicity studyMean maternal food consumption (range of SD values 5 1.934.34).
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50 Water Consumed (ml/rat/day)

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20 Control 0.50% 1.25% 2.50%

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0 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 17-18 18-19 Gestation Day (GD)

Fig. 2. Developmental toxicity studyMean maternal water consumption (range of SD values 5 1.7315.82). Birth Defects Research (Part B) 83:459476, 2008

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330

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270 Body Weig ht (g)

250

230

210

190

170

Control 0.50% 1.25% 2.50%

150 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Gestation Day

Fig. 3. Developmental toxicity studyMean maternal body weights (range of SD values 5 10.118.2).

Table 1 Maternal Parameters of Rats Given 02.5% IPA in the Drinking Water on GD 616 Developmental Toxicity Study
Isopropanol (% in drinking water) Maternal parameters No. mated females Maternal mortality (no.) No. females with litters GD 20 Mean maternal body weight (g) GD 207SD Mean maternal food intake a Mean maternal water intake a Mean calculated IPA intake GD 616 (mg/kg/day)
po0.05; po0.01; po0.001.
a

0 20 0 17 304744.9 19.870.72 33.872.30 0

0.5 20 0 18 308733.2 19.570.91 36.772.82 596

1.25 20 0 18 302739.7 17.370.80 29.473.30 1242

2.5 20 0 19 291732.7 14.870.81 18.172.08 1605

Mean (7SD) from GD 616 inclusive.

water consumption during and after treatment in the midand high-dose groups. Prior to exposure to IPA in the drinking water, mean body weights of all groups were not significantly different (Fig. 3). From GD 68, those animals consuming 2.5% IPA in the drinking water lost weight; for the remainder of the treatment period, those animals gained weight at a lower rate than the control group. After cessation of treatment (GD 1720), weight gain was greater in the group consuming 2.5% IPA in the drinking water than the control group value. Overall, mean body weights of animals in the 2.5% IPA group were lower than the control group from GD 7 to termination. Effects on weight gain in the low- and mid-dose groups were limited to a failure to gain weight during the first two days of treatment in the mid-dose group and during the first day of treatment in the low-dose group. All subsequent body weight measurements in these two dose groups were similar to controls. Intake of IPA in relation to body weight was calculated from the data for water intake, body weight, and the nominal concentrations in the test solutions. The intakes during GD 616 were 596, 1242, and 1605 mg/kg/day for the low-, mid-, and high-dose groups, respectively (Table 1).
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Litter and Fetal Effects

Litter parameters and fetal weights are summarized in Table 2. Endpoints for events that occurred prior to IPA exposures included pregnancy rate, total number of corpora lutea, and total numbers of preimplantation loss. There were no effects related to IPA exposure in postimplantation loss, mean number of implantation sites or live fetuses. Significant findings included a slight dose-dependent decrease in mean litter weight and a statistically significant decrease in mean fetal weight in groups consuming 1.25% and 2.5% IPA in the drinking water. A summary of fetal skeletal and visceral abnormalities is shown in Table 3. No gross abnormalities were observed. The only major skeletal changes were an absence of caudal vertebrae and short forelimb and hindlimb bones in a single control fetus. A statistically significant increase in variations was indicative of a lower degree of ossification in the treated animals. There was a dose-dependent decrease in the number of fetuses with the 4th sacral arch and a dose-dependent increase in the number of fetuses with less than 2 caudal arches. The sternum also showed reduced ossification because there

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Table 2 Litter and Fetal Data from Rats Given 02.5% IPA in the Drinking Water on GD 616 Developmental Toxicity Study
Isopropanol (% in drinking water) Litter data No. females with litters GD 20 Total corpora lutea Mean corpora lutea7SD Total implantations Mean implantation7SD Total preimplantation losses Mean preimplantation losses7SD Mean live fetuses7SD Total postimplantation losses Mean postimplantation losses7SD Mean % implantations Mean early resorptions7SD Mean late resorptions7SD No. of females with postimplantation losses Mean litter weight (g)7SD Mean fetal body weight (g)7SD Mean male fetal weight (g)7SD Mean female fetal weight (g)7SD Fetal sex ratio (M:F)
po0.01; po0.001.

0 17 226 1372.54 202 11.971.50 24 1.472.83 11.172.34 14 0.871.38 92.9 0.871.20 0.170.24 7 40.178.99 3.5970.202 3.7170.205 3.4770.214 1.05

0.5 18 255 14.272.23 204 11.372.19 52 2.974.32 10.273.19 21 1.271.58 85.0 1.171.37 0.170.24 13 38.777.87 3.5870.252 3.6970.180 3.4870.260 1.26

1.25 18 228 12.771.41 210 11.771.88 18 1.071.61 11.072.11 12 0.770.84 93.9 0.670.85 0.170.24 9 37.676.77 3.4370.221 3.5470.233 3.3270.228 1.23

2.5 19 231 12.272.93 210 11.173.15 21 1.1171.52 10.673.31 9 0.570.77 90.8 0.470.60 0.170.32 6 37.076.49 3.3570.282 3.4470.254 3.2470.319 1.35

were increased numbers of fetuses with small, absent, or incompletely ossified sternebrae. Other statistically significant skeletal findings were not dose dependent. A reduction in ossification of bones of the skull was only observed in the 0.5% IPA group, and there were more fetuses in the mid-dose group with forelimb proximal phalanges, less than 3 metacarpals or unilateral sternebrae. In both the low and mid-dose group, there were increased numbers of fetuses with dumbbellshaped sternebrae or 14 pairs of ribs. An examination of the viscera of the offspring of dams receiving 2.5% IPA showed no differences between controls and treated animals.

RESULTSOne-generation/Embryotoxicity Study F0 Generation

There were no deaths, abortions, early deliveries, or dams removed from the study. Administration of IPA in the drinking water caused an immediate, statistically significant dose-dependent decrease in water intake in the male rats (Fig. 4). Although intake returned to normal for males in the 0.5% group, intake was approximately 5%14% lower during the premating phase for the 1.0% group males and approximately 30% lower from days 711 until study termination for the 2.0% group males. For male rats consuming 2.0% IPA in the drinking water, there was a marked decrease in feed intake over the first four days of treatment (Fig. 5), and feed intake remained significantly less than control values over the remainder of the premating period. Smaller (but still significant) reductions in feed consumption were also seen in the low- and mid-dose groups. The overall mean feed consumption for all three treatment groups was statistically significantly lower than the control (Table 4). Body

weights of male rats receiving 2.0% IPA were 4%5% lower than control prior to start of treatment. At day 1, the difference was 8% and remained statistically significantly lower (8%10%) throughout the study (Fig. 6). Male rats consuming water containing 0.5% or 1.0% IPA in the drinking water had initial slight reductions in weight gain but recovered after one week of exposure. An initial decrease in water consumption was observed in 1.0% and 2.0% females during the premating phase (Fig. 7a). Although the 1.0% group returned to control levels after day 3, intakes in the 2.0% group recovered to about 70% of the control value from 50% of the control value on day 1. Whereas intake during gestation was 15%30% lower in those animals given 2.0% IPA, both the 0.5% and 1.0% group consumed more water than controls (Fig. 7b). During lactation, water consumption for the control and two lower doses increased dramatically, whereas intake by the 2.0% group was markedly reduced (Fig. 7c). For female rats consuming 1.0% or 2.0% IPA in the drinking water, there was a similar initial reduction in feed intake during the premating phase (Fig. 8a). The 1.0% female group had a return to control values; however, feed intake for the 2.0% group remained significantly lower than control values. Although females in the 1.0% and 2.0% IPA groups showed reduced feed intake on the first day of gestation, only animals in the 2.0% group continued to consume less feed than the control group, and this was generally within 10% of the control value (Fig. 8b). Similar reductions in feed intake for 2.0% females were also seen during the postpartum phase, whereas the 0.5% and 1.0% groups were unaffected (Fig. 8c). During the premating phase, all treated female groups showed an immediate weight loss (Fig. 9a), with recovery of weight gain after one week of exposure. The exposed female groups had mean body weight
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Table 3 Incidence of Findings in Fetuses of Rats from the Developmental Toxicity Study
Isopropanol (% in drinking water) Findings Visceral malformations No. examined viscerally Brain, hematoma in subarachnoidal space Brain, hematoma in dorsal region of cerebrum Brain, small right lateral ventricle Brain, enlarged lateral ventricles Head, enlarged nasal cavities Kidneys, enlarged Kidneys, small Kidneys, misshapen (major) Testes, displaced Skeletal malformations No. examined (fetuses) Vertebrae, caudal absent Forelimbs, humerus, radius, and ulna short Hind limbs, femur, fibula, and tibia short Skeletal variations Skull, frontals incompletely ossified Skull, parietals, incompletely ossified Skull, interparietals, incompletely ossified Skull, supraoccipital incompletely ossified Skull, nasals incompletely ossified Vertebrae, any thoracic centra 113 not ossified Vertebrae, any thoracic bicentral Vertebrae, extra cervical Vertebrae, 4th sacral arch present Vertebrae, 4th sacral arch bicentral Vertebrae, less than 2 caudal with arches Vertebrae, more than 2 caudal with arches Ribs, wavy ribs Ribs, 14 pairs of ribs Ribs, 14 ribs on one side Sternebrae, any sternebrae 15 small Sternebrae, any sternebrae 15 asymmetrical Sternebrae, any sternebrae 15 unilateral Sternebrae, any sternebrae 15 absent Sternebrae, any sternebrae 15 bicentral Sternebrae, any sternebrae 15 dumbell shaped Sternebrae, any sternebrae 15 incompletely ossified Sternebrae, any sternebrae 15 maligned Sternebrae, large Xiphisternum, incompletely ossified Xiphisternum, small Xiphisternum, bicentral Xiphisternum, absent Xiphisternum, unilateral Pelvic girdle, pubis small Pelvic girdle, pubis asymmetrical Pelvic girdle, ischium small Pelvic girdle, ischium incompletely ossified Pelvic girdle, ilium incompletely ossified Forelimbs, 31 metacarpals Forelimbs, less than 3 metacarpals Forelimbs, metacarpal incompletely ossified Forelimbs, proximal phalanges present Hindlimbs, proximal phalanges present Hindlimbs, distal phalanges absent Hematoma b
po0.05; po0.01; po0.001.
a b

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0 93 1/1 a 1/1 1/1 1/1 2/2 1/1 1/1 1/1 2/2 95 1/1 1/1 1/1 2/2 2/2 2/2 4/3 1/1 0/0 1/1 1/1 66/17 1/1 2/2 10/8 2/2 3/3 16/8 31/14 18/9 3/3 1/1 1/1 4/3 0/0 0/0 0/0 37/15 6/5 1/1 17/12 1/1 1/1 0/0 1/1 0/0 1/1 4/3 0/0 0/0 1/1 0/0 0/0 0/0

0.5 94 0/0 0/0 0/0 0/0 13/6 18/7 12/6 0/0 0/0 0/0 0/0 42/14 0/0 5/4 2/2 0/0 12/8 8/7 38/16 16/10 4/4 3/3 3/3 11/7 3/3 0/0 0/0 42/16 0/0 0/0 22/13 0/0 0/0 0/0 0/0 0/0 0/0 1/1 1/1 1/1 5/4 0/0 2/2 0/0

1.25 97 0/0 0/0 0/0 0/0 6/5 8/5 3/3 0/0 0/0 0/0 0/0 20/8 0/0 9/5 2/2 0/0 13/8 11/7 36/18 14/10 16/10 6/4 4/3 14/9 3/2 0/0 1/1 49/17 0/0 0/0 16/10 0/0 0/0 0/0 0/0 0/0 0/0 0/0 13/6 0/0 17/9 1/1 0/0 0/0

2.5 99 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 103 0/0 0/0 0/0 2/2 1/1 2/2 4/3 0/0 0/0 0 0 20/10 0/0 14/6 2/2 2/2 3/3 13/7 62/17 11/7 9/7 20/11 6/4 7/6 9/6 1/1 0/0 32/14 2/2 0/0 23/13 0/0 0/0 0/0 0/0 2/2 0/0 3/2 0/0 0/0 3/2 0/0 0/0 3/2

Incidence data is presented as (no. of fetuses/no. of litters). Found during skeletal examination

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Water Intake (ml/rat/day)

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15

10

Day of study (d)

Fig. 4. One-generation/embryotoxicity studyMean water intakes of male rats (range of SD values 5 0.556.63).

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25 Food intake (g/rat/day)

23

21

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Day of study (d)

Fig. 5. One-generation/embryotoxicity studyMean food intakes of male rats (range of SD values 5 0.285.86).

values lower than the control group value at the start of gestation, with partial recovery during the gestation period except in the group consuming 2.0% IPA in the water (Fig. 9b). Overall weight gain during gestation in these groups was similar to the control value. Following parturition, body weights for all dose groups were initially similar to the control; however, from PND 4 onward, weights of animals consuming water containing 2.0% IPA were significantly lower (Fig. 9c). The administered dosages of IPA were calculated from the data for water intake, body weight, and the nominal concentrations in the test solutions (Table 4). For the male groups, the mean dose level(s) of IPA were 383, 686, and 1107 mg/kg bw/day during the premating period, and 347, 625, and 1030 mg/kg bw/day for the 18 weeks of treatment. Mean values for the groups females were 456, 835, and 1206 mg/kg bw/day during the premating phase; 668, 1330, and 1902 mg/kg bw/day during the

gestation period; and 1053, 1948, and 2768 mg/kg bw/ day for the postpartum phase. There were no infertile males in any group (Table 5), and there was no effect of IPA exposure on female fertility. The length of gestation in females consuming water with IPA was comparable to the control values. The number of pups/litter on gestation day 1 was comparable to the control value for the 0.5% and 1.0% groups, but reduced in the 2.0% group. This decrease in litter size in the 2.0% group was not replicated in the embryotoxicity portion of the study, suggesting an increase in pup mortality during parturition or GD 0, followed by cannibalism of the dead pups by the dam. This explanation is also supported by a decrease in pup survival from postnatal day 1 onward (see below). There was a slight dose-dependent decrease in red blood cells in males given 2.0% IPA in the drinking
Birth Defects Research (Part B) 83:459476, 2008

95 -9 8 10 210 5 10 911 2 11 611 9 12 312 6

28 -3 2

63 -6 7

-3

-1

56 -6 0

42 -4

88 -9 1

21 -2 5

-2

34

35 -3 9

49 -5 3

12

71

14 -1

to

to

95 -9 8 10 210 5 10 911 2 11 611 9 12 312 6

Control 0.50% 1.00% 2.00%

28 -3 2

35 -3 9

42 -4 6

49 -5 3

14 -1

-3

-1

56 -6

88 -9 1

63 -6 7

-2

34

12

21 -2 5

to

71

to

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467

Table 4 F0 Generation Parameters of Rats Given 02.0% IPA in the Drinking Water One-Generation Reproductive Toxicity Study
Isopropanol (% in drinking water) F0 generation parameters Males Mean water intake (ml)7SD Mean food intake (g)7SD Mean calculated IPA intake (mg/kg/day) Mean calculated IPA intake (mg/kg/day) Females Mean water intake (ml)7SD Mean water intake (ml)7SD Mean water intake (ml)7SD Mean food intake (g)7SD Days 3 to 126 Days 3 to 126 Premating phase Days 3 to 126 0 31.171.74 25.070.85 0 0 0.5 31.271.75 23.571.09 383 347.0 1.0 28.171.49 24.071.07 686 625.4 2.0 22.271.11 22.271.22 1107 1030.4

Premating phase Gestation phase Postpartum phase Premating phase Gestation phase Postpartum phase Premating phase Gestation phase Postpartum phase

22.970.93 38.374.55 64.0727.23 15.370.66 19.772.12 39.7719.09 0 0 0

23.871.51 43.775.84 68.8730.13 15.270.94 20.072.01 42.1720.08 455.6 668.4 1052.8

22.572.22 42.076.40 63.0727.46 14.571.17 19.671.76 40.7719.93 835.1 1329.6 1947.8

15.772.12 29.573.62 40.3713.61 13.371.65 18.671.00 27.679.26 1206.1 1901.8 2768.3

Mean calculated IPA intake (mg/kg/day)

po0.05; po0.01; po0.001.

550

500

450

Bodyweight (g)

400

350

300

Control 0.50% 1.00%

250

2.00%

200
28 42 56 77 1 3 7 14 35 11 2 10 5 11 9 12 6 -1 49 70 84 91 -3 63 21 98

Day of study (d)

Fig. 6. One-generation/embryotoxicity studyMean body weights of male rats (range of SD values 5 5.155).

water and females in the 1.0% and 2.0% groups (data not shown). Mean values were still within 8% of the control values. For males only, there was also a slight but statistically significant increase in mean cell volume in the mid- and high-dose groups. There were no treatment-related effects on
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hematocrit, hemoglobin, or numbers of leucocytes in either sex. No macroscopic abnormalities were seen at necropsy in females in either the embryotoxicity or one-generation reproductive phases of this study (data not shown). In addition, no treatment-related histopathological changes

468
(a)

FABER ET AL.

(b)

(c)

Fig. 7. a. One-generation/embryotoxicity studyMean water intakes of female ratspremating phase (range of SD values 5 0.375.24). b. One-generation/embryotoxicity studyMean water intakes of female ratsgestation (range of SD values 5 3.9314.72). c. Onegeneration/embryotoxicity studyMean water intakes of female ratspostpartum phase (range of SD values 5 5.4449.45).

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(a)

469

(b)

(c)

Fig. 8. a. One-generation/embryotoxicity studyMean food intakes of female ratspremating phase (range of SD values 5 0.272.32). b. One-generation/embryotoxicity studyMean food intakes of female ratsgestation phase (range of SD values 5 2.3410.86). c. Onegeneration/embryotoxicity studyMean food intakes of female ratspostpartum phase (range of SD values 5 5.1628.99).

Birth Defects Research (Part B) 83:459476, 2008

470
(a)

FABER ET AL.

(b)

(c)

Fig. 9. a. One-generation/embryotoxicity studyMean body weights of female ratspremating phase (range of SD values 5 7.714.1). b. One-generation/embryotoxicity studyMean body weights of female ratsgestation (range of SD values 5 9.0230.8). c. Onegeneration/embryotoxicity studyMean body weights of female ratspostpartum phase (range of SD values 5 11.327.3).

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Table 5 Litter Parameters for the One-Generation Reproductive Toxicity Study
Isopropanol (% in drinking water) Litter parameters No. of infertile males Length of gestation (days) No. of females with litters on GD 1 No. of pups/litter on GD 1 Total no. pup survival (%) Pup survival/litter (%) Pup body weight (g) on GD 1 Pup body weight (g) on GD 4 Pup body weight (g) on GD 7 Pup body weight (g) on GD 14 Pup body weight (g) on GD 21 Pup body weight gain (g) GD 121
po0.05; po0.01; po0.001.
a

471

0 0 22.170.36 a 15 7.9 84.0 87.5 5.971.54 8.972.07 13.072.86 27.675.71 47.477.22 42.376.35

0.5 0 22.270.49 20 9.5 89.4 85.3 5.671.09 8.671.90 12.472.35 25.775.04 44.278.08 38.577.14

1.0 0 22.170.24 17 8.3 89.4 86.0 5.371.10 8.371.50 12.272.04 26.373.74 44.075.38 38.674.68

2.0 0 22.370.45 16 6.1 71.4 58.6 5.770.81 8.170.92 10.571.23 23.772.71 38.375.06 32.775.14

Mean7SD.

Table 6 Litter Parameters of Rats from the One-Generation Reproductive Toxicity Study Embryotoxicity Phase
Litter parameters No. females with litters GD 20 Corpora lutea Implantations Preimplantation losses Live fetuses Postimplantation losses Percent implantations Early resorptions Late resorptions Litter weight (g) Fetal body weight (g)
po0.05.
a

0 9 13.271.09 a 13.170.78 0.170.33 12.171.62 1.071.22 92.2 0.971.27 0.170.33 25.373.34 2.0970.135

Isopropanol (% in drinking water) 0.5 1.0 9 13.872.05 13.372.29 0.770.73 12.471.88 0.971.36 94.1 0.871.39 0.170.33 26.773.78 2.1570.095 7 12.770.76 13.070.82 0.170.38 12.070.82 1.070.82 92.4 1.070.82 0 26.577.13 2.1970.461

2.0 8 13.371.04 12.471.19 1.071.31 12.171.55 0.470.52 96.8 0.470.52 0 22.972.04 1.9070.133

Mean7SD.

were seen in tissues of the reproductive system of parental animals at the highest dose tested (data not shown). There was a statistically significant increase in absolute kidney weight and relative kidney, liver, and spleen weights in F0 high-dose group males (Table 7a). For F0 high-dose group females, there was a statistically significant increase in absolute liver and kidney weights and relative liver weights. The only significant findings from the embryotoxicity study portion of the study was an increase in preimplantation loss, a decrease in mean litter weight, and a decrease in mean fetal body weight in the 2.0% group.

F1 Generation
Litter parameters, including pup survival data and weights, are summarized in Table 5 (embryotoxicity phase) and Table 6 (one-generation reproductive toxicity phase). In the embryotoxicity study, there was a statistically significant increase in the total number of
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preimplantation losses in animals given 2.0% IPA and a slight but not statistically significant decrease in mean litter and mean fetal weights in this group (Table 6). The only other finding in this phase of the study was whole body edema seen in 40% of the fetuses in three of eight litters in the high-dose group. No macroscopic abnormalities of the viscera of these fetuses were detected, and the incidence of edema was not related to gender. In the one-generation reproductive phase, there was a statistically significant decrease in postnatal pup survival (Table 5) and in the average pup weight (by PND 7) in the 2.0% group. Although mean pup weight and mean pup weight gain were also lower for the 0.5% and 1.0% treatment groups, there was no dose-response relationship and the differences were not statistically significant. F1 generation animals of both sexes showed a significant increase in relative liver weights at all dose levels, with the effects being very highly significant at the 2.0% IPA level (Table 7b). High-dose group males also had a significantly higher relative kidney weight. In the F1 generation group receiving 2.0% IPA, there was a slight but statistically significant decrease in absolute brain

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FABER ET AL.
Table 7a F0 Generation Organ Weights

Weights Males No. in group Terminal Body weight (g) Liver (g) (g/100g BW) Spleen (g) (g/100g BW) Kidney (g) (g/100g BW) Gonads (g) (g/100g BW) Females No. in group Terminal Body weight (g) Liver (g) (g/100g BW) Spleen (g) (g/100g BW) Kidney (g) (g/100g BW) Gonads (g) (g/100g BW)
po0.05; po0.01; po0.001.
a

0 10 454.5745.80 a 11.6471.317 2.5770.276 0.8470.148 0.1970.031 2.7970.279 0.6270.059 3.5770.213 0.8070.116 15 234.1711.73 6.7470.712 2.8870.273 0.5370.071 0.2370.027 1.5470.085 0.6670.031 0.09670.0184 0.0470.008

Isopropanol (% in drinking water) 0.5 1.0 10 459.9734.35 11.4971.137 2.5070.137 0.8770.119 0.1970.032 2.7170.180 0.5970.045 3.5370.155 0.7770.074 20 239.8710.70 6.7770.521 2.8370.249 0.5570.056 0.2370.024 1.5370.092 0.6470.035 0.08970.0203 0.0470.009 10 458.5752.58 11.9071.774 2.5970.143 0.8270.124 0.1870.013 2.8570.418 0.6270.060 3.5670.223 0.7870.064 17 237.6714.50 7.3570.928 3.1070.345 0.5870.096 0.2470.035 1.5970.161 0.6770.062 0.09970.0247 0.0470.009

2.0 10 426.4728.51 12.2071.046 2.8670.171 0.9170.103 0.2170.019 3.0770.187 0.7270.030 3.5970.161 0.8470.060 16 243.4720.90 8.0071.327 3.2970.543 0.5270.078 0.2170.023 1.6670.153 0.6870.055 0.08370.0132 0.0370.006

Mean7SD.

Table 7b F1Generation Organ Weights

Isopropanol (% in drinking water) Weights Males No. in group Terminal body weight (g) Brain (g) (g/100g BW) Liver (g) (g/100g BW) Spleen (g) (g/100g BW) Kidney (g) (g/100g BW) Gonads (g) (g/100g BW) Females No. in group Terminal Body weight (g) Brain (g) (g/100g BW) Liver (g) (g/100g BW) Spleen (g) (g/100g BW) Kidney (g) (g/100g BW) Gonads (g) (g/100g BW)
po0.05; po0.01; po0.001.
a

0 12 173.3734.91a 1.6470.110 0.9870.184 6.6871.255 3.8770.260 0.6270.131 0.3670.041 1.5170.251 0.8870.066 2.1970.550 1.2670.154 12 128.4716.50 1.5470.125 1.2170.130 4.8470.716 3.7370.243 0.4770.071 0.3770.039 1.1570.130 0.9070.045 0.0770.020 0.0670.011

0.5 17 179.8724.46 1.6770.082 0.9470.100 7.3171.022 4.0770.182 0.6070.067 0.3470.052 1.5570.186 0.8670.067 2.4170.332 1.3470.082 18 128.3714.45 1.5770.079 1.2370.126 5.1570.621 4.0270.252 0.4770.061 0.3770.041 1.1670.170 0.9070.060 0.0770.010 0.0570.008

1.0 13 178.5731.44 1.6370.064 0.9570.194 7.3871.446 4.1370.332 0.6370.118 0.3670.068 1.5070.214 0.8570.065 2.3470.489 1.3070.115 14 132.1717.57 1.5370.082 1.1870.159 5.2370.667 4.0070.325 0.4670.073 0.3570.041 1.1770.117 0.9070.074 0.0870.024 0.0670.013

2.0 9 167.0722.74 1.5370.066 0.9370.115 7.2870.872 4.3770.116 0.5770.107 0.3470.039 1.5470.223 0.9270.059 2.3170.325 1.3870.068 9 124.4715.91 1.4170.106 1.1570.173 5.4070.707 4.4870.200 0.4570.094 0.3670.050 1.1470.124 0.9270.050 0.0770.025 0.0570.017

Mean7SD.

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REVIEW OF REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

weight and increase in relative empty cecum weights in both sexes. No treatment-related gross abnormalities were observed in the F1 generation animals at necropsy.

473

DISCUSSION
The following discussion presents an overall weight of evidence regarding male fertility and decrease in postnatal pup survival in rats exposed to isopropanol. The discussion draws on the BIBRA studies, the Nelson study, and those performed for the EPA TSCA Section 4 Test Rule data call-in. Administration of IPA via the drinking water presents palatability problems at the levels used in these studies. Large decreases in water and food consumption can cause adverse reproductive and developmental outcomes in laboratory animals. In these studies (BIBRA, 1987, 1988), it was difficult to separate the developmental effects due to IPA exposure from those due to the decreases in water and food consumption, secondary to palatability problems. In the developmental toxicity study, exposure to 0.5% or 1.25% IPA, low and intermediate dose levels, in the drinking water resulted in smaller decrements in water consumption, allowing analysis of results from those dose levels without the confounding effect of reduced water and food consumption. Previous investigations of the developmental toxicity of IPA were conducted by the inhalation and oral gavage routes. Nelson et al. (1988) exposed groups of pregnant rats to 3500, 7000, or 10,000 ppm IPA for 7hr/day on gestation days (GD) 119. Maternal toxicity was observed in the mid- and high-exposure dams with narcosis and/ or unsteady gait; a decrease in food consumption and a corresponding decrease in maternal weight gain. Fertility was adversely affected in female rats exposed to 10,000 ppm isopropanol. Six of 15 animals failed to become pregnant and 4 of the remaining 9 litters were totally resorbed. There was a dose-dependent reduction in fetal body weights at all three exposure concentrations and an increase in skeletal malformations (primarily rudimentary cervical ribs) at the 7000 and 10,000 ppm exposure levels. There was no dose-related increase in other malformation rates or in number of litters with skeletal or visceral variations. Comparison of the inhalation study results with those from the oral gavage and drinking water studies is complicated as the lowest exposure concentration (3500 ppm) is calculated to provide a daily area-underconcentration curve of approximately 2.7-fold higher than the 400 mg/kg/day dose level from the oral gavage

study (Gentry et al., 2002). In the oral gavage study, Tyl et al. (1994) exposed groups of pregnant rats to 0, 400, 800, or 1200 mg/kg/day of IPA from GD 615. Maternal toxicity (deaths) occurred at the 800 and 1200 mg/kg/ day dose levels, but no evidence of reductions in body weight or food consumption were found. Although there was no evidence of teratogenic effects due to IPA exposure, indications of fetotoxicity were evident, with decreases in fetal body weights at the 800 and 1200 mg/ kg/day dose levels. This suggests that the decrements in fetal body weights from dams consuming 1.25% or 2.5% IPA in this drinking water study (BIBRA, 1987), providing 1242 and 1605 mg/kg/day, respectively, were also related to IPA exposure rather than from reductions in water or food consumption per se. However, the increased incidence of variations presenting with reduced ossification in the current drinking water study was not replicated in the oral gavage study, suggesting that these effects may be related to the reductions in maternal water and food consumption noted here. Examination of the BIBRA study and the Tyl study suggests that when IPA is administered at dose levels of 800 mg/kg/day by the oral route, evidence of fetotoxicity, but not teratogenicity, is found. Fetotoxicity, as manifested by reduced fetal body weights, only occurred at dose levels that also caused maternal toxicity, either significant reductions in water and food consumption (drinking water study) or death (oral gavage study). Dose levels of 400 mg/kg/day (oral gavage) or 596 mg/ kg/day (via the drinking water) were without adverse effects. Comparison of the results from the BIBRA onegeneration study reported here with the oral gavage two-generation study previously reported (Bevan et al., 1995) resulted in some interesting contrasts. Table 8 summarizes the available evidence for the male fertility indices from these studies. Dose levels of 11001200 mg/kg/day in the BIBRA Probe and Definitive drinking water studies, did not affect the fertility of the male or female animals, even with significant reductions in water and food consumption in the group consuming water with 2.5% IPA. Specifically, in these studies the male mating index was 100% for all of the male animals in each dose group in contrast to the reported reductions in this parameter in the Bevan paper. Because the male animals in the current study were housed with three female rats during the mating period and the Bevan paper reported a 1:1 male to female mating paradigm, we went to individual data from the study reports in an attempt to identify possible causes for these differing outcomes.

Table 8 Isopropanol Dose Levels and Male Fertility Indices

Study BIBRA Probe Drinking Water Study Main Drinking Water Study Bevan et al. P1 Generation P2 Generation Statistically significantly reduced. Birth Defects Research (Part B) 83:459476, 2008 Dose levels (mg/kg bw/day) 0, 317, 711, 1001, 1176 0, 383, 686, 1107 0, 100, 500, 1000 0, 100, 500, 1000 Male fertility indices (%) All groups 5 100 All groups 5 100 80, 83, 83, 70 80, 82, 72, 62

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FABER ET AL.
propose that the weight of evidence suggests that IPA does not affect male mating or fertility at levels up to at least 1000 mg/kg/day. The BIBRA study is the more robust study to evaluate these functional endpoints. The decrease in postnatal pup survival noted in the BIBRA one-generation study replicates similar findings from previously reported papers. Increases in water consumption during pregnancy and lactation resulted in very high levels of IPA exposure, with values as high as 1902 mg/kg/day during gestation and 2768 mg/kg/day during the lactation period. Decreases in postnatal pup survival and decreases in mean pup body weights at the 2768 mg/kg/day level in the current study replicate, albeit at a much higher dose level, the results noted in the 1000 mg/kg/day group from the Bevan study. However, the 1.25% group in the BIBRA study provided a dose level of 1330 mg/kg/day during gestation and 1948 mg/ kg/day during lactation, with no decrease in postnatal pup survival. Again, a discrepancy was noted between the results from the present study with what was reported in the Bevan paper. The difference between the postnatal pup survival indices reported here and those reported in the Bevan paper may be simply due to the difference in how the oral dose was delivered. In this paper, IPA was administered in the drinking water, whereas in the Bevan et al. (1995) study, the IPA was delivered by oral gavage bolus dosing, using a water vehicle. In an attempt to better understand the reported decreases in postnatal pup survival in the Bevan study, we investigated other studies that used similar dosing paradigms to see whether these results had been replicated by others. One other study investigated postnatal pup survival following oral gavage bolus dosing of the dams with IPA. The developmental neurotoxicity study (Bates et al., 1994) conducted by oral gavage with dose levels of 0, 200, 700, and 1200 mg/kg bw/day also examined litter size and postnatal survival of pups from dams exposed to IPA. Dams (3135/dose level) were exposed to oral gavage bolus doses of IPA in water from GD 6 through PND 21. Litters were culled on PND 4 to eight pups/ litter, resulting in group sizes of 2631 dams/litter. There was a slight reduction in the percentage of live pups/ litter in the 1200 mg/kg/day group on PND 0 and 4 (86%87% vs. 93%95% in the control group), although the authors did not consider this decrease to be significant. The 700 mg/kg/day group had pup survival values equal to or better than the control values (see Table 2 in Bates et al., 1994). The majority of the decreased postnatal pup survival noted in the Bevan study occurred between PND 1 and 4, although there was also significant toxicity in postweaning pups when they started to receive IPA by oral gavage. To understand the differing results from the Bevan study and those reported by Bates et al. (1994), the original study reports were reviewed. The decrease in postnatal survival in individual litters correlated with the severity of the clinical signs (body weight loss, decreases in food consumption) in the individual dams and the size of the litter. Gross observations in the dead or dying pups included a lack of milk in their stomachs, suggesting a lack of maternal care. For example, in the 500 mg/kg/ day groups P2 group, three dams that lost a large percentage of their litter had reduced food consumption and lost body weight between PND 1 and 4. Because
Birth Defects Research (Part B) 83:459476, 2008

Examination of the original study report that was the basis for the Bevan paper revealed an interesting finding, a problem with how the mating index and fertility indices for males were calculated.1 During the breeding period of the two-generation study, male and female animals from the same dose level were cohabitated for one week. If mating did not occur, then another male (that had not bred the first week) was cohabitated with the female rat for another week. If mating still did not occur, then the procedure was repeated for a third week. When there are equal numbers of male and female animals, the method of mating works well. However, in the two-generation study, there were treatment-related deaths in the 1000 mg/kg/day parental groups (both generations) and in the 500 mg/kg/day P2 generation. These deaths occurred primarily in female animals. Therefore, there were a larger number of male animals than female animals available for breeding in that study. As the breeding was conducted, certain male animals within the 500 and 1000 mg/kg/day groups were given up to three opportunities to prove/disprove their fertility, whereas others were provided with a single opportunity. Because positive male fertility was determined by a single successful mating, it is obvious that not all of the male animals were provided with equal opportunities to prove or disprove their fertility. For this reason, the male mating index results reported in the Bevan et al. (1995) paper is suspect, and recalculation of the male mating index is not possible given the method of breeding used. The problem was exacerbated in the second (P2) generation because more female animals had died, creating a greater disparity between the number of male and female animals available for breeding. There is another important difference between the BIBRA study and the Bevan study. The male rats in the BIBRA study received treatment with isopropyl alcohol after they had attained sexual maturity, whereas the P2 generation male animals from the Bevan study were exposed throughout gestation, lactation, postweaning, and after reaching sexual maturity. Could IPA have caused a subtle effect in the P2 generation male animals that could be expressed as a reduced mating index or fertility index? Evidence against this hypothesis is that there were no other indications of altered male reproductive capacity in the 1000 mg/kg/day group from the P2 generation. There were no effects on weights or histopathology of male reproductive organs in this group. This is also true of the 1000 mg/kg/day group from the P1 generation and from all of the male animals from the BIBRA studies. Given the lack of effects of IPA on male fertility in the BIBRA study, even with dose levels greater than the 1000 mg/kg/day used in the Bevan study, and the problems associated with the methods used to derive the male mating index in the Bevan study, we therefore
1 The protocol followed then current U.S. EPA guidelines for reproductive toxicity studies, which provides: (A) For each mating, each female shall be placed with a single male from the same dose level until pregnancy occurs or 1 week has elapsed. If mating has not occurred after 1 week, the female shall be placed with a different male. Paired matings should be clearly identified. (B) Those pairs that fail to mate should be evaluated to determine the cause of the apparent infertility. This may involve such procedures as additional opportunities to mate with proven fertile males or females, histological examination of the reproductive organs, and examination of the estrus or spermatogenic cycles (40 CFR 798.4700(c)(6)).

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Table 9 Comparison of Live Pups in the 2-Generation and Developmental Neurotoxicity Studies by Number and Percent
Study Bevan et al., 1995 2-generation F1 2-generation F2 Dose (mg/kg bw/day) 0 100 500 1000 0 100 500 1000 Affected dams 3/24 2/25 5/25 10/20 02/24 3/24 3/21 5/16 Live pups PND 0 (x/%) 12/97% 12.9/96.7% 13.4/94.4% 12.3/80.9% 12.9/97.8% 14.1/96.7% 13/92% 13.2/91.3% Live pups PND 4 (x/%) 12/99.7% 12.7/98.7% 13/96.9% 11.2/91.4% 12.8/99% 14.1/98.4% 12.6/96.9% 12.7/96% Survival PND 421 (%) 99.4 99.5 99 92.2 100 97.8 94.4 91

Bates et al., 1994 Developmental neurotoxicity probe

Developmental neurotoxicity main study

0 200 400 800 1200 0 200 700 1200

0/15 0/7 0/7 0/6 0/5 0/34 0/35 0/31 0/35

11.6/90.8% 10.3/89.1% 10.7/92.8% 11.5/92.5% 10.8/80.5% 11.48/95.2% 11.03/94.1% 11.87/94.0% 11.38/87.7%

11.2/88% 10.3/89.1% 10.6/91.8% 11.3/91.2% 10.2/76.2% 11.21/93% 11.18/92.5% 12.24/97% 11.29/86.4%

100 100 100 100 100 99.5 98.2 100 99.57

Affected dams are dams with 42 pups born dead and/or 42 pups dying postnatally.

there were deaths due to IPA in the 100, 500, and 1000 mg/kg/day dose levels in the Bevan study, maternal toxicity in these lactating dams, although not reported, was not unexpected. The lack of clinical signs prior to death due to IPA administration was noted by Tyl et al. in the 1994 paper and corroborated what they observed and reported in the 1992 developmental toxicity paper conducted by the same group. A complete lack of clinical signs preceding death following repeated oral gavage exposures was noted in both the developmental toxicity and developmental neurotoxicity studies. However, the additional stress of parturition and lactation may have allowed the signs of IPA toxicity to become evident in susceptible dams with resulting pup deaths during the postnatal period. The Bates studies confirm that large oral doses of IPA can affect postnatal pup survival. Table 9 was compiled from the study reports from the Bevan two-generation and Bates developmental neurotoxicity studies. The criteria for a dam to be affected was arbitrarily set as 42 pups born dead or 42 pups dying postnatally.

suggests that the LOAEL for decreased postnatal survival from studies where IPA is administered by oral gavage is 1000 mg/kg/day and the NOAEL is 700 mg/ kg/day. The LOAEL from studies where IPA was administered in the drinking water is 2278 mg/kg/day, and the NOAEL from these same studies would be 1947 mg/kg/day.

REFERENCES
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CONCLUSIONS
Based on all of the available evidence, we conclude that IPA exposure does not affect male mating ability or fertility at dose levels up to 1000 mg/kg/day when administered by oral gavage. We conclude that IPA starts to affect early postnatal survival of pups when oral gavage administration reaches dose levels of 1000 1200 mg/kg/day, with no effect being observed at 700 mg/kg/day. Maternal effects (including lethality) may be noted at levels below 700 mg/kg/day, and offspring from affected dams may suffer indirectly from manifestations of this toxicity. The weight of evidence
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Tyl RW, Masten LW, Marr MC, Myers CB, Slauter RW, Gardiner TH, Strother DE, McKee RH, Tyler TR. 1994. Developmental toxicity evaluation of isopropanol by gavage in rats and rabbits. Fund Appl Toxicol 22:139151. Tyl RW. 1996. Personal correspondence to Kathryn Rosica, Chemical Manufacturers Association, Arlington,VA, from Rochelle Tyl, Center for Life Sciences and Toxicology, Research Triangle Institute, Research Triangle Park, NC, February 12. U.S. EPA. 1989. Isopropanol; Final Test Rule. Fed Reg 54(203): 43252 43264. U.S. EPA. 1992. Memorandum to Keith Cronin, Chemical Testing Branch from Jennifer Seed, Toxic Effects Section, July 1. Wilson JG. 1973. Environment and Birth Defects. New York: Academic Press.

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