Relative Developmental Toxicities of Acrylates in Rats following Inhalation Exposure

Transcription

1 TOXICOLOGICAL SCIENCES 48, (1999) Copyright 1999 by the Society of Toxicology Relative Developmental Toxicities of Acrylates in Rats following Inhalation Exposure A. M. Saillenfait, 1 P. Bonnet, F. Gallissot, J. C. Protois, A. Peltier, and J. F. Fabriès Institut National de Recherche et de Sécurité, Vandoeuvre, France Received May 31, 1998; accepted August 24, 1998 The developmental toxicities of seven acrylates were studied in Sprague-Dawley rats after inhalation exposure for 6 h/day, during days 6 to 20 of gestation. The exposure concentrations were: for acrylic acid, 50, 100, 200, or 300 ppm; for methyl acrylate, 25, 50, or 100 ppm; for ethyl acrylate, 25, 50, 100, or 200 ppm; for butyl acrylate, 100, 200, or 300 ppm; for ethylhexyl acrylate, 50, 75, or 100 ppm; for hydroxyethyl acrylate, 1, 5, or 10 ppm; and for hydroxypropyl acrylate, 1, 5, or 10 ppm. No treatment-related increases in embryo/fetal mortality or fetal malformations were observed after exposure to any of these acrylates. Fetal toxicity, indicated by reduced fetal body weight, was observed after exposure to 300 ppm acrylic acid, 100 ppm methyl acrylate, 200 ppm ethyl acrylate, and 200 or 300 ppm butyl acrylate in the presence of overt signs of maternal toxicity. While there was evidence of maternal toxicity, no significant developmental toxic effects were observed after exposure to ethylhexyl acrylate, hydroxyethyl acrylate, or hydroxypropyl acrylate at any concentration. These results indicate that inhaled acrylic acid, methyl acrylate, ethyl acrylate, butyl acrylate, ethylhexyl acrylate, hydroxyethyl acrylate, and hydroxypropyl acrylate are not selectively toxic to the embryo or fetus. Key Words: acrylates; inhalation exposure; developmental toxicity; rat. Acrylic acid esters are high-volume chemicals used in the manufacture of thermoplastics, acrylic resins, and emulsion polymers. They are also used as plasticizers, latex coating, adhesives, fibers, floor finishers, and lubricant additives (Bisesi, 1994). The production of acrylic ester monomers in the U.S. was approximately 450,000 tons in 1989 (Novak, 1991). This represented about 45% of the worldwide production. Published reports on the developmental toxicity of basic acrylic esters are few. When ethyl acrylate was administered by gavage to groups of 10 to 23 Wistar rats at doses of 25, 50, 100, 200, or 400 mg/kg/day from day 7 to 16 of gestation (day 1 of gestation being the day of insemination), maternal body weight was reduced in all treated groups. The total number of resorptions was significantly increased at the three highest doses, but the average number of live fetuses per litter was not 1 To whom correspondence should be addressed. Fax: E- mail: saillenfait@inrs.fr. significantly affected. Skeletal examination of about half of the fetuses revealed an increase in the overall incidence of skeletal defects (delayed ossification of sternebrae, skull anomalies, lack of ribs or shortened ribs) in all the treated groups. Soft tissues were not examined (Pietrowicz et al., 1980). Murray et al. (1981) exposed time-pregnant Sprague-Dawley rats to concentrations of 50 or 150 ppm ethyl acrylate for 6 h/day on days 6 15 of gestation. Maternal effects, evidenced by reduced body weight gain and food consumption and increased water consumption were observed at 150 ppm. There were no statistically significant increases in the incidences of individual or total malformations, although multiple malformations including hypoplastic tail were observed in only three fetuses from three different litters at 150 ppm, among the 338 fetuses from 29 litters examined. Butyl acrylate was administered by inhalation to Sprague-Dawley rats, 6 h/day, from day 6 to 15 of gestation at 25, 135, or 250 ppm (Merkle and Klimisch, 1983). Maternal toxicity, expressed by a decrease in body weight gain, occurred at 135 and 250 ppm. No evidence of teratogenic effects was found. There was a concentration-related decrease in the numbers of live fetuses per litter, but these differences were not statistically significant. The percentage of resorptions was also significantly increased at 135 and 250 ppm. Thus, only ethyl acrylate and butyl acrylate have been studied, and definite conclusions regarding their developmental toxicity potential are difficult to draw from the limited and somewhat divergent data available. This study was undertaken to provide further information on the developmental toxicity of six acrylic esters, in rats, after inhalation exposure throughout the embryonic and fetal periods. The test compounds included methyl acrylate, ethyl acrylate, butyl acrylate, ethylhexyl acrylate, hydroxyethyl acrylate, and hydroxypropyl acrylate. Acrylic acid was also included for comparative purposes. MATERIALS AND METHODS Chemicals. Acrylic acid (99 % pure), methyl acrylate (99 % pure), and ethyl acrylate (99 % pure) were obtained from Fluka Chemie AG (Buchs, Switzerland). n-butyl acrylate (99 % pure) was obtained from Aldrich Chemical Co. (Milwaukee, WI). 2-Hydroxyethyl acrylate (95.8% pure, GC) and hydroxypropyl acrylate (97.5% pure, GC, mixture of isomers) were a gift from 240

2 ACRYLATES EMBRYOTOXICITY 241 Röhm (Germany). 2-Ethylhexyl acrylate (99.7% pure, GC) was a gift from Elf Atochem (France). Animals. After two weeks of acclimatization, nulliparous female ( g) Sprague-Dawley rats supplied by IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l Arbresle, France) were housed overnight with adult males (one male:two or three females) from the same strain and supplier. The day that vaginal smears were found to be sperm-positive was considered day 0 of gestation (GD). Mated females were randomly assigned to treatment groups using a randomization system stratified by body weight on GD 0. Mated females were singly housed in clear polycarbonate cages with stainless-steel wire lids and hardwood shavings as bedding in rooms maintained at 21 2 C, a relative humidity of 50 5%, and a 12-h light-dark photocycle. For exposures, the females were transferred to stainless-steel wire mesh exposure cages, and the cages were moved into the chambers. After each exposure, the animals returned to their original cages and home rooms. Food pellets (UAR Alimentation Villemoisson, France) and filtered tap water were available ad libitum, except during exposures. Experimental design. Each of the above chemicals was tested in a separate experiment, following the same general protocol: groups of bred female rats (17 25 pregnant) were exposed to the compound 6 h/day on days 6 through 20 of gestation. The concentrations of acrylic acid were 50, 100, 200, and 300 ppm; of methyl acrylate, 25, 50, and 100 ppm; of ethyl acrylate, 25, 50, 100, and 200 ppm; of butyl acrylate, 100, 200, and 300 ppm; of ethylhexyl acrylate, 50, 75, and 100 ppm; of hydroxyethyl acrylate, 1, 5, and 10 ppm; and of hydroxypropyl acrylate, 1, 5, and 10 ppm. Control animals were exposed concurrently to filtered room air in an adjacent chamber with characteristics identical to those of the treatment groups. Exposure concentrations were based on preliminary studies in which severe maternal toxicity (i.e., weight loss during GD 6-13 and pronounced reduction in weight gain during GD 13-21) was observed at 200 ppm methyl acrylate, 25 ppm hydroxyethyl acrylate, and 25 ppm hydroxypropyl acrylate. Maternal mortality also occurred at 200 ppm methyl acrylate and 25 ppm hydroxyethyl acrylate. Selection of ethyl- and butyl-exposure levels was based, in part, on preliminary experiments in which marked decreases in maternal weight gain were observed at 200 and 300 ppm of ethyl and butyl acrylate. Results from previous prenatal inhalation toxicity studies on ethyl and butyl acrylate were also considered (Murray et al., 1981; Merkle and Klimisch, 1983). The high concentrations of ethyl and butyl acrylate for the definitive study (200 and 300 ppm, respectively) were chosen to maximize the opportunity of identifying embryolethal or teratogenic potential. Only minimal maternal toxicity was observed after exposure to 90 ppm ethylhexyl acrylate. Nevertheless, 100 ppm ethylhexyl acrylate was used as the highest concentration for the definitive developmental toxicity study, since preliminary level-setting studies have indicated that 100 ppm was the highest reliable vapor concentration technically possible. Generation of test atmospheres. Exposures were conducted in 200-L glass/stainless-steel inhalation chambers with dynamic and adjustable laminar air flow (6 20 m 3 /h). In order to prevent any leakage of the test atmospheres, the chambers were maintained at a negative pressure of no more than 3 mm water. The chamber temperature was set at 23 2 C, and the relative humidity at 50 5%. Food and water were withheld during exposure. Two vaporgeneration systems were used depending on the chemical tested. The first system, used for acrylic acid, 2 hydroxyethyl acrylate, and hydroxypropyl acrylate, delivered, with an infusion pump, a constant rate of liquid chemical from the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a countercurrent fashion to the liquid flow. In the second system, used for methyl acrylate, ethyl acrylate, n-butyl acrylate, and 2-ethylhexyl acrylate, an additional air-flow rate passed through the fritted disk of a heated bubbler containing the test chemical. In both systems, the vaporized compounds were introduced into the main air inlet pipe of the exposure chambers. Atmosphere sampling and analysis. Concentrations of acrylate esters were monitored continuously with a gas-chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. In addition, exposure Compound TABLE 1 Mean Analytical Concentrations of Compounds in Exposure Chambers Target concentrations (ppm) Analytical concentrations (ppm) Mean S.D. Low Range High Acrylic acid Methyl acrylate Ethyl acrylate Hydroxyethyl acrylate n-butyl acrylate Hydroxypropyl acrylate Ethylhexyl acrylate levels were determined once during each 6-h exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal. The charcoal samples were then desorbed with carbon disulfide, except for the 2-hydroxyethyl acrylate and 2-hydroxypropyl acrylate samples, which were desorbed with dichloromethane. The resulting samples were then analyzed by gas chromatography using appropriate internal standards. Internal calibrations of acrylates were performed using isobutyl acetate, n-butyl acetate, hydroxypropyl acrylate, n-hexyl acetate, 2-hydroxyethyl acrylate and 5-nonanone, for methyl acrylate, ethyl acrylate, 2-hydroxyethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, and 2-ethylhexyl acrylate analyses, respectively. Concentrations of acrylic acid were determined 3 times, at regular intervals, during each 6-h exposure period. Atmosphere samples were collected through quartz fiber filters impregnated with a Na 2 CO 3 and glycerol solution. The filters were then desorbed with deionized water. The resulting samples were analyzed by a Dionex ion chromatograph equipped with AG9-SC and AS9-SC columns. Concentrations of acrylic acid were not monitored continuously. Because the concentrations determined by analyses were essentially the same as the target concentrations, the target concentrations will be referred to throughout this paper (Table 1). In the case of acrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, and ethylhexyl acrylate, which have low vapor pressures (3.2, 0.07, 0.07,

3 242 SAILLENFAIT ET AL. and 0.14 mmhg at 20 C, respectively), the presence of liquid particles was evaluated at the highest concentration generated (i.e., 300, 25, 25, and 100 ppm, respectively). Airborn particles were measured with an Aerodynamic Particle Sizer (APS 3300 model, TSI, USA), with a minimum detection limit of 0.5 m. No differences in particle counts were observed between the clean filtered air (control) and the vapor-laden air in the exposure chambers. Maternal and fetal evaluations. Food consumption was measured for the intervals GD 6-13 and Maternal body weights were recorded on GD 0, 6, 13, and 21. On GD 21, the females were euthanized with an intrapulmonary injection of T61 (Hoechst, Frankfurt, Germany) and the uteri were removed and weighed. The number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide (10%) to detect very early resorptions (Salewski, 1964). Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin s solution and examined for internal soft tissue changes (Barrow and Taylor, 1969; Wilson, 1965). The other half were fixed in ethanol (70%), eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination (Staples and Schnell, 1964). Statistical analysis. Whenever possible, the data were presented as mean SD. The number of implantation sites and live fetuses and the various body weights were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett s test if differences were found. The percentages of non-live implants and resorptions and the proportions of fetuses with alterations in each litter were evaluated by using the Kruskal-Wallis test, followed by the Dixon-Massey test where appropriate. Rates of pregnancy, fetal sex ratio, and percentages of litters with malformations or external, visceral, or skeletal variations were analyzed by using Fisher s test. Where applicable, least-squares analysis was carried out. For all statistical tests, the level of significance was set a priori at Acrylic Acid RESULTS All the animals survived this exposure. Maternal body weight gain was significantly reduced during the first half of exposure at 200 ppm, and thoughout the whole exposure period at 300 ppm (Table 2). Absolute weight gain was significantly reduced at 200 and 300 ppm. A decrease in maternal food intake was observed during the first half of exposure at 50 and 100 ppm (8% less than control), and throughout exposure at higher concentrations (Table 3). The mean numbers of implantation sites and of live fetuses and the incidence of non-live implants and resorptions were comparable across groups (Table 4). Acrylic acid induced a concentration-related decrease in fetal body weights that were significantly different from those of control at 300 ppm (9% lower than control). Several external and visceral malformations were observed sporadically with no indication of an adverse effect related to exposure (Table 5). The occurrences of fetuses with external or skeletal variations did not differ between control and acrylic acidtreated groups. A significant increase in the percentage of litters with visceral variations was observed at the low concentration, together with an increased incidence of distended ureter, a common variant in rat fetuses. In contrast, the mean percentage of fetuses with any variations per litter was significantly lower at the highest concentration. These findings appeared to be random and to have no biological significance related to acrylic-acid exposure. Methyl Acrylate Exposure to methyl acrylate up to 100 ppm did not cause maternal death. Significant decreases in maternal weight gain and in food consumption were observed at 50 and 100 ppm during the entire exposure period (Tables 2 and 3). Exposure to 50 or 100 ppm was associated with maternal weight loss when gravid uterus weights were substracted from the body weight gains. No significant effect on the implantation sites, live fetuses, incidence of non-live implants and resorptions, or on fetal sex ratio was discerned in any of the groups exposed to methyl acrylate (Table 4). Methyl acrylate induced a concentrationrelated decrease in fetal body weights that achieved significance at 100 ppm (17% lower than control). A single case of malformation was observed at 100 ppm (Table 6). There were no statistically significant increases in the incidences of external, visceral, or skeletal variations in any treatment group relative to controls. Ethyl Acrylate No maternal deaths were observed during this study. Exposure to 200 ppm led to significant decreases in maternal body weight gain throughout exposure and in absolute weight gain (Table 2). No data on food consumption are available because of a technical failure. There was no significant difference in the numbers of implantation sites and live fetuses, in the incidence of non-live implants and resorptions, or in the fetal sex ratio (Table 4). Fetal body weights were significantly reduced at 200 ppm (7 8% lower than control, p 0.01). Single occurrences of visceral malformations were seen in control and in the 50 and 200 ppm-treated groups (Table 7). The incidences of external, visceral, and skeletal variations were scattered, with no indication of adverse effects in any of the exposed groups when compared to the controls. Butyl Acrylate All female rats survived the test period. For the entire exposure period, maternal weight gains were markedly lower in the 200- and 300-ppm groups than in the control group, with body weight loss occurring at 300 ppm between GD 6 and GD 13 (Table 2). Absolute weight gains were depressed in a concentration-related manner at all exposure levels. Food consumption was significantly reduced during the first half of the exposure at 100 ppm and throughout the exposure period at 200 and 300 ppm (Table 3). The maximum decrease was 40 50% at the highest concentration. No adverse effects on the numbers of implantation sites and live fetuses, or the incidence of non-live implants and resorp-

4 ACRYLATES EMBRYOTOXICITY 243 TABLE 2 Change in Weight During Gestation in Sprague-Dawley Rats Inhaling Acrylic Acid, Methyl Acrylate, Ethyl Acrylate, Butyl Acrylate, Ethylhexyl Acrylate, Hydroxyethyl Acrylate, or Hydroxypropyl Acrylate on Days 6 to 20 of Gestation a Compound Concentration ppm/6 h/day No. of dams Body weight (g) on GD6 Body weight gain (g) on GD Absolute weight gain (g) b Acrylic acid c ** ** 5 12** ** 75 15** 88 18** 13 14** Methyl acrylate ** 73 20** 90 25** 8 21** ** 47 38** 51 48** 40 36** Ethyl acrylate ** 70 15** 87 21** 17 16** Butyl acrylate * ** 76 26** 84 34** 16 20** ** 27 24** 18 34** 60 26** Ethylhexyl acrylate ** Hydroxyethyl acrylate * ** Hydroxypropyl Acrylate * * ** a Includes all dams pregnant at euthanization. b (Day 21 body weight) (gravid uterus weight) (Day 6 body weight). c Values are expressed as means SD. *,** Significant differences from the control (0 ppm) value, p 0.05, and p 0.01, respectively. tions were noted among litters exposed to butyl acrylate (Table 4). Fetal body weight was significantly reduced at 200 ppm (all and males) and at 300 ppm (all, males, females). These decreases amounted to 7 8% and 26 28% of the control values at 200 ppm and 300 ppm, respectively. Few sporadic malformations were seen in the control and 300 ppm groups (Table 8). There was no evidence of treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations (mainly incomplete ossification of sternebrae and of vertebral centra) was similar in the control and treated groups. Ethylhexyl Acrylate No test dams died. Except for a significant decrease in absolute weight gain at 100 ppm, there were no significant changes in maternal weight gain of females exposed to ethylhexyl acrylate, compared to those of controls (Table 2). Rats from the 100-ppm group showed a significant decrease in food intake throughout the entire exposure period (8 11% reduction) (Table 3). No adverse effects were observed on the mean number of implantations and live fetuses among litters exposed to ethyl-

5 244 SAILLENFAIT ET AL. TABLE 3 Food Consumption of Sprague-Dawley Rats Inhaling Acrylic Acid, Methyl Acrylate, Butyl Acrylate, Ethylhexyl Acrylate, Hydroxyethyl Acrylate, or Hydroxypropyl Acrylate on Days 6 to 20 of Gestation a Compound Concentration ppm/6 h/day No. dams Food consumption (g/dam/day) on GD Acrylic acid b c d c d 23 2 d 21 1 d d 20 2 d 19 2 d Methyl acrylate d 20 3 d 20 3 d c 16 2 d 16 6 d 16 4 d Butyl acrylate c c d 23 3 d 21 3 d d 15 4 d 16 4 d Ethylhexyl acrylate d 26 3 d 24 3 d Hydroxyethyl acrylate d 25 2 d 23 2 d Hydroxypropyl acrylate c 24 2 c 22 2 c a Includes all dams pregnant at euthanization. b Values are expressed as means SD. c,d Denote significant differences from the control (0 ppm) value, p 0.05 and p 0.01, respectively. hexyl acrylate (Table 4). The statistically significant reductions in the incidence of non-live implants and resorptions at 50 and 100 ppm were not considered to be of toxicological significance. There was a statistically significant trend toward decreased fetal body weights (p 0.05), but the pairwise comparisons to the concurrent control group were not significantly different. No significant differences were observed between control and treated groups in the incidence of fetal malformations or variations (Table 9). Hydroxyethyl Acrylate No test dams died. Maternal body weight gain during GD 6-13 and absolute weight gain were significantly reduced at 10 ppm (Table 2). A slight but statistically significant decrease in maternal food consumption was seen at 10 ppm for the entire exposure period (Table 3). There were no significant changes in the numbers of implantations and live fetuses, incidence of non-live implants and resorptions, or fetal body weights across groups (Table 4). The only malformation observed was a unilateral microphthalmia at 1 ppm (Table 10). There were no significant changes in the incidence of external, visceral, or skeletal variations. Hydroxypropyl Acrylate No maternal deaths were observed. Maternal weight gain was significantly less than control during the first half of exposure at 10 ppm and absolute weight gain at 5 and 10 ppm. Food consumption was slightly reduced during treatment at 10 ppm. The number of implantation sites and live fetuses, and the incidence of non-live implants and resorptions were comparable among groups (Table 4). There was no effect on fetal

6 TABLE 4 Reproductive Parameters in Sprague-Dawley Rats Inhaling Acrylic Acid, Methyl Acrylate, Ethyl Acrylate, Butyl Acrylate, Ethylhexyl acrylate, Hydroxyethyl Acrylate, or Hydroxypropyl Acrylate on Days 6 to 20 of Gestation Litters with implants Litters with live fetuses Compound Concentration ppm/6 h/day No. Females on study No. of litters No. of implantation sites per litter % of nonlive implants per litter a %of resorption sites per litter No. of litters No. of live fetuses per litter Fetal sex ratio M:F Average fetal body weight (g) per litter All Males Females Acrylic acid b c c c d d d Methyl acrylate d d d Ethyl acrylate d d d Butyl acrylate c c d d d Ethylhexyl acrylate c c c c Hydroxyethyl acrylate Hydroxypropyl acrylate ACRYLATES EMBRYOTOXICITY a Resorptions plus dead fetuses. b Values are expressed as means SD. c,d Denote significant differences from the control (0 ppm) value, p 0.05 and p 0.01, respectively. 245

7 246 SAILLENFAIT ET AL. TABLE 5 Incidence of Malformations and Variations in Fetuses of Sprague-Dawley Rats Inhaling Acrylic Acid on Days 6 to 20 of Gestation a Concentration (ppm/6 h/day) Total No. fetuses (litters) examined External 330 (23) 271 (20) 307 (22) 290 (21) 337 (23) Visceral 165 (23) 135 (20) 154 (22) 145 (21) 169 (23) Skeletal 165 (23) 136 (20) 153 (22) 145 (21) 168 (23) Malformations b Anasarca (1) 0 Hydrocephaly 1 (1) Microphthalmia (unilateral) (1) 0 Omphalocele 1 (1) (1) 0 Interventricular septum defect 1 (1) Ectopic testis (bilateral) 1 (1) Anal atresia 1 (1) (1) 0 No. (%) fetuses with any malformations 2 (0.6) (1.0) 0 No. (%) litters with any malformations 2 (8.7) (14.3) 0 Mean % fetuses with any malformations per litter c External variations Club foot 0 1 (1) No. (%) fetuses with external variations 0 1 (0.4) No. (%) litters with external variations 0 1 (5.0) Mean % fetuses with external variations per litter Visceral variations Dilated renal pelvis 1 (1) 1 (1) 1 (1) 1 (1) 0 Distended ureter 19 (8) 30 (14) f 16 (11) 15 (8) 4 (3) No. (%) fetuses with visceral variations 19 (11.5) 30 (22.2) 16 (10.4) 16 (11.0) 4 (2.4) No. (%) litters with visceral variations 8 (34.8) 14 (70.0) e 11 (50.0) 8 (38.1) 3 (13.0) Mean % fetuses with visceral variations per litter f Skeletal variations Skull Parietals, incomplete ossification, slight (1) 0 Hyoid, incomplete ossification or unossified d 3 (2) 0 1 (1) 0 0 5th and/or 6th sternebrae, incomplete ossification or unossified 8 (7) 8 (5) 6 (5) 3 (2) 6 (6) Rib(s) Floating (1) Cervical, rudimentary 3 (3) 6 (3) 0 2 (2) 1 (1) 14th, supernumerary 10 (7) 3 (2) 10 (8) 4 (4) 9 (6) Thoracic vertebral centra, incomplete ossification (one or two) 10 (8) 10 (7) 5 (5) 9 (5) 7 (6) No. (%) fetuses with skeletal variations 32 (19.4) 25 (18.4) 23 (15.0) 19 (13.1) 23 (13.7) No. (%) litters with skeletal variations 19 (82.6) 12 (60.0) 14 (63.6) 12 (57.1) 13 (56.5) Mean % fetuses with skeletal variations per litter No. (%) fetuses with any variations 51 (15.4) 56 (20.7) 39 (12.7) 35 (12.1) 27 (8.0) No. (%) litters with any variations 20 (87.0) 20 (100.0) 20 (90.9) 16 (76.2) 14 (60.9) Mean % fetuses with any variations per litter f a The incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined. A single fetus may be represented more than once in listing individual defects. b One fetus in the control group (air) had hydrocephaly, protruding tongue, interventricular septum defect, ectopic testis, and anal atresia. One fetus in the 200 ppm group had anasarca and anal atresia. c Mean SD. d Unossified: alizarine red S negative. e p 0.05, Fisher s test. f p 0.05, Dixon-Massey s test.

8 ACRYLATES EMBRYOTOXICITY 247 TABLE 6 Incidence of Malformations and Variations in Fetuses of Sprague-Dawley Rats Inhalating Methyl Acrylate on Days 6 to 20 of Gestation a Concentration (ppm/6 h/day) Total No. fetuses (litters) examined External 324 (25) 305 (21) 323 (23) 336 (23) Visceral 162 (25) 153 (21) 162 (23) 168 (23) Squelettal 162 (25) 152 (21) 161(23) 168 (23) Malformations b Craniorachischisis (1) Multiple skeletal malformations (1) No. (%) fetuses with any malformations (0.3) No. (%) litters with any malformations (4.3) Mean % fetuses with any malformations per litter c External variations Club foot (unilateral) 1 (1) No. (%) fetuses with external variations 1 (0.3) No. (%) litters with external variations 1 (4.0) Mean % fetuses with external variations per litter Visceral variations Ovary, displaced (1) Dilated renal pelvis 0 1 (1) 0 0 Distended ureter 10 (4) 8 (4) 4 (4) 6 (2) No. (%) fetuses with visceral variations 10 (6.2) 8 (5.2) 4 (2.5) 7 (4.2) No. (%) litters with visceral variations 4 (16.0) 4 (19.0) 4 (17.4) 3 (13.0) Mean % fetuses with visceral variations per litter Skeletal variations Hyoid, incomplete ossification 0 1 (1) 0 2 (2) 5 and/or 6th sternebra(e), incomplete ossification or unossified d 6 (5) 4 (2) 5 (4) 17 (5) Rib(s) Cervical, rudimentary 3 (1) 7 (5) 3 (3) 1 (1) 14th, supernumerary 11 (6) 9 (6) 11 (7) 9 (7) 13th, short (1) Thoracic vertebral centra, incomplete ossification (one or two) 11 (7) 5 (4) 6 (4) 6 (4) No. (%) fetuses with skeletal variations 29 (17.9) 24 (15.8) 25 (15.5) 32 (19.0) No. (%) litters with skeletal variations 15 (60.0) 12 (57.1) 13 (56.5) 15 (65.2) Mean % fetuses with skeletal variations per litter No. (%) fetuses with any variations 40 (12.3) 32 (10.5) 29 (9.0) 39 (11.6) No. (%) litters with any variations 16 (64.0) 13 (61.9) 16 (69.6) 17 (73.9) Mean % fetuses with any variations per litter a The incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined. A single fetus may be represented more than once in listing individual defects. b One fetus in the 100 ppm group had craniorachischisis, protruding tongue, and multiple skull and vertebral alterations including fused thoracic vertebral arches, thoracic hemicentrae, bilobed thoracic and lumbar vertebral centrae, and fused vertebral centrea and archs in the caudal and sacral regions. c Mean SD. d Unossified: alizarine red S negative. weights at any exposure level. Visceral malformations were observed in single fetuses in the control and the 5 ppm groups (Table 11). Several visceral and skeletal variations were observed, with no significant differences between treated and control groups. DISCUSSION Acrylic acid and six of its esters were assessed for their prenatal inhalation toxicity in rats. Exposure to 200 or 300 ppm acrylic acid caused obvious maternal toxicity with significant decreases in body weight gain and food consumption throughout the exposure period. At 50 and 100 ppm acrylic acid exposure, a decrease in food consumption was observed only during the first half of exposure and was not accompanied by a significant decrease in body weight changes. Marked maternal toxicity was demonstrated at 50 and 100 ppm methyl acrylate, 200 ppm ethyl acrylate, and 200 and 300 ppm butyl acrylate. Thus, there were pronounced decreases in maternal

9 248 SAILLENFAIT ET AL. TABLE 7 Incidence of Malformations and Variations in Fetuses of Sprague-Dawley Rats Inhaling Ethyl Acrylate on Days 6 to 20 of Gestation a Concentration (ppm/6 hr/day) Total No. fetuses (litters) examined External 237 (17) 225 (17) 255 (19) 271 (19) 261 (17) Visceral 119 (17) 112 (17) 128 (19) 135 (19) 131 (17) Skeletal 118 (17) 113 (17) 127 (19) 136 (19) 130 (17) Malformations b Retina, folded (1) Brain: lateral ventricle, blood filled (1) 0 0 Diaphragmatic hernia 1 (1) (1) No. (%) fetuses with any malformations 1 (0.4) 0 1 (0.4) 0 1 (0.4) No. (%) litters with any malformations 1 (5.9) 0 1 (5.3) 0 1 (5.9) Mean % fetuses with any malformations per litter c External variations Club foot (unilateral) (1) No. (%) fetuses with external variations (0.4) No. (%) litters with external variations (5.9) Mean % fetuses with external variations per litter Visceral variations Dilated renal pelvis 4 (2) (1) 1 (1) Distended ureter 4 (4) 8 (5) 11 (7) 8 (7) 2 (2) No. (%) fetuses with visceral variations 7 (5.9) 8 (7.1) 12 (9.4) 9 (6.7) 2 (1.5) No. (%) litters with visceral variations 5 (29.4) 5 (29.4) 8 (42.1) 8 (42.1) 2 (11.8) Mean % fetuses with visceral variations per litter Skeletal variations Skull Parietals, incomplete ossification, slight 1 (1) Supraoccipital, incomplete ossification, slight (1) 0 Hyoid, incomplete ossification or unossified d 2 (2) 6 (2) 2 (2) 0 0 Sternebra(e) Incomplete ossification or unossified 6 (6) 9 (7) 8 (6) 5 (5) 8 (6) 1st and 2nd, fused 0 1 (1) Rib(s) Cervical, rudimentary 2 (2) 2 (2) 1 (1) 2 (2) 2 (2) 14th, supernumerary 2 (2) 6 (2) 6 (5) 9 (7) 6 (5) Thoracic and/or lumbar vertebral centra, incomplete ossification (one to three) 8 (5) 8 (4) 8 (7) 7 (6) 15 (8) No. (%) fetuses with skeletal variations 19 (16.1) 27 (23.9) 23 (18.1) 22 (16.2) 27 (20.8) No. (%) litters with skeletal variations 11 (64.7) 11 (64.7) 11 (57.9) 13 (68.4) 13 (76.5) Mean % fetuses with skeletal variations per litter No. (%) fetuses with any variations 26 (11.0) 35 (15.6) 35 (13.7) 31 (11.4) 30 (11.5) No. (%) litters with any variations 13 (76.5) 12 (70.6) 15 (78.9) 16 (84.2) 14 (82.3) Mean % fetuses with any variations per litter a The incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined. A single fetus may be represented more than once in listing individual defects. b One fetus in the 200 ppm group had folded retina, protruding tongue, and diaphragmatic hernia. c Mean SD. d Unossified: alizarine red S negative. body weight gain and food consumption over the entire exposure period. Less severe maternal toxicity was observed at 100 ppm butyl acrylate and was expressed as a transient decrease in food intake and a reduced absolute weight gain. Exposure to 100 ppm ethylhexyl acrylate resulted in maternal toxicity, evidenced by decreased absolute weight gain and food consumption during the period of treatment. This was the highest concentration possible without generating aerosol. Exposure to 10 ppm hydroxyethyl or hydroxypropyl acrylate caused overt maternal toxicity. This was evidenced by a transient decrease in body weight changes, a decrease in absolute weight gain, and a continuous reduction of food consumption during exposure. Maternal effects produced by 5 ppm hydroxypropyl acrylate were limited to a significant decrease in absolute weight gain. No evidence of teratogenic effects was found after exposure

10 ACRYLATES EMBRYOTOXICITY 249 TABLE 8 Incidence of Malformations and Variations in Fetuses of Sprague-Dawley Rats Inhaling Butyl Acrylate on Days 6 to 20 Gestation a Concentration (ppm/6 h/day) Total No. fetuses (litters) examined External 353 (25) 353 (24) 347 (24) 367 (25) Visceral 177 (25) 177 (24) 174 (24) 183 (24) Skeletal 176 (25) 176 (24) 173 (24) 183 (24) b Malformations c Anophthalmia (unilateral) 1 (1) Microphthalmia (unilateral) 1 (1) Palate, cleft 1 (1) Omphalocele 1 (1) Diaphragmatic hernia (1) Ectopic testis (bilateral) 1 (1) Sternum, entire unossified (1) Thoracic and lumbar vertebral centra unossified (1) No. (%) fetuses with any malformations 2 (0.6) (0.5) No. (%) litters with any malformations 2 (4.0) (4.0) Mean % fetuses with any malformations per litter d External variations Club foot 1 (1) (1) No. (%) fetuses with external variations 1 (0.3) (0.3) No. (%) litters with external variations 1 (4.0) (4) Mean % fetuses with external variations per litter Visceral variations Dilated renal pelvis 0 2 (2) 1 (1) 0 Distended ureter 16 (9) 11 (7) 6 (5) 1 (1) No. (%) fetuses with visceral variations 16 (9.0) 11 (6.2) 6 (3.4) 1 (0.6) No. (%) litters with visceral variations 9 (36.0) 7 (29.2) 5 (20.8) 1 (4.2) f Mean % fetuses with visceral variations per litter Skeletal variations Skull Supraoccipital, incomplete ossification (1) Hyoid, incomplete ossification 0 1 (1) 0 1 (1) 5 and/or 6th sternebra, incomplete ossification or unossified e 4 (4) 5 (5) 7 (7) 14 (8) Rib(s) Cervical, rudimentary 3 (3) 1 (1) 0 2 (2) 14th, supernumerary 4 (3) 6 (6) 13 (6) 9 (7) 13th, short (1) Floating 1 (1) Thoracic vertebral centra, incomplete ossification (one or two) 11 (9) 18 (12) 10 (8) 21 (11) No. (%) fetuses with skeletal variations 22 (12.5) 28 (15.9) 27 (15.6) 46 (25.1) No. (%) litters with skeletal variations 15 (60.0) 19 (79.2) 16 (66.7) 18 (75.0) Mean % fetuses with skeletal variations per litter No. (%) fetuses with any variations 39 (11.0) 39 (11.0) 33 (9.5) 48 (13.1) No. (%) litters with any variations 21 (84.0) 22 (91.7) 17 (70.8) 20 (80.0) Mean % fetuses with any variations per litter a The incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined. A single fetus may be represented more than once in listing individual defects. b One fetus was inadvertently not examined skeletally, and therefore the N is reduced. c One fetus in the control group (air) had microphthalmia, cleft palate, and omphalocele. One fetus of another litter in the control group had anophthalmia, ectopic testis, and bilateral club foot. d Mean SD. e Unossified: alizarine red S negative. f p 0.05; Fisher s test.

11 250 SAILLENFAIT ET AL. TABLE 9 Incidence of Malformations and Variations in Fetuses of Sprague-Dawley Rats Inhaling Ethylhexyl Acrylate on Days 6 to 20 of Gestation a Concentration (ppm/6 h/day) Total No. fetuses (litters) examined External 355 (25) 357 (24) 351 (23) 352 (23) Visceral 178 (25) 179 (23) 175 (23) 176 (23) Skeletal 177 (25) 178 (24) 176 (23) 176 (23) Malformations b Head: tongue protruding and edema 0 1 (1) 0 0 Aorta, transposed 0 1 (1) 0 0 Diaphragmatic hernia 0 1 (1) 2 (2) 0 Hydronephrosis (unilateral) 1 (1) 2 (1) 1 (1) 0 No. (%) fetuses with any malformations 1 (0.3) 4 (1.1) 3 (0.8) 0 No. (%) litters with any malformations 1 (4.0) 3 (12.5) 3 (13.0) 0 Mean % fetuses with any malformations per litter c External variations Club foot (unilateral) 0 2 (2) 0 0 No. (%) fetuses with external variations 0 2 (0.6) 0 0 No. (%) litters with external variations 0 2 (8.33) 0 0 Mean % fetuses with external variations per litter Visceral variations Dilated renal pelvis 0 1 (1) 3 (3) 1 (1) Hydroureter (unilateral) (1) 0 Distended ureter 5 (4) 12 (8) 11 (8) 8 (5) No. (%) fetuses with visceral variations 5 (2.8) 11 (6.1) 14 (8.0) 8 (4.5) No. (%) litters with visceral variations 4 (16.0) 8 (34.8) 9 (39.1) 5 (21.7) Mean % fetuses with visceral variations per litter Variations Skull Parietals, incomplete ossification, slight 1 (1) (1) Interparietal, incomplete ossification, slight 0 1 (1) 0 0 Hyoid, incomplete ossification or unossified d 1 (1) 0 1 (1) 2 (2) Sternebrae, incomplete ossification or unossified 9 (6) 14 (7) 10 (4) 7 (7) Rib(s) Cervical, rudimentary 1 (1) 3 (1) 1 (1) 2 (2) 14th, supernumerary 14 (7) 12 (8) 13 (7) 9 (6) Thoracic vertebral centra, incomplete ossification (one or two) 11 (8) 16 (12) 14 (7) 9 (7) No. (%) fetuses with skeletal variations 36 (20.3) 40 (22.5) 35 (19.9) 28 (15.9) No. (%) litters with skeletal variations 17 (68.0) 19 (79.2) 12 (52.2) 17 (73.9) Mean % fetuses with skeletal variations per litter No. (%) fetuses with any variations 41 (11.5) 53 (14.8) 49 (14.0) 36 (10.2) No. (%) litters with any variations 19 (76.0) 22 (91.7) 16 (69.6) 18 (78.3) Mean % fetuses with any variations per litter a The incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined. A single fetus may be represented more than once in listing individual defects. b One fetus in the 50 ppm group had head edema, protruding tongue, and transposed aorta. c Mean SD. d Unossified: alizarine red S negative. to any of the tested compounds, up to concentrations which were toxic to the mothers. Fetal toxicity was evident from the statistically significant decreases in the mean fetal body weight at 300 ppm acrylic acid, 100 ppm methyl acrylate, 200 ppm ethyl acrylate, and 200 and 300 ppm butyl acrylate. These effects occurred only in the highest-dose groups, where there was also marked maternal toxicity (i.e., severe decreases in maternal weights associated with reduced food consumption). Thus, the developmental toxicity observed here may be related to the altered maternal status. Although there was some evidence of maternal toxicity, no adverse developmental effects were noted at concentrations up to 100 ppm ethylhexyl acrylate, and 10 ppm hydroxyethyl or hydroxypropyl acrylate. Therefore, the no-observable-adverse-effect-level (NOAEL) for developmental toxicity was 200 ppm for acrylic acid, 50 ppm for methyl acrylate, 100 ppm

12 ACRYLATES EMBRYOTOXICITY 251 TABLE 10 Incidence of Malformations and Variations in Fetuses of Sprague-Dawley Rats Inhaling Hydroxyethyl Acrylate on Days 6 to 20 of Gestation a Concentration (ppm/6 h/day) Total No. fetuses (litters) examined External 274 (21) 263 (18) 304 (22) 301 (21) Visceral 137 (21) 132 (18) 152 (22) 150 (21) Skeletal 137 (21) 131 (18) 152 (22) 151 (21) Malformations b Microphthalmia (unilateral) 0 1 (1) 0 0 No. (%) fetuses with any malformations 0 1 (0.4) 0 0 No. (%) litters with any malformations 0 1 (5.5) 0 0 Mean % fetuses with any malformations per litter c 0 0 External variations Palate (rugae mishappen) 0 1 (1) 0 0 Club foot (unilateral) 1 (1) 0 1 (1) 2 (2) No. (%) fetuses with external variations 1 (0.4) 1 (0.4) 1 (0.3) 2 (0.7) No. (%) litters with external variations 1 (4.8) 1 (5.6) 1 (4.5) 2 (9.5) Mean % fetuses with external variations per litter Visceral variations Dilated renal pelvis (2) 0 Hydroureter (unilateral) (2) 0 Distended ureter 5 (3) 7 (5) 19 (8) 15 (6) No. (%) fetuses with visceral variations 5 (3.6) 7 (5.3) 19 (12.5) 15 (10.0) No. (%) litters with visceral variations 3 (14.3) 5 (27.8) 8 (36.4) 6 (28.6) Mean % fetuses with visceral variations per litter Skeletal variations Skull Parietals, incomplete ossification, slight (1) 0 Hyoid, incomplete ossification (2) 5th sternebra, incomplete ossification or unossified d 12 (8) 4 (3) 5 (4) 3 (3) Rib(s) Cervical, rudimentary 1 (1) 2 (2) th, supernumerary 7 (4) 6 (3) 16 (7) 6 (5) 13th, short 0 1 (1) 0 1 (1) Thoracic and/or lumbar vertebral centra, incomplete ossification (one to three) 10 (6) 12 (8) 18 (11) 12 (10) No. (%) fetuses with skeletal variations 28 (20.4) 23 (17.6) 38 (25.0) 22 (14.6) No. (%) litters with skeletal variations 11 (52.4) 12 (66.7) 16 (72.7) 13 (61.9) Mean % fetuses with skeletal variations per litter No. (%) fetuses with any variations 34 (12.4) 31 (11.8) 57 (18.7) 39 (13.0) No. (%) litters with any variations 13 (61.9) 13 (72.2) 17 (77.3) 16 (76.2) Mean % fetuses with any variations per litter a The incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined. A single fetus may be represented more than once in listing individual defects. b One fetus in the 1 ppm group had microphthalmia and mishappen palate ruggae. c Mean SD. d Unossified: alizarine red S negative. for ethyl acrylate and butyl acrylate 100 ppm for ethylhexyl acrylate, and 10 ppm for hydroxyethyl and hydroxypropyl acrylate. Neeper-Bradley et al. (1997) reported no evidence of developmental toxicity in pregnant New Zealand White rabbits exposed to 25, 75, or 225 ppm acrylic acid on GD Klimisch and Hellwig (1991) exposed rats on GD 6 15 of gestation to acrylic acid at 40, 120, or 360 ppm. There were clear signs of maternal toxicity at 360 ppm. Body weight gain and food consumption were reduced

13 252 SAILLENFAIT ET AL. TABLE 11 Incidence of Malformations and Variations in Fetuses of Sprague-Dawley Rats Inhaling Hydroxypropyl Acrylate on Days 6 to 20 of Gestation a Concentration (ppm/6 h/day) Total No. fetuses (litters) examined External 286 (21) 260 (20) 294 (21) 285 (21) Visceral 143 (21) 130 (20) 147 (21) 143 (21) Skeletal 143 (21) 130 (20) 147 (21) 142 (21) Malformations Microphthalmia (bilateral) (1) 0 Diaphragmatic hernia 1 (1) No. (%) fetuses with any malformations 1 (0.3) 0 1 (0.3) 0 No. (%) litters with any malformations 1 (4.8) 0 1 (4.8) 0 Mean % fetuses with any malformations per litter b External variations No. (%) fetuses with external variations No. (%) litters with external variations Mean % fetuses with external variations per litter Visceral variations Subclavian branching variation 0 1 (1) 1 (1) 0 Dilated renal pelvis (2) 0 Distended ureter 5 (4) 8 (5) 10 (5) 7 (7) No. (%) fetuses with visceral variations 5 (3.5) 9 (6.9) 11 (7.5) 7 (4.9) No. (%) litters with visceral variations 4 (19.0) 6 (30.0) 6 (28.6) 7 (33.3) Mean % fetuses with visceral variations per litter Skeletal variations Sternebra(e) 5th incomplete ossification or unossified c 5 (4) 2 (2) 0 5 (4) 1st and 2nd, fused 0 1 (1) 0 0 Rib(s) Cervical, rudimentary 4 (4) 0 1 (1) 1 (1) 14th, supernumerary 4 (4) 11 (6) 18 (8) 4 (3) 11 and 12th, wavy (1) 0 Thoracic vertebral centra, incomplete ossification (one to three) 3 (3) 3 (2) 15 (8) 1 (1) No. (%) fetuses with skeletal variations 16 (11.2) 17 (13.1) 33 (22.4) 11 (7.7) No. (%) litters with skeletal variations 11 (52.4) 9 (45.0) 16 (76.2) 9 (42.9) Mean % fetuses with skeletal variations per litter No. (%) fetuses with any variations 21 (7.34) 26 (10.0) 44 (15.0) 18 (6.3) No. (%) litters with any variations 13 (61.9) 13 (65.0) 17 (80.9) 13 (61.9) Mean % fetuses with any variations per litter a The incidence of individual defect is presented as number of fetuses (number of litters). Only live fetuses were examined. A single fetus may be represented more than once in listing individual differences. b Mean SD. c Unossified, alizarine red S negative. throughout exposure at 360 ppm and at the beginning of the exposure period at 120 ppm. Indices of maternal toxicity were considered minimal at 40 ppm since only the absolute weight gain was significantly decreased. No adverse effects on fetal viability, morphology, or growth were observed at any concentration. In the current study, exposure to 50 ppm acrylic acid produced only slight maternal toxic effects, as indicated by the transient decrease in food consumption. Thus, the NOAEL is probably somewhat less than 50 ppm acrylic acid for the dams. Ethyl acrylate was administered by inhalation to pregnant Sprague-Dawley rats from GD6 to GD 15 at concentrations of 50 or 150 ppm (Murray et al., 1981). Maternal toxicity, expressed as decreased body weight gain, reduced food consumption, and increased water intake, was seen at 150 ppm. Growth and survival of the progeny was not affected. The incidences of individual or total anomalies were not significantly increased. However, three of the 29 litters exposed to 150 ppm ethyl acrylate produced three fetuses with multiple malformations, including hypoplastic tail. This alteration, which had been noted to occur among controls in past studies, was not considered as indicative of a