FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

Reproductive

19,228-237 (1992)

Study of Acrolein on Two Generations

RICHARD A. PARENT,*” Y‘onsultos,

Ltd.,

HALINA E. CARAVELLO,? AND ALAN M. HOBERMAN+

P. 0. BO.Y 14082. Baton Rouge, Louisiana 70898; TBaker and $Argus Research Laboratories. Horsham. Received

August

8, 199 1; accepted

Reproductive Study of Acrolein on Two Generationsof Rats. PARENT, R. A., CARAVELLO,

H. E., AND HOBERMAN, (1992). Fundam. Appl. Toxicol. 19,228-237.

A. M.

Four groups of 30 male and 30 female rats were intubated with 70 daily dosesof acrolein at levels of 0, 1, 3, or 6 mg/kg in a dosing volume of 5 ml/kg. Rats within each dosinggroup (FOgeneration) were then assignedto a 21-day period of cohabitation and dosing for femalescontinued through cohabitation gestation and lactation. Males were euthanized after cohabitation. F, generation rats were chosen from pups, and a similar pretreatment,cohabitation, gestation,and lactation regimenwas accomplishedresulting in F2 generation pups. Reproductive parameters, body weights, food consumption, and clinical signs were recorded and necropsies were carried out on all treated

animals. Histopathologic

exams were accomplished on selected

reproductive tissues.In addition, gross lesions,target tissues, stomachs,and lungs were examined. For the most part, reproductive parameters were unaffected by acrolein treatment with the exception of reducedpup weights in the F, generation pups at the high-doselevel (6 mg/kg/day). Gastric lesionswere noted consistently in high-doseanimalsand somemid-dose(3 mg/kg/ day) rats. Erosions of glandular mucosa and hyperplasia/hyperkeratosisof the forestomach were the most frequent stomach lesionsobserved.Effects on body weight gains were noted frequently for the high-doseanimals and achieved statistical significancein the mid-doseanimalson severaloccasions.Mortality in all high-doseanimalswaselevatedrelative to control animals. Acrolein, therefore, cannot beconsidereda selectivereproductive toxin in the rat, but doesproduce toxicological effects down to a dosinglevel of 3 mg/kg/day. o 1992 Society of Toxicology.

Acrolein is not only an important industrial chemical and herbicide (Bowmer and Higgins, 1976; Bowmer and Sainty, 1977; Hopkins and Hattrup, 1974; Unrau et al., 1965), but it is also found naturally in a number of foods, in wine, in tobacco smoke, and even in the air that we breathe (Anonymous, 1978, 1979, 1980, 1985, 1986; Carson et al., 1981; Beauchamp et al.. 1985; Izard and Libermann, 1978). Considering the opportunities for exposure to acrolein, it is surprising how little is known about the toxicity of this material. ’ To whom 0272-0590/92 Copyright 8 All rights of

correspondence

should

be addressed.

$5.00 1992 by the Society of Toxicology. reproduction in any form reserved.

of Rats

228

January

Pecformance Chemicals, Pennsylvania

Inc.. Houston,

Te.ras:

24, 1992

Early work dealt mainly with the acute toxicity of acrolein (Anonymous, 1978, 1979, 1980, 1985, 1986; Carson et al., 1981; Beauchamp et al., 1985; Izard and Libermann, 1978) and its irritating properties (Anonymous, 1986: Catilina et al., 1970; Gosselin et al., 1979; Nielsen et al., 1984; WeberTschopp et al., 1977). A few longer-term studies including a subchronic inhalation study reported mortality, upper respiratory tract irritation, and growth depression in rats (Feron et al., 1978). A chronic inhalation study reporting no carcinogenic activity in hamsters (Feron and Kruysse, 1977), a chronic drinking water study in F344 rats (Lijinsky and Reuber, 1987; Lijinsky, 1988) reporting increased adrenal cortical adenomas, a chronic skin painting study in mice resulting in no increase in benign or malignant skin lesions in mice (Salaman and Roe, 1956), and a chronic study using subcutaneous injections reporting no increase in subcutaneous sarcomas in treated mice (Steiner et al., 1943) have been reported, but designs of the longer-term studies were inadequate (Anonymous, 1985) and interpretation of results was sometimes erroneous (see Parent et al., 1992b). Mutagenicity studies have been carried out with acrolein and have reported conflicting results (Hemmininki et al., 1980; Marnett et al., 1985; Rosen et al., 1980; Izard, 1973; Lijinsky and Andrews, 1980; Ssaki and Endo, 1978; Anderson et al., 1972; Zimmering et al., 1985; Ellenberger and Mohn, 1977; Au et al., 1980; Lutz et al., 1982; Curren et al., 1988; Hales, 1982; Hayworth et al., 1983; Parent et al., 199 lb). We have found acrolein to be mutagenic in Salmonella typhimurium but within a very narrow range of concentration (R. A. Parent and H. E. Caravello, unpublished results). The cytotoxic properties of acrolein may explain the diversity of mutagenic findings reported. The only reproduction studies that have been reported are a negative dominant-lethal assay carried out on mice (Epstein et al., 1972) and a single generation-limited reproduction study carried out by the inhalation route (Bouley et al., 1975). Neither reproductive nor fetal parameters were effected. Several studies involving direct treatment of embryos with acrolein (Schmid et al., 198 1; Mirkes et al., 198 1; Korhonen et al., 1983; Slott and Hales, 1986, 1987; Mirkes et al., 1984; Chhibber and Gilani, 1986) have also been conducted and generally result in embryo lethality and embryo malforma-

REPRODUCTIVE

TOXICITY

tions. One study using intravenous injection of acrolein in rabbits demonstrated one of the few observations of embryo lethality in whole animals (Claussen et al., 1980). In addition, retarded fetal growth and malformations were noted to increase with dose but never reached a level of statistical significance. There have been many reports of acrolein being the toxic metabolite of the drug cyclophosphamide (Ohno and Ormstad, 1985; Alarcorn and Meierhoffer, 197 1; Patel, 1990; Lablanc and Waxman, 1990; Schmid et al., 198 1; Mirkes et al., 198 1, 1984; Slott and Hales, 1987; Wrabetz et al., 1980); it has been suggested that acrolein is the metabolite responsible for the teratogenic effects of cyclophosphamide (Hales, 1983). As part of a major effort to better understand the toxicity of acrolein, we have undertaken a number of studies. These include chronic studies in rats (Parent and Caravello, 1990; Parent et al., 1992b), mice (Parent et al., 1992a), and dogs (Parent et al., 1992c), teratogenicity studies in rats and rabbits (R. A. Parent and H. E. Caravello, unpublished results), mutagenicity studies (Parent et al., 199 lb), and metabolism studies (Parent et al., 199 I a). The two-generation reproduction study reported here is part of that overall effort to fill the data gaps relating to acrolein. Although some work has been done to elucidate various aspects of the reproductive toxicity of acrolein (see above), to this point, nothing has been adequate. The purpose of this study was to provide information concerning the effects of acrolein on gonadal function, estrous cycles, mating behavior, conception, parturition, lactation, and weaning. The study was also designed to provide information about the effects of acrolein on neonatal morbidity and mortality. MATERIALS

AND METHODS

E.yperimental Design: Main Study F. generation. One hundred thirty Sprague-Dawley Crl:CD (SD)BR virgin male rats and 130 virgin female rats of the same strain were obtained from Charles River Breeding Laboratories, Inc. (Raleigh, NC), acclimated for approximately 2 weeks, and randomly (computer-generated random units; weight ordered) assigned to four groups of 30 males and 30 females. On the first day of dosing, all rats were 58 days of age and weighed 278 to 375 g for the males and 187 to 25 1 g for the females. All rats were intubated daily for 70 days with acrolein at dosing levels of 0 (water), 1, 3, or 6 mg/kg in a dosing volume of 5 ml/kg. Dosing levels were chosen based on a dose range-finding study involving dosing of six groups of 16 rats (8 males, 8 females) 7 days prior to cohabitation through either Day 25 of presumed gestation or postnatal Day (PND) 4. Physical signs, body weight, food consumption, pup viability, and various reproductive parameters were observed. Many mortalities were noted at the higher dosing levels (IO, 15, and 20 mg/kg/day), while clinical signs (excess salivation, decreased motor activity, labored breathing, bradypnea, tales, gasping, thin appearance) were noted at the 5 mg/kg/day dosing level. Gastric lesions were also noted at 5 mg/kg/day levels while both decreased body weight gains and feed consumption were noted at 2.5 mg/kaJday. Mating and fertility parameters as well as reproductive parameters were unaffected at 2.5 mg/ kg/day.

OF ACROLEIN

229

In the main study, rats within each dosing group were assigned to a 2 Iday cohabitation period (computer-generated randomized assignment). During this period, one male rat was paired with one female rat for a maximum of 14 days. Female rats that did not mate during this ICday period were assigned a different male rat (using a table of random units) from the same dosage group that had previously mated. These female rats remained in cohabitation for a maximum of 7 additional days. Female rats with spermatozoa observed in a smear of vaginal contents and/or copulatory plugs observed in situ2 were considered to be at Day 0 of presumed gestation and were assigned to individual housing. All F0 generation female rats were permitted to naturally deliver their F, generation litters. Intubation of the female rats continued daily through the cohabitation, gestation, and lactation (Day I of lactation = day of parturition) periods. Daily intubation continued for all F0 generation female rats until the day of or the day prior to scheduled termination on either Day 25 of presumed gestation (female rats that did not deliver offspring) or Day 2 1 of lactation (female rats that delivered offspring). From 96 to 130 daily doses of the test substance were given to each F0 generation female rat. Necropsy of the F0 generation female rats also occurred after the cohabitation period on either Day 25 of presumed gestation (female rats that did not deliver offspring) or Day 2 1 of lactation (female rats that delivered offspring: Day I of lactation = day parturition occurred). Male rats were intubated daily during cohabitation and until the day prior to scheduled termination (93 to 94 total doses). The F0 generation male rats were euthanized (carbon dioxide asphyxiation) and necropsied following completion of the cohabitation period. F, generation. F, generation pups were selected for continued study at weaning on Day 2 1 of lactation. A table of random units was used to identify 40 pups per sex from as many litters as possible in each dosage group. Whenever possible, a male and a female pup from each F, generation litter were retained. If a litter did not contain a male and/or a female pup, an equal number of male and female pups was obtained by randomly selecting pups from other litters in the same dosage group. The selected F, generation male and female rats were intubated with appropriate dosages of the test substance beginning on Day 22 postpartum and continuing for at least 72 days prior to the 2 1-day cohabitation period. A computer-generated randomization procedure was used to assign 30 rats per sex per dosage group to a cohabitation period. F, generation rats that were not selected for cohabitation were euthanized and necropsied. Pairing procedures and other procedures relative to gestation were identical to those employed for the FOgeneration. Daily intubation of the F, generation male rats was continued until the day prior to scheduled necropsy following completion of the 2 l-day cohabitation period (totals of 106 to 125 daily dosages). Daily intubation of the F, generation female rats was continued until the day of or the day prior to scheduled necropsy following the 2 l-day cohabitation period (totals of 104 to 149 daily dosages) on (1) Day 25 ofpresumed gestation (female rats that did not deliver pups). or (2) Day 21 of lactation (female rats that delivered pups). All F, generation female rats were permitted to naturally deliver their F2 generation litters. Fl generation. The pups resulting from the F, generation were not intentionally dosed with test material but may have been exposed in utero (during gestation) and during lactation (through maternal milk). These F2 pups were all terminated and necropsied on PND 2 1. Animal Housing Adult rats were housed (one or two rats per sex per cage), in hanging wirebottomed stainless-steel cages suspended above absorbent paper liners (Techboard; Shepard Specialty Paper Co., Kalamazoo, MI) except during ’ The placental sign (red substance observed in the vaginal smear) was used to identify pregnancy when mating was not identified during the initial 14 days of the 2 1-day cohabitation period. F,, generation female rats with placental sign observed were removed from cohabitation and assigned to individual housing.

230

PARENT.

CARAVELLO,

the cohabitation and lactation periods. Cages were changed approximately every other week, and cage pan liners were changed approximately three times a week. During the cohabitation period (a maximum of 2 1 days), male and female rats of corresponding dosage groups were housed together, one male per female in wire-bottomed cages. Beginning no later than Day 14 of presumed gestation. the female rats were individually housed in nesting boxes. Each dam and delivered litter were housed in a common nesting box during each 2 I -day postpartum period. Nesting boxes contained Bed-o’cobs bedding (Andersons Co., Delphi. IN). The bedding was changed approximately three times a week. Study rooms were maintained under positive pressure and independently supplied with a minimum of 10 air fresh changes per hour of 100% HEPA filtered air in AiroClean rooms (Airo Clean, Inc., West Chester, PA). Room temperatures were maintained at 74 f 4°F and relative humidity at 40 to 70%. A 12-hr light/dark cycle was maintained throughout the study period. Animals were provided with Certified Rodent Chow 5002 in meal form (Ralston-Purina Co., St. Louis, MO) which was available ad libitum throughout the study. Local water processed through a reverse osmosis membrane and treated with up to 1 ppm chlorine was available ad lib&m via an automatic watering system (when in wire cages) or individual sipper bottles (when in nesting boxes). Feed was analyzed for nutrient content and contaminants (heavy metals and pesticides) by the supplier. Water was analyzed by Purity-Standard Laboratories (Chalfont, PA) and Lancaster Laboratories, Inc. (Lancaster, PA) for bacterial content, hardness, pH, iron, chlorine. and pesticides. Both feed and water were found to be acceptable for use in the conduct of toxicological studies in accordance with accepted standards. Test Material

The acrolein used in this study was distilled and stabilized with 0.25% hydroquinone and was supplied in two lots having activities of 96.05 and 96.72% (Baker Performance Chemicals, Houston, TX). The test material also contained about 3% water and was stored refrigerated, blanketed by nitrogen, and protected from light. Dosing solutions were prepared daily at concentrations of 0 (vehicle), 0.2, 0.6, and I .2 mg/ml by dilution with reverse osmosis membrane-processed deionized water. All dosing was done within 3 hr of stock solution preparation. Each stock solution was analyzed prior to dosing for the first 4 days and approximately weekly thereafter. The stability of 0.2 and 1.2 mg/ml (the low- and high-dose solutions) was carried out at 6 hr postpreparation and indicated a 7% loss for the low-dose solution and a 4% loss for the highdose solution. Dosing solutions were generally within 1% of their intended concentrations. All analyses were carried out in triplicate using a Cary 118 twin beam spectrophotometer (Varian, Instrument Division, Sunnyvale, CA) setat nm.

F. and F, generation parental rats. All rats were observed for viability at least twice each day during the acclimation and study periods. The F,, generation rats were observed for clinical signs and general health four times during the acclimation period, and body weight was recorded at least once weekly. Body weights for the F0 and F, generation rats were recorded daily during the dosage period. The F0 and F, generation rats were observed for clinical signs, general health, and/or pharmacotoxic effects at least twice each day during the dosage period (once immediately prior to intubation and within approximately 1 hr postdosage). Feed consumption for the F0 and F, generation male rats was recorded weekly throughout the study, except during cohabitation when feed consumption was observed and/or recorded but not tabulated. Feed consumption for the F, and F, generation female rats was recorded weekly prior to cohabitation, on Days 0. 6, 12. 15, 20, and/or 25 of presumed gestation and on Days I, 4, 7, 10, 14. 16, 18, and 2 1 of lactation. Body weight data were not tabulated during cohabitation. Maternal behavior of the F0 and F, generation dams that naturally delivered litters was recorded on the days the pups were weighed during PND

AND HOBERMAN 2 1. Nursing behavior, care of pups, and other dam-pup interactions were observed daily and deviations from expected maternal behavior were noted. Estrous cycling (vaginal cytology) of the F, and F, generation female rats was evaluated during the cohabitation periods (a maximum of 21 days, 14 days with the first male rat and 7 days with the second male rat) until Day 0 of presumed gestation was identified (spermatozoa were observed in a smear of vaginal contents and/or a copulatory plug was observed in situ.’ Mating performance of F0 and F, generation female rats was evaluated daily during the cohabitation period and was confirmed by (1) observed delivery of offspring, (2) a confirmed date of mating, or (3) implantation sites present at necropsy. The F0 and F, generation female rats were evaluated for duration of gestation (Day 0 of presumed gestation to the day delivery was completed), litter size (live and dead pups, as well as live pups only), and pup viability. Pups that either appeared stillborn or died before initial examination of the litter for viability were examined for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Each litter was examined subsequently at least twice daily for pup viability. F0 and F, generation female rats that were not observed to have delivered offspring were euthanized by carbon dioxide asphyxiation on Day 25 of the presumed gestation period and examined for gross lesions and implantation sites.Uteri from female rats that appeared nonpregnant were pressed between two glass plates and transilluminated to confirm lack of implantation sites. F0 and F, generation dams that had delivered offspring were terminated by carbon dioxide asphyxiation on Day 2 1 of lactation. Complete necropsies were performed on any F, and F, generation rat that was found dead and on all F0 generation rats at scheduled necropsy. The F0 and F, generation female rats were also examined for the presence of implantation sites at necropsy to identify pregnancy in female rats that did not deliver a litter. F, and F2 generation litters. Vital status at birth was determined for pups that either appeared stillborn or died before initial examination of the litter, as previously described. Viability of each litter was evaluated at least twice each day during the 2 l-day postpartum observation period. Dead pups observed at these times were removed from the nesting box. When not precluded by autolysis and/ or cannibalization by the dam, dead pups were necropsied and examined for the possible cause of death. Tissues with gross lesions were saved in neutral-buffered 10% formalin. The numbers of live and dead pups present in each litter were recorded once each day. Physical signs (including gross external physical anomalies) were recorded for the pups once each day during the 21-day postpartum period. Pup body weights for the F, and F2 generation litters were recorded on Days I (day of parturition), 4,7, 14, and 2 1 postpartum. Nursing behavior and nesting activity (pups and dams) were observed and recorded on the days pup body weights were recorded. The F, and F2 generation litters were culled on Day 4 postpartum to five male and five female pups, whenever possible. If five pups of either sex were not present, then all pups of that sex were retained, and the litter was culled to a total of ten pups. In litters with ten or fewer pups, no culling occurred. Pups culled on Day 4 postpartum were euthanized by carbon dioxide asphyxiation and necropsied. The remaining F, generation pups were weaned on Day 2 1 postpartum. With the exception of F, generation pups selected as the next parental generation and pups culled on Day 4 postpartum, all F, and FZ generation pups were euthanized by carbon dioxide asphyxiation and necropsied on Day 21 postpartum. The head of each pup was cross sectioned (single cut at the suture of the frontal parietal bones) (Staples, 1974) and the brain was examined for hydrocephaly. The remainder of the carcasswas processed as indicated below. Tissues with observed gross lesions were preserved in neutral-buffered 10% formalin for possible histopathological evaluation.

REPRODUCTIVE

TOXICITY

The F, generation pups selected as the next parental generation were weaned on Day 21 postpartum. Daily intubation via gavage with the vehicle or appropriate dosage of the test substance was initiated on Day 22 postpartum. Histopathology Evaluation Gross examination of all rats at necropsy was performed. Tissues retained included testes, prostate, seminal vesicles. epididymides, ovaries, uterus, vagina, heart, lungs, kidneys, liver, stomach, spleen, thyroid (with parathyroids), adrenal glands, pituitary gland, brain, and spinal cord (thoracic-lumbar). Organs from all vehicle control and high-dosage group rats were microscopically examined. Organs with abnormalities or equivocal results were examined for the low- and middle-dosage group rats. Gross lesions from other tissues were also saved for histopathologic examination. Statistical Methods Parental. maternal, and pup incidence data’ were analyzed using the variance test for homogeneity of the binomial distribution (Snedecor and Cochran, 1967). Body weights and feed consumption data, organ weight data, and litter averages for pup body weight and percentage male pups were analyzed using Bartlett’s test of homogeneity of variances (Sokal and Rohif, 1969) and the analysis of variance (Snedecor and Cochran, 1967), when appropriate [i.e., Bartlett’s test was not significant (p > O.OS)].If the analysis of variance was significant (p < 0.05). Dunnett’s test (Dunnett, 1955) was used to identify the statistical significance ofthe individual groups. Ifthe analysis of variance was not appropriate [i.e., Bartlett’s test was significant (p Q 0.05)], the Kruskal-Wallis test (Sokal and Rohif, 1969) was used. If there were greater than 75% ties, then Fisher’s exact test was used (Siegel, 1956). In caseswhere the Kruskal-Wallis test was statistically significant (p < 0.05), Dunn’s method of multiple comparisons (Dunn, 1964) was used to identify the statistical significance of the individual groups. Count data obtained at natural delivery were evaluated by the KruskalWallis test (Sokal and Rohlf, 1969). In caseswhere statistical significance occurred (p d 0.05) Dunn’s method of multiple comparisons (Dunn, 1964) was used to identify the statistical significance of the individual groups.

231

OF ACROLEIN

dences of these signs were significant only for the high-dose animals in both generations. Other clinical signs which occurred in both generations in mid- and high-dose were sometimes noted to be significant at the high dose. These include ungroomed coat, chromorrhinorrhea, yellow to redbrown perioral substance, excess salivation, bradypnea, alopecia, reddish-brown urine, abdominal distension, emaciated appearance, soft or liquid feces, cold to the touch, paleness, head tilt, and no feces. Body Weights and Feed Consumption Relating to body weight gain, some significant effects were noted as shown in Figs. 1 through 6. High-dose F0 males demonstrated a significant decrement in weight gain initially and continued to show a decrement throughout the study (see Fig. 1). The mid-dose males also showed a trend toward lower body weight gain throughout, but this was not of statistical significance. Figures 2, 3, and 4 show weight gains for F0 females during premating, gestation, and lactation. There is clearly demonstrated a trend toward lower body weight gain at the high dose during all three periods and a slight indication of a decrement for mid-dose females during premating. For the F, generation, high-dose males show a body weight gain decrement throughout the study (see Fig. 5) but other dosing groups appear unaffected. Female premating body weights do show scattered significant effects at the high dosing levels (Fig. 6) but again other dosing levels appear unaffected. Although there appears to be a trend toward lower body weights for high-dose F, females during gestation and lactation, the data do not indicate significance; and the lactation data are scattered.

RESULTS

Mortality

650

,

300

’

A statistically significant (p G 0.01) increase in mortality was noted for high-dose (6 mg/kg/day) female rats of both the F0 (9/30 deaths) and F, (7/40) generations. High-dose males also showed increased mortality for both F0 (3/30) and F, (8/40) generations, the latter being statistically significant. The only other mortality in the F0 generation was a low-dose (1 mg/kg) male. Additional mortalities did occur at mid dose (3 mg/kg) in the I;, males (l/40) and females (3/40) and at low dose in the females (2/40). No control rats died during this study. Clinical Signs Clinical signs in both generations were noted at both midand high-dose levels and included rales, labored breathing, gasping, hyperpnea, and/or irregular breathing. The inci3 The individual rat was the unit of analysis for adult clinical sign data. The litter was the unit of analysis for gross litter observation data.

1

6

15

22

DAY

29

36

OF THE

43

50

57

64

70

STUDY

FIG. 1. Body weights of F0 generation male rats versus days on study. *Significantly different from the vehicle control group value (p < 0.05). **Significantly different from the vehicle control group value (p < 0.01).

PARENT,

232

CARAVELLO,

AND HOBERMAN

260 $ m 240 320

200

1 1

300 I 6

I 15

I 22

I 29

DAY

I 36

OF THE

13

I 50

I 57

I 64

1 1

,

,

,

4

7

&

/

LACf4ATION

,

,

16

16

DAY

STUDY

FIG. 2. Premating body weights of F0 generation female rats versus days on study.

FIG. 4. Lactation body weights (FOgeneration female rats) versus days of lactation.

Both absolute and relative feed consumption for F,, males tended to be reduced in a dose-dependent manner for the first 3 weeks of the study, but were increased for the remainder of the study. These increases were statistically significant for the high-dose males and were present, but not significant, for the mid-dose males. Similar findings were noted for FO females during premating but not during gestation or lactation. For the F, generation high-dose males, feed consumption was depressed for the first 3 weeks then increased beyond

controls for the remainder of the premating period, sometimes achieving statistical significance. The F, females showed some variability of feed consumption during premating, but no consistent trend was noted. No differences from controls were noted during gestation and lactation. Necropsv and Histopatho1og.v The animals that died during the study consistently exhibited effects on glandular and forestomach thought to be

0

.. I 1

/ I 8

I 16

I 22

I 29

DAY 0

6

16

DAY”OF

20

GEST&DN

FIG. 3. Gestational body weights (FO generation female rats) versus days of gestation.

A6

43

I 50

I 67

I 64

;1

I 76

65

POSTWEANING

FIG. 5. Body weights of F, generation male rats versus day of postweaning. *Significantly different from the vehicle control group value (p < 0.05). **Significantly different from the vehicle control group value (p < 0.01).

REPRODUCTIVE 400

300 3 % x

3 2 0

200

TOXICITY

for the high-dose F, males (80.0 versus 88.0% for controls) is not considered to be related to treatment since it is not statistically significant relative to the control group. There is no dose-response relationship, and the value is within the range for historical controls.4 Similarly, no effects on reproductive performance were noted for either FOand F, female rats. Fertility indices for FOfemale rats were also within the ranges observed for controls.5

!

Litter Data

.. 0

I 1

I 8

233

OF ACROLEIN

I 15

I 22

:9

DAY

I 36

I 43

I 50

I 57

I 64

I 71

I 78

85

POSTWEANING

FIG. 6. Premating body weights of F, generation female rats versus days of postweaning. **Significantly different from the vehicle control group value (p < 0.01).

related to the test material. Necropsy and histological evaluation revealed glandular and forestomach lesions at an incidence of l/3 in the F,, high-dose males that died, 819 in the females, 8/8 in the F, high-dose males, and 5/7 in the F, high-dose females. In addition, l/3 of the F, mid-dose females had stomach lesions. These lesions consisted of ulcer(s) and/or hyperplasia/hyperkeratosis of the mucosa of the forestomach, increased incidences of erosion(s), ulcer(s), and/or hemorrhage of the glandular mucosa and focal hyperplasia of the glandular mucosa. Increased amounts of submucosal inflammation and edema plus mononuclear-cell infiltrates occurred in areas of erosion and ulcer(s). The combined histopathological findings relating to the stomach are indicated below in Table 1. No treatment-related gross or microscopic changes were observed in the reproductive tissues of any of the F0 or F, generation male or female rats. Absolute and relative weights of testes and epididymides were likewise unaffected by treatment of both the F0 and F, generation males. Of the animals that died during the study, there were extensive respiratory findings at necropsy, including lung congestion, frothy material in trachea, and mottled lungs. There was at least one case of focal lung hemorrhage. Another frequent observation in these animals was abdominal distension, gas-filled stomachs and intestines. Reproductive Performance

Administration of doses of acrolein as high as 6 mg/kg/ day for 70 consecutive days did not affect the mating performance of either the F. or F1 generation male rats (see Table 2). Fertility indices (pregnant rats/rats mated) were similar across all groups. The slight reduction in the fertility index

Although one low-dose FO dam had a litter of 12 early resorptions and one high-dose FOdam was terminated moribund on Day 10 of the lactation period and delivered nine pups, none of which survived to PND 1, no other FO dam lost its litter during the 2 1-day postpartum period. There were no statistical differences among the groups for still births, pup deaths, or viability indices. Pup weights tended to be reduced in the FOhigh-dose group on PND 4 and were significantly reduced (p < 0.01) on PNDs 7, 14, and 21 (only PND 21 shown in Table 2). The significant reductions in F, high-dose female pup weights persisted during the postweaning period. Significant reductions (p < 0.0 1) in body weight gains occurred on Days 1 to 8 and 7 1 4 In 10 studies conducted at the test facility between 1988 and 1989, 2 17 (88.2%) F, generation control rats sired litters of 246 that mated (range, 75.0 to 96.3%). 5 In I1 studies conducted at the test facility between 1988 and 1989, 286 (90.8%) F0 generation control rats were pregnant of 3 15 that mated (range, 79.3 to 100%).

TABLE 1 Incidences of Stomach Lesions”

Congestion Glandular mucosa Edema/inflammation Submucosa Erosion(s) Glandularmucosa Focal hyperplasia Glandular mucosa Hyperplasia/ hyperkeratosis Forestomach Mononuclear infiltrations Submucosa Ulcers Forestomach Ulcers Glandular mucosa

F0 Males (n = 30)

F0 Females (n = 30)

F, Males (n = 40)

F, Females (n = 40)

0, 0, 0, 1

0, 0, 0. 0

0, 0. 2, 1

o,o, l,O

o,o, 0, 3

0, 0, 0, 1

o,o, 0, 7

l,O, I, 11

O,O,O,ll

l,O, 1, 10

0,0,0,17

2, 1, I,15

0, 0.0, 4

0, 0. 0, 5

0, 0, 0, 7

0. 0, 1. 1

0,0,0,24

0,0,4, 28

0, 0, 0, 40

O,O, 2, 33

0. 0, 0. 4

0, 0, 0. 2

0. 0, 0, 6

0, 0. 0, 0

0, 0, 0, 3

(lo, 0, 0

0, 0, 0, 3

0. 0, 0, 1

0, 0. 0. 4

0. 0, 0, 0

0, to,

0, 0, 0. 0

1

n Incidences are presented in order of control, low, mid, and high dose.

234

PARENT,

CARAVELLO,

AND HOBERMAN

TABLE 2 Mating, Fertility, and Pup Data

Rats tested Female fertility index” (%) Male fertility index” (%) Gestation indexb (W) Implantation sites per dam (mean + SD) Pups delivered livebom/litter (mean + SD) Viability index’(%) Lactation index’ (%) Male pups/total live pups (W k SD)’ Pup weight/litter in grams (mean k SD) PND 1 Pup weight/litter in grams (mean -t SD) PND21

FO F, FO F, Fo F, Fo F, Fo F, Fo F, Fo F, Fo F, Fo F, Fo F, Fo F,

Control (vehicle)

Low dose

Mid dose

High dose

30 30 26/29 (89.6) 23127 (85.2) 24/27 (88.9) 22/25 (88.0) 26/26 (100) 22123 (95.6) 14.9 +I 2.9 15.7 t 3.3 13.2 k 3.0 14.4 L 3.5 340/343 (99.1) 302/3 18 (95.0) 250/250 (100) 210/210 (100) 48.1 ?I 14.2 55.1 f 14.4 6.6 + 0.4 6.3 + 0.6 51.2 +_ 4.4 46.8 k 5.9

30 30 26130 (87.7) 26/30 (86.7) 22126 (84.6) 24/28 (85.7) 25126 (96.2) 26/26 (100) 16.5 + 2.0 15.5 + 4.7 15.0 k 2.0 14.5 * 4.1 370/375 (98.7) 3641316 (96.8) 2481249 (99.6) 2451246 (99.6) 49.6 + 12.6 48.9 f 14.3 6.5 + 0.4 6.4 zk 0.5 51.4 Xk 3.0 46.7 + 4.1

30 30 29130 (96.7) 26129 (89.6) 28129 (96.9) 23127 (85.2) 29/29 (100) 25126 (96.2) 15.4 I 1.7 16.3 f 3.0 14.0 + 1.9 14.2 + 3.6 401/407 (98.5) 324/356 (9 1.O)d 287/288 (99.6) 2291230 (99.6) 51.4 IL 13.7 51.4 + 14.4 6.6 k 0.5 6.5 IL 0.7 50.5 * 3.7 46.9 + 4.3

23 29 21/23 (91.3) 23/28 (82.1) 20/22 (90.9) 20/25 (80.0) 21/21 (100) 20/23 (87.0) 15.1 t 2.0 15.4 f 2.3 13.9 k 2.1 13.5 t 2.8 27 l/278 (97.5) 2641270 (97.8) 194/194 (100) 194/194 (100) 54.3 * 14.5 46.7 -+ 10 6.5 i 0.4 6.4 f 0.5 47.0 f 4.7” 45.0 f 4.5

’ Pregnant rats/rats mated. b Live litters delivered/number of pregnant rats. ’ Number of pups alive on PND 4 (preculling)/number of liveborn pups on PND 1 d Significantly different from the vehicle control group value (p G 0.05). e Number of pups alive on PND 2 l/number alive on PND 4 (postculling). /Number of pups born alive but found dead on PND 1. g Significantly different from the vehicle control group value (p d 0.01).

to 78 postweaning and body weight gains frequently tended to be reduced for this dosage group at other intervals during this period (see Fig. 6). F, male pups treated with the high dose of acrolein showed a significant (p < 0.0 1) weight decrement from Days 1 to 85 postweaning (see Fig. 5). Viability, growth, and morphology of pups were otherwise unaffected by treatment during the 2 1-day postpartum period. Implant sites, duration of gestation, and parturition, litter sizes, numbers of dams with stillborn pups, number of dams with all pups dying, viability, and lactation indices were similarly unaffected by treatment of FO females with acrolein. The percentage of males observed in the F, highdose animals appears lower than controls (46.7 versus 55.1%). No trend is obvious, and the observation is not considered significant. Dosages of acrolein as high as 6 mg/kg/day did not affect the natural delivery or pup litter observations for the F1 generation dams and their Fz generation offspring. Differences that occurred were not dosage dependent. Significant increases (p G 0.0 1) in F2 pups dying on PND 1 and significant decreases (p i 0.05) in the viability index (see Table 2) occurred for the 3 mg/kg/day (mid) dosage

group. These values were considered unrelated to the test substance because the values were not dosage dependent. Dosages of acrolein as high as 6 mg/kg/day did not affect the average number of implantation sites per dam, duration of gestation and parturition, mean litter size, pup body weight, or sex ratio (percentage male pups per total pups) with few exceptions noted above. There were no statistically significant differences in the number of dams with stillborn pups, the number of dams with all pups dying, or the lactation indices. There were no statistically significant differences among the groups in the number of F, litters with gross abnormalities for the pups (F2) during lactation or gross lesions identified in the pups at necropsy. DISCUSSION Animals dosed at 6 mg/kg/day, both males and females, suffered statistically significant increased mortality in both the F. and FI generations. The pattern of mortality continued into the F, mid-dose animals and low-dose females. Most of these animals showed signs of respiratory distress (rales,

REPRODUCTIVE

TOXICITY

labored breathing, gasping, hyperpnea, and irregular breathing) and histopathologic findings that included congested lungs, gastric distension, and stomach lesions. Although the lung findings raises questions about possible aspiration of test material, stomach lesions are noted regularly in these animals as well as those that have gone on to complete the study (see Table 1). Clearly, there is an association between stomach lesions and administration of acrolein. This is particularly true for diagnoses of erosion(s) of glandular mucosa and hyperplasia/hyperkeratosis of forestomach and is found in both sexes of F0 and F, animals. The effects continue into the mid-dose females. Although dosing of dogs with acrolein-containing capsules resulted in gastrointestinal effects (mainly vomiting), no stomach lesions were observed up to levels of 2 mg/kg/day (Parent and Caravello, 1992~). Similarly, a 2-year rat study dosed at up to 2.5 mg/kg/day (Parent et al., 1992b) and an 18-month mouse study dosed up to 4.5 mg/kg/day (Parent et al., 1992a) all failed to produce stomach lesions. A significant difference between this study and those cited is the concentration of the dosing solution. In this study, the dosing volume used was 5 ml/kg, whereas those of the other studies were dosed at 10 ml/kg. In the rat and mouse studies cited, early deaths were also noted and similar findings were frequently made relating to respiratory system involvement (Parent et al., 1992a,b). For this study, it is not clear whether these deaths were due to a frank toxicity effect during an adaptive period or to some mechanical effect relating to dosing. Body weight gain was affected in this study (see Figs. 1 to 6) in a dose-dependent manner, and again the mid-dose animals were sporadically involved. Pathological evaluation was confined, for the most part, to reproductive organs with the exception of stomach and lungs. These were identified as potential targets from prior studies (Parent et al., 1992a,b,c). No effects of acrolein or any reproductive organs were noted, nor were any effects found on reproductive performance or pup viability. The no-observable-adverse-effect-level (NOAEL) of acrolein for the parental (FO and F,) generations is 1 mg/kg/ day based on the stomach lesions noted and the decreased body weight gains. The NOAEL for male reproductive performance is greater than 6 mg/kg/day (no effects were produced by the highest dosage tested). The NOAEL for female reproductive performance is 3 mg/kg/day, if the reduced pup body weights that occurred for the F. generation offspring (F, generation pups) during the lactation period are considered an effect on female reproductive performance. The NOAEL for the offspring is 3 mg/kg/day, because the gastric lesions that occurred for the 3 and 6 mg/kg/day dosage groups were considered a measure of toxicity in the adults. (The pup body weights of the 6 mg/kg/day dosage group F, generation litters were reduced during the lactation period; this effect did not recur for the second generation offspring.)

OF

ACROLEIN

235

Dosage-dependent increases in clinical observations and reduced body weight gains were caused by the 3 and 6 mg/ kg/day dosages of acrolein. Feed consumption was also reduced by the 6 mg/kg/day dosage of the test substance. In both generations, the 3 mg/kg/day dosage caused a low incidence of gastric lesions in the female rats; the 6 mg/kg/day dosage of the test substance caused gastric lesions in male and female rats in both generations. The 3 mg/kg/day dosage of acrolein caused a small increase in mortality of male and female rats in the second generation, and the 6 mg/kg/day dosage significantly increased (p G 0.01) mortality of male and/or female rats in both generations. Dosages of acrolein as high as 6 mg/kg/day did not affect mating or fertility of the F. or F, generation male or female rats or the viability and morphology of their offspring. The 6 mg/kg/day dosage of acrolein reduced body weights of the first generation pups throughout the lactation period. Thus, interrelated effects on female reproductive performance and development of the offspring (reduced F, generation pup body weights during the F. generation lactation period) occurred only at the higher of two dosages that were toxic to the parental generations. Therefore, acrolein should not be identified as a selective reproductive toxin. These results are consistent with other studies carried out previously (Parent et al., 1992a,b; R. A. Parent and H. E. Caravello, unpublished results) in that few systemic effects are observed. Weight gain decrements in this study are thought to be due to stomach lesions, resulting in decreased food consumption. Pulmonary effects noted are also thought to be local effects in that they may have resulted from aspiration of small amounts of test material. Similar findings were noted for the rat and mouse studies above cited. Acrolein is an extremely reactive chemical which will bind to readily available amine and sulthydryl groups on protein (Draminski et al., 1983; Esterbauer et al.. 1976; Wilmer et al., 1988; Hemmininki et al., 1980). Direct contact with target cells will produce positive mutagenic responses under certain conditions (Marnett et al.. 1985; Lutz et al., 1982; Curren et al., 1988). Indeed, protocols involving direct contact with rat embryo cells in culture (Schmid et al., 1981; Mirkes et al.. 1981: Korhonen et al., 1983; Slott and Hales, 1986, 1987) under some conditions will produce embryo lethality. Direct injection of acrolein into the yolk sack of rabbit embryos (Claussen et al., 1980) or intraamniotic administration to rats (Hales, 1983; Slott and Hales, 1985) has been reported to produce embryo lethality. Intravenous administration of acrolein in rabbits also results in dose-dependent embryo lethality (Claussen et al., 1980). When administered to both male and female Swiss mice by intraperitoneal injection in an assay designed to measure a dominant-lethal mutagenic effect, acrolein failed to produce a positive response (Epstein et al., 1972). In another study involving inhalation exposure up to as much as 0.55 ppm of acrolein for 26 days including a period of cohabitation,

236

PARENT,

CARAVELLO,

no effects were noted for number of animals pregnant, number of live fetuses, and mean weight of fetus (Bouley et al., 1975). There have been suggestions that acrolein may be a toxic metabolite of cyclophosphamide and may be responsible for the teratogenic effects noted for this drug (Schmid et al.. 198 1; Mirkes et al., 198 1; Slott and Hales, 1987; Mirkes et al., 1984; Claussen et al., 1980; Hales, 1983). The possible in viva generation of acrolein could place the agent near target tissues that could very well be affected, but dosing as in this study allows ample opportunity for acrolein to react with numerous proteins in feed and stomach and also to decompose within the gastrointestinal tract. Under the conditions of the experiments reported herein, acrolein cannot be considered as a reproductive toxin but does demonstrate other toxic effects down to dosing levels of 3 mg/kg/day. ACKNOWLEDGMENTS We acknowledge Ronald J. McCarty, BS, Diane Sembello, BS, and Christopher Turner, BS, for technical assistance; W. Ray Brown, DVM, Ph.D. for his pathological evaluation of this study: Baker Performance Chemicals for allowing the publication of this study: and Michael Siener for preparing the manuscript for publication.

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