Document bO45d4KJBdEB5zKxgodJOmp5D

m 'Toy. (\fe 1977 PAPER SYNTHETICS CONFERENCE September 26-28 The Drake Chicago, Illinois TECHNICAL ASSOCIATION OF THE PULP AND PAPER INDUSTRY One Dunwoody Pork, Atlanta, Ga. 30338 U.S.A. Telephone (404)394-6130 TWX 810--757-014S "''Copyright 1977 by TAPPI SL 040753 TOXICOLOGICAL AND PHARMACOKINETIC STUDIES ON INHALED AND INGESTED VINYLIDENE CHLORIDE IN LABORATORY ANIMALS J. M, Norris Associate Scientist The Dow Chemical Company P. 0. Box 1706 Midland, Michigan 48640 ABSTRACT The toxicity of vinylidene chloride (VDC) was evaluated in 90-day and 2-year studies incor porating VDC in the drinking water of rats or by inhalation of the vapors. The levels of VDC in the drinking water studies were 0, 60, 100 or 200 ppm (mg/O and in the inhalation studies 0, 25 or 75 ppm. The toxicity of inhaled and ingested VDC in rats exposed for 90 days was limited to minimal reversible liver changes at 25 and 75 ppm by inhalation and at 200 ppm only in the drinking water study. VDC was nontumorigenic in rats exposed for 2 years by either route. Pharmacokinetic studies on inhaled and ingested VDC indicated VDC is metabolized in the rat to a reactive metabolite(s) which is normally detoxified by conjugation with gluta thione. At relatively high exposure levels of VDC, as liver glutathione becomes depleted, toxicity is elicited. Preliminary data on the metabolism of VDC in mice indicate increased production of the reactive metabolite over that in the rat rendering the mouse more suscepti ble to the toxic effect of VDC. Daily administration of VDC in peanut oil to dogs for 90 days caused no adverse effects at dosages .of 0, 6.25, 12.5 or 25 mg/kg. The reproductivity of rats on a three generation study, incorporating 0, 60, 100 or 200 ppm VDC in the drinking water, was not adversely affected. VDC was not teratogenic in rats or rabbits exposed on gestation days 6-15 or 6-18, respectively, to vapor concentrations up to 160 ppm for 7 hours/day or in rats on drinking water containing 200 ppm. A cytogenetic study in rats inhaling 0, 25 or 75 ppm VDC for 6 hours/day, 5 days/week for 6 months revealed no chromosomal aberrations. A multi-study program to investigate the toxi city of vinylidene chloride (VDC) in laboratory animals has been in progress since 1974 in the Toxicology Research Laboratory of The Dow Chemical Company. The program is being con ducted under the auspices of the Manufacturing Chemists Association and sponsored by fif teen companies*.* i American Cijanamid Company, Aaahc-Pow Ltd,, EASF-Wyaudo t te Corporation, 8. F. Goodrich Che.mic.al Company, The Dow Chemical Company, E.l. duPont de hemoun Company, W. R. Grace i Company, Karelia Chemical Induttry Company, Monsanto Company, Morton Chemical Company, Otin Corporation, PPG Induitriei, Rohm and Haai Company, Staley Chemical, and Tennei&ee Eastman Company. The various studies in this program are: 1) 90-Day Toxicoloqical Study Incorporating VDC in the Drinking Water of Rats 2) 2-Year Toxicological Study in Conjunction With a 3-Generation Reproduction Study Incorporating VDC in the Drinking Water of Rats 3) 90-Day Toxicological Study on VDC in Pea nut Oil Administered to Dogs 4) 90-Day VDC Vapor Inhalation Study in Rats 5) 2-Year VDC Vapor Inhalation Study in Rats 6) Teratology Studies on Ingested or Inhaled VDC in Rats and Rabbits 7) Pharmacokinetic Study on Ingested or In haled VDC in Rats 8) Cytogenetic Study on Bone Marrow Cells of Rats The 90-day and 2-year toxicity studies, in gestion and inhalation, were supplemented by pharmacokinetic: studies whereby the rate of vinylidene chloride in the body of the animal was determined following exposure by both routes at high and low dose levels. The cytogenetic study was conducted in conjunction with* the long-term inhalation study. The 3 generation reproduction study was con- 45 ducted to evaluate the possible effects of VDC on the reproductivity pf the animals. The teratology studies were conducted to evaluate the possible effects on the unborn by -exposing pregnant animals, by ingestion or by ".inhalation, to vinylidene chloride. The "vinylidene chloride used in the various toxi city studies was sales grade material obtain ed from The Dow Chemical Company. An epidemiology study which was not a part of the MCA program has been reported1 by The Dow Chemical Company on 138 workers exposed to work'atmospheres containing VDC starting in 1944, There were no findings statistically related or individually attributable to VDC exposure in this cohort. The average TWA, depending upon the job performed by the workers ranged from <5 to 70 ppm. The design of the 90-day and 2-year studies in corporating VDC in the drinking water of rats is given in Table 1. Groups of male and fe male Sprague-Dawley rats, 6-7 weeks of age at the start of the study, were supplied drinking water containing 0, 60, 100 or 200 ppm (mg/), nominal concentrations, of VDC. The various parameters monitored during the studies included nonprotein sulfhydryl con tent of the liver and kidneys of the animals on the 90-day study. The purpose of these analyses ,was to help elucidate the biochemical effects and possible mechanism of toxic action of vinylidene chloride. Parameters also in cluded were clinical observations for changes in appearance and demeanor, body weight, food 'Consumption, water consumption, periodic amatology determinations, clinical chemis tries (liver and kidney enzyme studies) and urinalyses. The weight of the major organs was measured and complete gross and microscopic examinations of organs and tissues were made on those animals dying during the study, those sacrificed in moribund condition or after 90 days or those surviving until the termination of the 2-year study. The highest concentration (200 ppm) of VDC in corporated in the drinking water in these studies was determined by the limit of solu bility of VDC in water. The daily mg7kg body weight dosage to the animals on the 90-day and 2-year studies are given in Table 2. All of the animals on the 90-day study sur vived. There was no evidence of a toxicolo gical effect associated with the ingestion of VDC as monitored by the various parameters. Microscopic pathological examination showed that minimal liver changes had occurred at the 200 ppm concentration only. The microscopic findings at the two lower levels were compara ble to those in the control animals. corporation of VDC in the drinking water of for 2 years did not adversely affect body weight, food consumption or water consumption. There was a nondose related decrease in sur vival of male rats at 18 and 24 months. Gross Pathological examination of those VDC-treated .'-imals that died during the course of the -i-udy, those that were sacrificed in moribund condition or those surviving to the termina tion of the 2-year study showed no compoundrelated changes. Microscooir pathological examination of the tissues and organs from these animals revealed no compound-related tumorigenic changes. The only significant nontumorigenic change seen in the VDC-treated animals was a statistically significant greater incidence of fatty liver changes and hepatocellular hypertrophy (portal area) at all dose levels in the females and at the high dose level in the males, and central lobular atrophy at the high dose level in the females only. The 90-day and 2-year VDC vapor inhalation studies in rats were initiated at the same time. Twenty rats/sex/dose level were used for the 90-day Study and 100 rats/sex/dose level were used for the 2-year study. The exposures were 6 hours/day, 5 days/week. The design of the studies is given in Table 3. Initially the exposure levels used for these studies were 0, 10 and 40 ppm with the pro vision that if no adverse effects were seen among a group of animals sacrificed after 30 days, the levels would be increased to 25 and 75 ppm, respectively. Interim kills were conducted after 6 months and 12 months for the evaluation of possible pathologic compoundrelated changes. The parameters monitored were the same as in the drinking water study. A cytogenetic study to evaluate the possible mutagenic effect of vinylidene chloride was conducted on a group of animals exposed for 6 months. No compound-related adverse effects from ex posure to vinylidene chloride vapors were seen among the rats exposed to 10 or 40 ppm for 30 days. The only significant finding seen in the rats exposed for 30 or 90 days to 25 or 75 ppm of vinylidene chloride was minimal microscopic changes in the liver (characterized by a minimal increase in the degree of vacuoLation in the cytoplasm of the hepatocytes). These minimal changes have been interpreted as being reversible. Sub sequent interim kills at 6 and 12 months in the 2-year study continued to show minimal microscopic changes in the liver. The in cidence of occurrence of these changes was greater in the-animals exposed to 75 ppm vinylidene chloride after 90 days on the study. An increased incidence was no.t seen in the animals exposed to 25 ppm until the 12-month interim kill. No treatment-related adverse effects on de meanor or appearance, hematologic parameters or clinical chemistry values were observed in the animals on the 2-year study. There was a decrease in body weight gain in male rats only exposed to both concentrations during months 8-12 or 13 of the study. There was a slight increase in mortality in the female rats at both dose levels after 12 months on the study and in the males in the last months of the study. Gross pathological examination of animals dying during the study, thuse--__ sacririced in moribund condition a'- inter im kills or those survivi^^ unt-il the ter mination of the study showed no compound-re lated changes in the liver, urinary system, respiratory system or lymphorcticular sys tem. A final statement on the pathological SL 040755 examination must await the completion of the microscopic examination of the tissues and organs from these animals. Results of the cytogenetic study in rats ex posed for 6 hrs/day, 5 days/week for 6 months to 0 (control), 25 or 75 ppm VDC revealed no chromosomal aberrations in control or VDCoxposoJ animals. The study in which VDC was administered daily to beaqle dogs for 90 days showed no compoundrelated effect at dose levels of 6.25, 12.5, or 25 mg/kg as judged by clinical observations of demeanor and appearance, body weights, food consumption, hematologic or clinical chemistry determinations, organ weights and gross and microscopic pathological examinations of tissues and organs. The three generation reproduction study was started in groups of rats on the 2-year study after they had been exposed to the various concentrations of VDC in the drinking water for 90 days (refer to Table 1).' Each group of VDC exposed animals used in the reproduc tion study consisted of 10 males and 20 fe males, with 15 males and 39 females used for controls. The females of the first mating (F. dams) were remated because of decreased fertility in all groups including the controls. The pups in the F.,, litter were mated to pro duce the 2nd generation. The pups from the F, litter were mated to produce the F,,r ,, and litters. The parameters monitored In thiscstudy were the litter size and sex ratio at birth and weaning, the growth and survival of the neonate, toxicity prior to mating, fertility, length of gestation, delivery of live litters and gross and microscopic path ological examination of tissues and organs from dams and pups. No clear cut effect due to VDC in the drinking water was observed in this reproduction study. There was a slight, but noncompound-related effect, on pup sur vival in the F- litters and a marked effect at all dose levels in the F.. litters but not in the F1Af F1b, F3b or F3C titters. The teratology study on ingested VDC in rats was conducted at the highest possible con centration of VDC in the drinking water, 200 ppm. The teratology study on inhaled VDC was conducted in rats and rabbits at vapor concentration of 0, 20, 80 and 160 ppm for 7 hours/day. The rats in both studies were exposed to VDC on days 6-15 of gestation and the rabbits on days 6-18 of gestation. Vinylidene chloride was not teratogenic in rats or rabbits inhaling up to 160 ppm of the compound for 7 hrs/day or in rats given water containing 200 ppm of the compound. Some evidence of embryotoxicity or fetotoxicity was observed in both rats and rabbits exposed to vinylidene chloride by inhalation; these effects were associated with maternally toxic levels of exposure. At concentrations of vinylidene chloride which had little or no effect on the dam, no adverse effect on embryonal or fetal development was discerned in either species. Pharmacokinetic studies were conducted as a part of this multi-study program to ascertain the fate of vinylidene chloride in the body of the rat and to give perspective to the toxicological findings. Using radioactive VDC, the rates at which VDC was absorbed, distributed and ultimately cleared from the body of the rat via metabolism and/or excre tion were determined for high and low dose levels. Thus the pharmacokinetic profile was obtained for a single oral dosage of l or 50 mg of radioactive VDC/kg body weight of the rats and after a single 6-hour inhalation exposure to 10 ppm or 200 ppm of the radio active material. The elimination of the radio activity was followed for 72 hours in both fed and fasted rats. This involved analysis for radioactivity of expired air, urine and feces. Subsequently the distribution or residual radioactivity in tissues and car cass was determined and an attempt was made to identify urinary metabolites. These Studies show that the fate of VDC following oral administration or inhalation exposure of rats is dependent upon the dose adminis tered and the nutritional state of the animals. As the dose level is increased from 1 to 50 mg/kg oral or from 10 to 200 ppm in halation, the metabolic pathway becomes satu rated so that, percentage-wise, less of the dose administered is metabolized and more is eliminated via the lungs as VDC. At the 1 mg/kg oral dose and the 10 ppm inhalation dose there was no difference in elimination by fed vs. fasted rats. However, at relatively high levels of 50 mg/kg oral or 200 ppm inhalation there was a significant increase in the ex cretion of VDC via the lungs and decrease in the urinary excretion of radioactivity in fed vs. fasted rats. The studies indicated that VDC is biotransformed to a reactive metabo lite (s) in the liver. At low levels, 1 mg/ kg or 10 ppm, the reactive metabolite(s) is detoxified by conjugation with glutathione in the liver. However, when the liver glutathione levels are lowered or depleted i.e., in fasted animals or at high doses of VDC, 50 mg/kg or 200 ppm, VDC or the reac tive metabolite (s) are free to react with tissue macromolecules and a toxic response is elicited. Two of the four major urinary metabolites that have been identified give evidence of the conjugation with glutathione. These are thiodiglycolic acid and N-ecetvlS-(2-hydroxyethyl)cysteine------ -- A pharmacokinetic study of inhaled VDC in Swiss mice was started in Dow's Toxicology Research Laboratory after recent reports by Maltoni2 and Lee3 of tumorigenic findi/.gs in mice exposed to VDC. Maltoni qualified the finding of kidney tumors in Swiss mice ex posed to 25 ppm, but not 10 ppm, indicating that (1) 25 ppm was near the acutely lethal dose (50 ppm) for Swiss mice; (2) 25 ppm caused toxic changes in the kidney early in the study, (3) Swiss mice have a high in cidence of kidney disease which could hjve enhanced the susceptibility to the tumorigenic changes, and, most significant, (!) the Swiss mice were far more susceptible to VDCinduced toxicity and/or lethality than the rat or man. SL 040756 Lee repotted that CD-I mice exposed for one year to 55 ppm VDC, a lethal level for the Swiss mouse, showed hepatomas, angiosarcoma of the liver and pulmonary adenomas. Prelimi nary pharmacokinetic data the Dow study suggest that production of reactive meta bolite (s) is greater in the mouse than in the rat, thus rendering the mouse more sus ceptible to the toxic effects of VDC. Com prehensive interpretation of the toxic and tumor-igenic effects seen in the mice must await the completion of this study. However, it is sufficiently evident that the use of mouse data for human hazard evaluation of exposure to VDC is inappropriate. To summarize - ingestion of drinking water containing VDC by rats produced minimal liver changes interpreted as being reversible in nature in a 90-day study at the highest con centration administered, 200 ppm, which was equivalent to daily dosage of 19-26 mg/kg body weight. The results of the pharmacokinetic study in rats, indicating saturation of meta bolic pathways at relatively high levels, tie in with the minimal toxicological findings at the highest level in the 90-day ingestion study. There was no grossly observable evi dence of progression of this effect-on the liver or an effect on any other organ in the animals in the 2-year ingestion study. Dose related toxic changes were seen on microscopic pathological examination of liver tissue at all dose levels in the females and only at the top dose level in the males. Ingestion and/or inhalation of VDC by rats on teratology, cyto genetic or reproduction studies showed the compound to be neither a teratogen, mutagen or one adversely affecting reproductivity. Dogs administered daily doses as high as 25 mg/kg body weight for 90 days showed no compound-related adverse effects. Inhalation of 25 or 75 ppm VDC by rats for 90 days produced the same kind of minimal liver changes seen at the highest d'ose level in the ingestion study. The results of the inhalation pharmacokinetic study, indicating diminished ability to metabolize VDC at a concentration in excess of 10 ppm, also tie in with the minimal toxic effects seen at 25 or 75 ppm. There was no grossly observable evidence of progression of the effect on the liver or an effect on any other organ in the animals on the 2-year inhalation study. 1. Ott, M. G., Fishbeck, W. A., Townsend, J. C. and Schneider, E. J., J. Occup. Med. 18(11) -.735-738 (November, 1976) ." 2. Maltoni. Cesare, "Carcinogenicity Bio assays ef Vinylidene Chloride" paper presented at TAFFI International FVDC Seminar, Hamburg, Germany; January 24-26, 1977 .3 3. Lee, Cheng-Chun, "Evaluation of the Envi ronmental Toxicants Vinyl Chloride (VC) and Vinylidene Chloride (VDC), Progress Report No. 10, MRI Project No. 3612-B for National Institute of Environmental Health Sciences (1977). SL 040757 48 66/sex/dose level 98/sex/controls 10 Days on Study 30 Hem. Chem. Urin. 48/sex/dose level 80/sex/controls V90 ISO 360 540 730 \ N Hem. N /k Chem. AK Urin. NA t - t. NA Reproduction Study j---------------------------- -- ------------------ / lA As / 1 i Males_________iFemales ^ 1st Generation 2nd-------->3rd CODE: N NA HEM Chera Urin Necropsy (study terminated) Necropsy for nonprotein bound SH determination Hematology determinations Clinical Chemistry determinations Urinalysis Generations TABLE 1 Design of 90-Day and 2-Year Toxicology Studies and Three Generation Reproduction Study on Vinylidene Chloride Incorporated into the Drinking Water of Rats 49 SL 040758 Concentration in water, ppm 0 _ (controls) 200-230 {1) 100-120 60-70 Calculated Equivalent Dose (mq/kg/dny) 90-Pay Study 2-Year Study 10-13 19-26 0 16-40 (3) 8-20 5-12 ^Maximum concentration based on water solubility. Range based on body weight of 305-487 grams for male rats and 213-313 grams female rats. ^Range based on body weight of 200-500 grams for male rats on 2-year study. Female dose range falls within range given. TABLE 2 90-Day and 2-Year Studies Incorporating Vinylidene Chloride in the Drinking Water of Rats Monitored Parameter Clinical Observation Body Weights Hematology Urinalyses Clinical Chemistries Organ Weights Cytogenetics Nonprotein Bound Sulfhydryl Gross Pathologic Exam Microscopic Pathologic Exam 90-Day Study 30 9Q xx xX X X XX X ,. X X X X X Days on Study 2-Ycar Study 30 180 365 X X X X XX XX X XX XX X X TABLE 3 Design of the 90-Day and 2-Year Vapor Inhalation Studies on Vinylidene Chloride in Rats 735 X X X X X X X X 50 SL 040759