Recent progress in safety evaluation studies on plasticizers and plastics and their controlled use in Japan.

Recent experimental studies in Japan on the evaluation of potential health hazards from phthalate esters used in manufacturing poly (vinyl chloride) as well as several plastics for medical devices and for food containers and packages were introduced. Development of pulmonary granuloma formation after intravenous injection of diethylhexyl phthalate was assumed to be dependent on the particle size of the phthalate in vehicle used. Dietary administration of large amount of diethylhexyl phthalate and dibutyl phthalate produced renal cysts in mothers and in descendants in reproduction studies in mice. Cytotoxicity and mutagenicity of the phthalates and several plastics and resins were also examined by in vivo and in vitro studies. Hematological parameters examined in rabbits after repeated intravenous injection of diethylhexyl phthalate and after implantation of plastics in aorta for 3--6 months did not show any significant change. A slow decrease of radioactivity was observed in adipose tissue of rats following oral administration of 14C-labeled diethylhexyl phthalate. tthe administrative action on phthalates by the Japanese Ministry of Health and Welfare is briefly reviewed.

Recent experimental studies in Japan on the evaluation of potential health hazards from phthalate esters used in manufacturing poly(vinyl chloride) as well as several plastics for medical devices and for food containers and packages were introduced. Development of pulmonary granuloma formation after intravenous iniection of diethylhexyl phthalate was assumed to be dependent on the particle size of the phthalate in vehicle used. Dietary administration of large amount of diethylhexyl phthalate and dibutyl phthalate produced renal cysts in mothers and in descendants in reproduction studies in mice. Cytotoxicity and mutagenicity of the phthalates and several plastics and resins were also examined by in vivo and in vitro studies. Hematological parameters examined in rabbits after repeated intravenous injection of diethylhexyl phthalate and after implantation of plastics in aorta for 3-6 months did not show any significant change. A slow decrease of radioactivity was observed in adipose tissue of rats following oral administration of 14C-labeled diethylhexyl phthalate. The administrative action on phthalates by the Japanese Ministry of Health and Welfare is briefly reviewed.
Plastics have found wide usage in medical and paramedical applications as well as for food containers and wrapping materials. In the past much attention was directed at the physical, chemical, and mechanical properties of these materials in meeting the requirements for developing suitable products. However, more recently, attention has been focused on the potential toxic liability of these products when they are used for medical purposes or as contaminants of the food or materials which may come into contact with the human body.
Since Jaeger and Rubin (1) pointed out that diethylhexyl phthalate (DEHP) accumulated in rat liver after perfusion and in the human spleen and abdominal adipose tissue after blood transfusion, evaluation of the safety of phthalates used as additives in manufacturing polyvinyl chloride products became a matter of * Department of Pharmacology, National Institute of Hygienic Sciences, Kami-Yoga, Setagaya, Tokyo, Japan 158. concern and interest in Japan. The Ministry of Health and Welfare and many institutes began to examine the effects of plasticizers on blood, their accumulation in tissues, and their other toxicological effects in experimental animals. Of the many plasticizers, DEPH has been mainly used for these studies. Acute Toxicity Studies Miyahara et al. (2) administered DEHP and dibutyl phthalate (DBP) to male adult mice orally and by intravenous injection. The mice treated orally tolerated 100 g/kg of DEHP, and the LD50 of DBP was 9.77 g/kg on day 7 of observation; for those treated intravenously the LD50 values were 1.37 g/kg and 0.72 g/kg, respectively. Yamada (3) administered DEHP orally to male and female mice at doses of 22.7, 69.4 ml/kg, and the LD50 after 7 days was 49.7 ml/kg; toxic symptoms including decrease of spontaneous move-  Yamada (6) ments, decrease of body temperature, and loss of body hair were observed. Oba and Suzuki (4) also administered DEHP orally to male and female mice and reported that both sexes tolerated 150 g/kg dose. Miyahara (2) intravenously injected 0.5 ml/kg of undiluted DEHP to a dog and found necrosis with embolism in the lung tissue after 6 days. He found granuloma with perivascular histocyte infiltration in the lung of rats 30 days after a single intravenous injection of 0.1 ml/ kg of DEHP. Miyahara assumed that these pathological changes may be due to the toxicity of the materials rather than their physical properties.
Oba and et al. (5) injected rats intraavenously with undiluted DEHP and emulsified DEHP which was sonicated in rat serum to an average particle size of less than 2 Mm. The dose given was 5, 10, and 50 ul/kg in the animals given undiluted DEPH and 50 ug/kg in the group receiving emulsified serum. Five animals in each group were sacrificed on days 1, 3, 6, 15 and 30 after the injection. In the groups of animals receiving undiluted DEHP the dose-dependent accumulation of droplets increased in the capillaries of alveolar wall with polymorphnuclear cell infiltration on day 1. These lesions developed to granuloma with a proliferation of histocytes and fibroblasts by day 3. After 15 days these lesions were found to have decreased. These findings were assumed to be nonspecific granuloma formation due to the injection of oily foreign materials. In the group injected with the DEHP emulsion serum, similar but less severe changes were observed. Autoradiographic examination of male rats 6 hr after 14C-DEHP injection with and without dilution of the serum, accumulation of radioactivity in the lungs was observed in the group receiving undiluted sample but not in the group receiving serum-emulsified DEHP. These findings were considered to support the histopathological findings.

Subacute and Chronic Toxicity
Yamada (6) fed male rats for 3 months on a diet containing 0.2, 1, and 5 % DEHP. A weight increase of the liver was observed in all treated groups, and an increase of drugmetabolizing enzyme activities of liver microsomes was noticed in the two lower dose groups. In the 5% group, atrophy of the tests was noted. In another experiment, Yamada (3) orally administered 1, 4, and 10 ml/kg DEHP to male and female rats (nine to ten animals in each group) for 3 weeks. In the 4 ml/kg group the increase in body weight was small while that in the liver weight was large, but it was noticed that liver weight returned to normal levels within 2 weeks after termination of the treatment. In the 10 ml/kg group, there was a great increase in the mortality rate and in the liver weight, with a proliferation of the bile duct and swelling of Kupffer's cells.
Ohta et al. (7) fed mice a diet including 0.25 and 2.5%o DEHP for three months and found an increase in the weight of the kidneys along with formation of renal cysts. Also Yamada (8) fed groups of three to five male rats with a diet including 0.5% and 2.5%o DEHP for 21 weeks and found that the weight gain was at a much lower rate. Both groups showed an enlargement of the liver, elevation of serum alkaline phosphatase activity, and cloudy swelling and necrosis of the liver cells and the renal tublule epithelial cells as well as an absence of spermatogenesis. Another study of chronic toxicity of DEHP and DBP is in progress at Environmental Health Perspectives the Departments of Toxicology and of Medical Supply of the National Institute of Hygienic Sciences.

Teratological and Reproduction Studies
Nakayama et al. (9) administered 2.5 and 5 ml/kg of DEHP orally to rats between days 7 and 13 of pregnancy. The mortality of the fetuses increased, and about 50% of the implants were resorbed in 2.5 ml/kg group, but no teratogenic effect was found. Onda et al (10) examined the effect of DEHP and DBP administration on two strains (ddY and ICR) of mice for three generations. Dietary administration of these esters at a level of 10 and 100 mg/kg-day increased the formation of renal cysts in the F1 and F2 generations. The occurrence of kidney damage in the offspring of the group of mothers fed DEHP was found to be higher than in those the DBP groups and F2 groups of ddY/JCL strain. It was assumed that these phthalate esters diffused across the placental barrier and caused the renal damage to the fetuses.

Cytotoxicity and Mutagenicity Studies
Odashima and Ishidate (11) examined the inhibition of cell colony formation and inci-dence of chromosmal aberrations induced by phthalate esters with cultured Chinese hamster cells. DEHP inhibited 50% of the colony formation with 0.125 mg/ml. The toxic effect ranged in the order: DBP, DEHP, DPBG (butyl phthalyl butylglycolate) and DEP (diethyl phthalate), with DBP having the greatest effect and DEP the least. Chromosomal aberration was not produced with 0.5 mg/ml of DEP and 0.16 mg/ml of DEHP. DEHP also did not induce any chromosomal change in the ratascites hepatoma cells (AH-13) and in the bone marrow cells of female rats (AC1/N) after intraperitoneal injection of 0.2-2 g/kg.
Saito and Itagaki (12,13) examined the inhibition of colony formation of cultured KB cells derived from human nasopharyngeal carcinoma. Silicon, polyethyene, polypropylene, polystyrene, polycarbonate, and acrylic resins did not inhibit the KB cell colony formation, but melamine and urea resins, especially flexible PVC, inhibited colony formation. With the plasticizers, concentration of 50 %7 inhibition was 36.9 ug/ml with DEP and 10 fAg/ml with DEHP, respectively.
Kasuya (14) examined the inhibition of the outgrowth of the cerebellar tissue cells of newborn rats by tissue culture method. DNBP (din-butyl phthalate) markedly inhibited the cell   (about 0.3 and 3 mg/ml) of these esters did Mutagenicity of the twelve samples of phthanot produce any bacteriocidal or mutagenic eflate esters including DBP and DEP was ex-fects, as shown in Table 3.  Pharmacological Studies Sasagawa (16) examined the hemolysis of human erythrocyte during the storage of plasma in PVC bags kept at 40C for 6 and 14 days and reported the rate of hemolysis after storage in plastic bags was lower than that observed in plasma kept in glass bottles. Miyahara et al. (2) injected undiluted 0.5 ml/kg of DEHP and 0.25 ml/kg of DBP intravenously into dogs and observed an increase of RBC counts and hematocrit values within 2 hr and in WBC counts after 4 hr. The elevations of serum LDH in both groups and, GOT and ALP in the DBP group were also observed. A decrease of arterial blood pressure, heart rate and a depression of respiration were noticed. An intraarterial injection of 0.5 ml of DBP depressed the perfusion pressure in hind leg preparation.
Nakaura et al. (17) injected male rabbits intravenously with DEHP emulsified with 5%o Tween 80 in saline. The particle size of the sample was less than 25 em. Hematological parameters such as RBC, WBC, platelets, PCV, hemoglobin, blood specific gravity, serium protein, serum calcium and clotting time were examined. After administration of 50 and 100 mg/kg of DEHP twice weekly, these parameters did not show significant deviation from those observed in the control during a 3-month period. The results obtained on blood coagulation are shown in Table 4. Also Nakaura et al. (18,19) examined the changes in these blood parameters for 3-6 months after an implantation of plastics for medical purposes into aortic arch of rabbits and allowed the blood to freely flow over the samples. The samples used were PVC, Teflon, polyacetal, and Hydron. Again, abnormal changes were not observed. Polycarbonate, polypropylene, and polyethylene bottles were cut into pieces and extracted with saline and 5% alcohol-saline solution. Using extracts of these materials, acute toxicity tests with mice, local irritation tests, and pyrogen tests with rabbits showed negative results. Implan-tation of these resins into dorsal rabbits also revealed the lack of effects. muscles of local toxic Absorption, Distribution, and Metabolism Tanaka et al. (20) administered 14C-DEHP orally to rats and examined the absorption, distribution, metabolism, and excretion. About 40-50% was recovered after 7 days in the feces and 30-35% in the urine. High accumulation and slow decrease in radioactivity was found in the liver and intestine after a single intravenous dose of 1.03 uCi of 14C-DEHP as shown in Table 5. A delayed excretion of DEHP was observed in particular in the adipose tissue after oral administration of 1.41 fACi ( Table   6). Orally ingested DEHP was excreted unchanged in the feces, and urinary metabolites such as monoethylhexyl phthalate and phthalic acid were found. Administrative Action on Phthalate Esters The Ministry of Health and Welfare has established the regulation on the migration of phthalate esters and other substances from the PVC container or packages for foodstuff, in 1973. This regulation demands limitation of such migration from the PVC containers or packaging material into the contents under the certain levels. As shown in Table 7, this regulation contains the examinations on three cases for contents: case I for fats, oils and fatty foodstuffs; case II for liquors, wines and alcoholic beverages, and case III for other than the above-mentioned foodstuffs.