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TOXICOLOGY OF DICHLOROMETHANE (METHYLENE CHLORIDE)* . . ' I. Studies on Effects or Daily Inhalation 'a Leon A. Heppel1, Paul A. Neal1, T. L. Perrin*, M. L. Ore4 and V. T. Porterfield* Research Section, Division of Industrial Hygiene, National Institute of Health, U. S. Public Health Service, Bethesia, Maryland V - ICHLOROMETHANE- (methylene chlo D ride), CHtCli, is finding wide and in creasing use as a solvent for cellulose of 6-7 mg.fl. (1700-2,000 p.p.m;), for 4 weeks. There were no deaths and no important changes in the blood picture, but the animals did experience esters, fats, oils, resins and rubber. It is aalsroeduction in body temperature and slight depres utilized in paint stripping operations. The com sion. No autopsy data were recorded. Gross (6) pound is a colorless liquid with a boiling point of subjected rats to repeated 8 hour exposures at ' 40-41C., a melting point of --96.7C., a molecu concentrations of 4-6 mg./l. (1300 p.p.m.). The lar weight of 84.93 and a specific gravity of 1.336 animals were sacrificed at intervals of 25 days. (20C./4C.). It is reported (1) to be practically No definite {pathological changes were found until noninflammable and non-explosive at ordinary after 75 days. These consisted of slight atrophy temperatures, but at higher temperatures under and central fatty degeneration of the liver with certain laboratory test conditions it is capable of sporadic round cell infiltration of the par forming combustible mixtures with air. Its vapor enchyma. Similar, although somewhat greater pressure at 20C. is 348.9 mm, Hg and so the changes were found in rabbits which had inhaled possibility exists in industry of exposures to high 10 mg./l. (2900 p.p.m.) for 50 and 75 days. Flury, concentration. Neumann and Miller (7) subjected cats to expo The acute toxicity of dichloromethane has been sures which lasted 4 to 8 hours on 6 days a week. i studied by a number of investigators.- Mice were They used a concentration of 25 mg./l. (7200 ii killed by a two hour exposure to 14,500 p.p.m. (2). p.p.m.) and the experiment was carried on for a ii Nuckolls (1) determined that dichloromethane was period of 4 weeks.. The animals showed dis \ toxic for guinea pigs. An exposure of 2 hours at a turbances of coordination after 6-8 hours in the l i concentration of 8,000 to 10,000 p.p.m. led to exposure chamber. A temporary increase in marked irritation, tremors and dyspnea with slow hemoglobin and red cell count was noted (amount recovery. At a level of 50,000-54,000 p.p.m. not stated). No abnormalities were discovered ~ maintained for 1 hour to 1J hours, there resulted in the urine. The animals were sacrificed 1, 3, progressive narcosis and death. Animals which and 9 weeks after the completion of the exposure. ; died showed edema of the lungs, cloudy swelling Marked fatty infiltration without degeneration ' of the kidneys and beginning yellow atrophy of the of the liver and kidney was found. liver. Muller (4) found that the pure preparation Some preliminary experiments were carried out caused no organic lesions in mice whereas the tech in this laboratory which had to be discontinued nical product caused fatty degeneration of the due to the transfer of key personnel to the armed liver. Maloff (5) found no such degeneration in forces. Two rhesus monkeys, 2 dogs, 2 pups, 4 the livers of dogs even after repeated anesthesias rabbits, 16 young guinea pigs and 16 young rats produced by ordinary commercial preparations. were simultaneously exposed to a concentration A few studies have been carried out on the of 500 p.p.m. of dichloromethane. Exposures % ~ effects of repeated exposures to dichloromethane. were carried out for 8 hours daily, 5 days a week, Lehmann and co-workers (3) exposed rabbits and over a period of 15 weeks. Three of the exposed cats 8 hours a day, 6 days a week, to concentrations rats died within the first 6 weeks of the experi- ' Jj ment, whereas none out of 16 control rats died. , * Received for publication November 18, 1943. ' * Assistant Surgeon (R). One of the exposed pigs died while there were 2 1 1 Surgeon. deaths among 16 control pigs. The young animals i 1P. A. Surgeon (Division of Pathology). * Assistant Chemist. gained weight rapidly, and all of the survivors ; 'Junior Chemist. appeared in excellent condition throughout the ASI 000015708 i Jin. 19441 TOXICOLOGY OF D [CHLOROMETHANE. I. 9 test period. Blood studies were carried out at weekly intervals on the rabbits. These included red cell, white cell and differential counts and hemoglobin determinations. No significant changes were noted except for a slight increase in eosinophils after the 8th week of exposure. In a second experiment 4 cats, 4 rabbits, 16 young rats and 16 young guinea pigs were exposed 8 hours daily, 5 day5 a week, for 15 weeks to 1100 p.p.m. of dichloromethane. There occurred 1 death among the experimental guinea pigs, while 2 out of animals produced by repeated daily exposures to dichloromethane are presented. Concentrations of 5,000 p.p.m. and 10,000 p.p.m. were employed. Experimental Procedure The exposure chamber used in these experiments was the same as described previously (9). It was of sufficient size (4' x 4' x 61) to permit a num ber of animals of different species to be exposed simultaneously. A metered 400 liters of air per minute were drawn through the chamber, giving a Fig. 1. Apparatus for volatilizing dichloromethane at a constant rate. _ Air from the storage tank passes through the rotameter and then to the fritted gas bubbler. The depth of immersion of the bubbler is kept , constant. The amount of dichloromethane which evaporates is measured by noting the volume of fluid which leaves the reservoir bottle. 16 control animals died. Two female rabbits died, both after giving birth to a litter. The young rats and guinea pigs made good weight gains. The cats, on the other hand, did not seem to thrive. No symptoms were noted except for sleepiness and reduced activity in the early part of the experiment The rats gave birth to many litters during the test period. Blood studies were nega tive except that both female rabbits showed a leucopenia shortly before death. ' In the present paper the effects on various complete change of air every seven minutes. : Inside the chamber were 3 electric fans, which served to mix completely the air apd dichloro methane vapors. The method for obtaining the concentrations of 5,000 p.p.m. and 10,600 p.p.m. is shown in Figure 1. The solvent was vaporized at a constant rate by passing a stream of air under uniform pressure through a fritted glass bubbler which was immersed . in the liquid. The temperature of the liquid was maintained within narrow limits by means of a 10 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [wrf. 26, no. 1 water bath, and a constant depth of immersion of the bubbler was achieved by means of a Mariotte flask arrangement. The vapors passed into a mixing bulb where they joined the main air stream entering the chamber. The concentrations of dichloromethane in the exposure chamber were checked by three independ ent methods. First, these values were calculated from measurements of the volume of solvent volatilized each hour and the air flow through the chamber. Sample calculations of this sort have been included in a paper by Dudley and Neal (9). Another check on the concentration of di chloromethane in the chamber atmosphere was obtained by withdrawing a gas sample each day and analyzing it by thermal decomposition and determination of the ``inorganic chlorides (8). TABLE/ 1 Physical Data on the Dichloromethane Which Was Used 1ST LOT 2hd lot 3U LOT Specific gravity 20720".................. Index of refraction, no*c................... 1.4241 Initial distillation temperature, *C.... 38 Vol. per cent boiling 39"-40* ................. 95 Vol. per cent boiling 40"-41".................. Dry point, *C........... 39.5 1.3287 1.4241 38 78 21 41 1.3288 1.4238 37 98 40 The third control procedure consisted of taking frequent measurements throughout the course of the day's exposure, using the Raleigh-Jeans interference refractometer. The dichloromethane was a commercial prod uct. Physical data on the various lots which were used is shown in Table 1. In addition, the three samples were tested for water, aldehydes, chlorides, acids and substances decomposed by sulfuric acid. The results were negative except that a faint yellow color developed in the sulfuric acid layer when the last named test was carried out, Nuckolls' directions (1) were followed. Unfortunately, the temperature and humidity prevailing inside the chamber could not be con trolled. They were measured each day. The exposures to 5,000 p.p.m. were carried out during the fall and winter months, but the second experi ment using 10,000 p.p.m. was done during a warm, < humid summer. Temperatures as high as 93F. and relative humidities as high as 80 per cent were recorded. i Diets: The dogs received a mixture consisting of 1 equal parts by weight of ground horse meat and ! ` Kippell biscuits. In addition each animal received . 100 c.c. of whole milk per day. Guinea pigs and ; rabbits received pressed pellets of the following composition: 15 per cent oat groats, 30 per cent pulverized % 1 whole wheat, 40 per cent alfalfa < leaf meal US #1 sun cured, 13 per cent soy bean meal, 0.25 per cent sodium chloride and 1 per cent calcium carbonate. Small amounts of irradiated yeast and wheat germ oil were also included in the { formula. Of the rats exposed to 5,000 p.p.m. of j dichloromethane, half received Purina dog check- I' ers while the remainder were fed the following j ration: 20 per cent wheat flour, 20 per cent com i meal, 31 per cent rolled oats, 13.5 per cent milk ! powder, 3.5 per cent brewer's yeast, 6 per cent pow dered hog's liver, 2 per cent dextrin and 4 per cent . salts. All of the rats exposed to 10,000 p.p.m. of dichloromethane were fed Purina dog checkers. The rodentp received supplements of fresh cab bage. The monkeys were fed Chim crackers, whole wheat bread, milk, eggs and a variety of fresh fruits and vegetables. Results op Vapor Exposures 17 milligrams per liter (approx. 5,000 p.p.m.) The first series of animals were exposed simul taneously to a concentration of 17 mg./l. (approx. 5,000 p.p.m.), 7 hours daily, 5 days a week, for ; periods up to 6 months. Fluctuations in the concentration of dichloromethane which prevailed in the chamber were small. A gas sample was withdrawn each day and analyzed by a combustion method (8). The, average value was 17 mg./liter , with a standard deviation of 2 mg./l. There was good agreement between the results of the gas analyses and the concentrations as calculated from the volume of dichloromethane which was f/- volatilized. Table 2 shows the number of animals exposed and the deaths which occurred. Weight Changes: The adult animals appeared well nourished at all times. Two pups were bom in the exposure chamber and exposed daily from birth. They made excellent weight gains. The young rats also gained weight rapidly, but the exposed guinea pigs lagged behind the controlj ,, Jan. 19441 TOXICOLOGY OF DICHLOROMETHANE. I. 11 animals. At the end of the exposure the experi mental pigs had an average weight of 820 gm. compared with 1025 gms. for the control pigs. Food consumption of the exposed rats was com parable with that of the control rats, whereas the exposed guinea pigs ate somewhat less than did the control pigs. Symptom: The rats appeared well throughout the experimental period. They showed no evi dence of narcosis while in the chamber. The rabbits also seemed to be unaffected by the re peated exposures to dichloromethane. Both fe males bore young once during the period of 6 months. The litters were not raised, but this TABLE 2 Anmals Exposed to 5,000 p.p.m. ANIMAL NXMBEl AVER innraEi AGE or EX PfcAlH* Mile Fe WEIGHT male POSURES Dogs................... i 5 8400 132 0 Puppies............... i 1 470 115 0 Rabbits............... 2 2 3400 124-131 0 Guinea pigs......... 8 550 131 3* Guinea pigs......... 6 400 . 65 0 Rats.................. 11 1 146 132 It Rats.................... 5 174 77 0 Rats.............. 4 160 42 0 *Died after 35, 90 and 96 exposures. Autopsy showed extensive pneumonia associated with moderate centro-lobular fatty degeneration of the liver. No deaths occurred among 14 control pigs. t Died shortly after giving birth to a litter (22 ex posures). Autopsy showed many thrombi in renal vessels associated with marked cortical infarction. No deaths among 28 control rats. may have been due to the disturbance resulting from daily transfers to the chamber. The surviv ing guinea pigs also appeared to thrive. The dogs showed no ill effects from the expo sures. Their appetite was usually excellent. . Periodically they were carefully examined and special attention was paid to the appearance of the cornea, sclera, eye grounds, eye movements, gait, condition of mucous membranes, state of deep reflexes and sensory perception. Nothing abnor mal was ever noted. Mean arterial blood pressures were determined on 5 dogs at intervals of one to two weeks, using the method of direct puncture of the femoral vessel of trained, unanesthetized dogs. All of the blood pressure readings for each dog were averaged, and these averages varied from 100 to 115 mm. of i mercury. Of 83 determinations, the results of all , but 3 fell between the normal value of 90 and 125. Studies on Urine and Blood: Catheterized urine specimens were examined at intervals of several weeks. They were tested for pH, specific gravity, presence of albumin, sugar, acetone, urobilin, urobilinogen and abnormal cellular constituents. The results of these examinations were uniformly negative. After the six adult dogs had been exposed over & period of 4 months blood samples were drawn in order to measure the level of prothrombin. All values were normal. Determinations of icterus index were carried out on the sera of dogs from time to time during the experimental period of 6 months. It was never elevated. Bromsulfalein excretibn was studied after the animals had been on test for 6 months. The results were compared with those obtained with normal, unexposed dogs. Five milligrams of dye per kilogram was injected intravenously and blood samples were obtained at 15 minutes and 30 minutes after the injection. In most instances there was a retention of 4 per cent after 30 minutes for the 12 observations on the experimental ani mals and the 12 determinations on the control dogs. Plasma protein levels were determined on the six adult dogs after they had been exposed to dichloromethane over a period of 6 months. Analytical methods were as follows: 1) for total . protein, macro-Kjeldahl digestion, using Wong's , digestion method (10); 2) separation of albumin fraction according to Kingsley's technique (11); 3) for non-protein nitrogen, nesslerization as de scribed in Hawk and Bergeim (12). The results were comparable with those obtained upon normal dogs in this laboratory and they are similar to the levels reported in the literature. Hematological Changes: Red and white cell1 counts, hemoglobin determinations and differential cell counts were carried out, first at weekly inter vals and then every two weeks, during the entire , exposure period on 5 of the adult dogs and on all 4 , rabbits. No abnormalities were encountered in the case of the rabbits. Three of the dogs showed a tendency for the red ceil count to rise but this trend was hardly significant. 34 milligram per liter (approx. 10,000 p.p.m.) As a pilot experiment, a group of animals . received a single exposure of 7 hours to 10,000 AS I 00001 57U i 12 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [not. 26, no. 1 p.p.m. These included 10 rats, 9 guinea pigs, and animals threw themselves about, bit one another, 4 rabbits. One of the rabbits died several days knocked their heads against the sides of the cage, after the exposure. All,of the other animals re and licked tire shavings on the floor. Frothing mained alive and well during an observation period appeared about their mouths and conjunctival j of one month. Sixteen mice (average weight 25 irritation develbped. gms.) were exposed for 4 hours to 10,000 p.p.m. Toward the end of the 4 hour period the dogs ' All but one survived. were still engaged in these wild, aimless movements ' Daily exposures on 5 days a week were then or else lay on the floor, apparently exhausted. begun and these were continued for 8 weeks. After,, the animals were removed to their living . The first exposure lasted for 6 hours, while all of cages they rapidly recovered from the effects of ` the others were only four hours in length. A gas the anesthesia and ate their food almost at once. 1 sample was withdrawn from the chamber each day They were all sacrificed after 6 exposures because and analyzed by the combustion method. The of the numerous bruises which they developed average value was 53.8 mg./liter with a standard while tossing about in the exposure chamber. deviation of 1.4 mg./liter. There was generally The two monkeys showed nothing during the TABLE 3 Animals Exposed to 10,000 p.p.m. first hour except decreasing activity. During the second hour they developed incoordination and had trouble in getting up from a lying position. AXtNAl tfTOOEK AVER Nuwm AGE Of EX- DEATHf i Male Fe WEIGHT rosuui male Dogs..................... Monkeys............... 4 7500 6 0 2 4050 36-37 0 Towards the end of the 4 hour period the animals lay prostrate with barely perceptible respirations. During the last two weeks it seemed that they developed a resistance to the vapors for they were able to sit up throughout the exposure period. The animals generally ate their food, appeared Rabbits................. 5 Guinea pigs........... 11 , 1 Rats...................... 9 7 3240 653 190 37 38 38 well and gained in weight during the course of the ' 3*, 0 experiment. This was especially surprising since one of the monkeys, at autopsy, was found to have 2t widespread tuberculosis. * Died after 1, 12, and 22 exposures. Autopsy - The rabbits showed excitement at the beginning : examination of 2 of the rabbits showed pulmonary congestion and edema with focal necrosis, and splenic congestion. t Died after 33 and 38 exposures. Autopsy on second rat showed marked pulmonary congestion and edema with focal extravasation of blood. of the exposure period followed by varying degrees : of inactivity. Some lay on their sides while others were able to itaaintain an upright position. Very few signs of narcosis were displayed by the guinea pigs, which showed only increasing somnolence. ; There was no evidence of mucous membrane irrita .good agreement between the results of the gas analyses, and the concentrations as calculated from the volume of dichloromethane which was volatilized. In Table 3 are shown the number of animals exposed and the deaths which occurred. Weight Changes: The young rats gained weight rapidly. The exposed guinea pigs lagged behind the control animals throughout the test period, and at the end of exposure their average weight was 88 per cent of that of the control pigs. tion. A few animals showed mild incoordination towards the end of the exposure period. The rats } developed difficulty with their gait during the first half hour in (he chamber. During the second hour ! they lay on their sides and at the end of 4 hours they were prostrate with very depressed respira- ; tion. Within one hour after removal from the chamber the animals appeared normal again. ; Hematological Changes: Only a few blood counts i. were carried out for this experiment because the i dogs were sacrificed after but a few exposures and i Symptoms: The dogs were more violently affected than any of the other animals. At first they showed only mild incoordination. Within a most of the rabbits died early in the experiment. ; No important changes were noted for any of the L animals. [ few minutes they seemed to go into an excitement. . Pathological Findings stage of anesthesia manifested by very energetic Autopsies were performed upon all of the experi- [ but purposeless movements of all sorts. The mental and many of the control animals. Gross i Jan, 1944] TOXICOLOGY OF DICHLOROMETHANE. I. 13 findings were negative except for pulmonary slight to moderate fatty degeneration. The fat changes described below. In the case of the dogs droplets were of small size and were observed the brain was also examined. Organ weights were chiefly in liver cells in congested areas and, to a obtained for the livers, kidneys, lungs, hearts, lesser extent, in irregularly scattered macrophages. and spleens of the exposed and control animals. One of the 2 monkeys revealed disseminated There were no significant differences between the tuberculosis lesions but no other histopathologic two groups. alterations were found in either of the two. Microscopic examination was done for most of The 2 rabbits which survived 37 exposures were the animals exposed to 5,000 p.p.m. Examination, negative, but the 2 which died showed marked was done on each of the 7 dogs, 4 rabbits, 14 guinea pulmonary congestion and edema with focal extrav pigs and 21 rats. Unexposed animals taken from asation of blood. One of these also exhibited stock at the same time as those on experiment marked congestion of the spleen with considerable served as controls; these included 4 rabbits, 10 pyknosis and katyorrhexis of the lymphoid cells guinea pigs, and 10 rats. In the group exposed to of the follicles. 10,000 p.p.m., examination was done on each of the Four of the 6 exposed guinea pigs revealed 4 dogs and 2 monkeys, and on 4 of the 5 rabbits, 6 slight to moderate fatty degeneration of the liver of the 12 guinea pigs and 6 of the 16 rats. The 4 characterized by fine fat droplet accumulation in rabbits included the 2 which died after 12 and 22 the liver cells in centrolobular areas. . The remain- exposures and the 2 which survived. The 6 guinea pigs studied represented a sample of the group TABLE 4 of 12, all of which survived. The 6 rats examined included the one which died after the 38th expo sure, together with a sample of 5 from the 14 Analyses or Blood Obtained From Does Imjuediaxely Following Exposure to 5,000 p.p.m. or Dichloromethane jor 7 Hours which survived. Four unexposed rats and 4 unexposed guinea pigs were used as controls. ' Only the liver was examined from the 4 dogs DOO NUMI11 HO* CAiaUM KG*. CHlCWlOO ML. Avenge Ringc exposed to 10,000 p.p.m., but from all other ani mals on both exposures, the heart, lungs, liver, spleen and kidneys were studied. In addition, the adrenals from the guinea pigs and skeletal muscle from the rats were examined. All tissues were fixed in a 10 per cent solution of formalin l 2 3 4 5. .7 7 7 7 7 9.6 10,5 8.7 9.5 10.7 8.1-11.7 9.5-12.6 7.4-12.0 7.2-13.2 8.7-12.5 and routine sections were stained with eosinmcthylcne blue. Sections from the liver, spleen and kidneys were stained for hemosiderin and frozen sections from the liver and kidneys were stained for fat. Among the animals exposed to 5,000 p.p.m. of dichloromethane, there were no lesions which appeared to be related to the exposure. The only animals showing pathological changes of any significance were the 2 guinea pigs which died after 90 and 96 exposures respectively, and the rat ing 2 were normal. One of the 4 controls exhibited moderate fatty degeneration of the liver, but the fat droplets were moderate in size and diffusely distributed. The rat which died after -the 38th exposure showed marked pulmonary congestion and edema with focal extravasation of blood. Similar but less marked changes were seen in the lungs of one of the rats which survived, but there were no significant lesions in the others. which died after giving birth to a litter. Each of these guinea pigs showed an extensive pneu monia associated with moderate centrolobular fatty degeneration of the liver. The rat exhibited many thrombi in renal vessels associated with marked certical infarction. Among those animals exposed to 10,000 p.p.m., no lesions were seen in the livers of 2 of the 4 dogs. The other 2 showed moderate centrolobular congestion with narrowing of liver cell cords and Analyses of Volatile Chloride in Blood and Urine Froth Animals Exposed to Various Concentrations of Dichloromethane The volatile chloride was determined by the combustion method developed in this laboratory(13). Volatile chloride released by the aeration of blood or urine and decomposed by passing over heated platinum was absorbed in an alkaline sulfite solution and titrated by a standard method. '^ J ' .. /: v 14 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [vol. 26, no. 1 1 TABLE 5 . Dog blood was withdrawn from the jugular vein Analyses of Blood and Urine Obtained from 2 and rabbit blood from the heart. Whenever neces- , Doos Following a 3 Hour Exposure to 5,000 P.P.M. of Dichloromethane sary the samples were stored in the refrigerator, in aeration tubes or glass stoppered volumetric . 1AMFUK OttAlMES DOO *6 DOO *7 Mgi. cmcii/ioo ai. flasks. Uncontaminated blood Containing volatile chloride has been stored five days without1 any loss of volatile chloride. The results are shown * Blood Immediately alter ex posure........................ 2 hours after exposure... 4 hours after exposure,.. 6 hours after exposure... 13.7 5.7 -- 0.2 Urine 3.1 1.9 T-- 0.2 Blood 5.1 -- 1.5 Urine 1.1 --' 0.6 in Tables 4, 5, and 6. The figures usually repre sent the average of 2 determinations. Blood withdrawn from a dog after a six hour exposure to 10,000 p.p.m. of dichlororaethane had a concentration of 21.8 mg. dichloromethane in 100 ml. while blood from 3 rabbits had the following concentrations: 18.6, 20.4 and 16.4 mg./lOO ml. One rabbit exposed to 10,000 p.p.m. of dichloro TABLE 6 methane for four hours was bled J, 1 J, 2}, and 3} Analyses of Blood Obtained from Rabbits Following Exposure to 10,000 p.p.m. of hours after the exposure was discontinued. The following concentrations of dichloromethane were Dichloromethane found in the blood at these intervals: 10.4, 9.0,. AKUtAL NO* PTOATIOM or nrotou Tttt SAMPLE WAS OBTADVU ATTXK DlSCONTUHttNO MGS. CHtCli/100 KXFOStflZ ML. 4.1, and 2.6 mg./lOO ml. A urine specimen was obtained from a rabbit three hours after a four hour exposure to 10,000 kturt p.p.m. dichloromethane. The concentration of 7 13 9 10 12 7 10 9 2 2 3 11 1 11 7 3 10 ' 3 11 . . .9 .2 7. 9 ' 10 10 3 3 .3 3 1 1 2 V 2 3 3 4 4 4 4 4 4 4 4, 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Immediately Immediately ' Immediately Immediately Immediately i Immediately Immediately Immediately 15 minutes 15 minutes 15 minutes 10 minut^i 30 minutes 1 hour 1 Lour 1 hour 1 hour 2 hours 2 hours 2 hours 3 hours ' 3 hours 4 hours 4 hours ' 5 hours ' 5 hours 6 hours 7 hours 1' 8 hours 8.7 . 9.0 14.9 ' 12.5 ' 17.0 14.5 13.9 17.1 13.4 14.5 14.6 19.9 7.3 5.8 10.7 7.0 6.6 C.2 ,3.7 6.0 3.6 3.0 1.7 2.6 , 1.1 1.1 ' 1.5 , 1.1, 0.9 dichloromethane in this urine was 3.4 mg./lOO ml. ' Effects of Skin Applications in Rabbits \ A pledget of cotton was soaked in dichlorome thane and then applied for one minute to the ear.: Applications were made once a day, 5 days a week for 4 weeks. The response to dichloromethane seemed to be ' that which would result from mild simple irritation maintained by repeated exposures. It had a fat. solvent action which made the hair follicles become more prominent. Then there was noted hyperemia followed by a scaly exfoliation. ) Discussion The data presented here confirm other reports on the low toxicity of dichloromethane for various experimental animals. A concentration of 5,000 p.p.m., one half per cent in the atmosphere, had no discernible ill effect on dogs, rabbits, rats or guinea pigs, with one exception. In the case of the guinea pigs there Was an adverse effect upon rate of: growth. Reproductive function was unimpaired and young rats and puppies grew normally. This is remarkable when one considers that the . animals were in the exposure chamber for 7 hoursf daily, 5 days a week, for 6 months. There was no r AS I 000015714 r- Jan. 1944] TOXICOLOGY OF DICHLOROMETHANE. I. 15 indication from the urine examinations of renal irritation nor did microscopic sections of the kid neys show any lesions. Absence of liver damage in the dogs is indicated by the following evidence: 1) repeated normal values for the icterus index of blood scrum, 2) normal levels of plasma protein and normal A/G ratios after 6 months of exposure, 3) normal prothrombin levels after 4 months .of exposure, 4) absence of urobilinogen in the urine on repeated examinations, S) normal bromsulfalein excretion values, 6) absence of pathological changes in the liver. Even when the animals were repeatedly exposed for periods of 4 hours to 10,000 p.p.m. of dichloromethane, which caused light to moderate anes thesia, there were few residual ill effects. Recovery was rapid and appetite was generally unim paired. Autopsy examination of the few animals which died showed pulmonary congestion and edema. It is possible that the high temperatures and humidities which prevailed during this part of the work may have been partly responsible for these deaths. Two out of 4 dogs showed slight to moderate fatty degeneration of the liver after 6 exposures. Four out of 6 guinea pigs also devel ops liver injury. There was no microscopic evidence of liver damage for the 2 monkeys, 2 rabbits and 6 rats which received almost 40 exposures. Concentrations as high as 5,000 p.p.m. would probably not be tolerated for very long by human beings. According to Lehmann and Flury (6) a concentration of 310 p.p.m. can just be detected by smell; 2300 p.p.m. causes nausea after 30 min utes; 7200 p.p.m. causes paresthesias of the ex tremities after 8 minutes, as well as congestion in the head, a sense of heat and slight irritation of the eyes. The analyses of volatile chloride in blood and urine show that appreciable quantities of dichloromethane arc excreted by the urinary route. The concentration in the blood of dogs after an exposure of 7 hours to 5,000 p.p.m. is approximately the same as that reported for rabbits by Moran (13). The data give some idea as to the rate of elimina tion of dichloromethane. It would be desirable to carry out a detailed study of the rateand extent of absorption and elimination of dichloromethane by various routes. Such an investigation must be deferred, however, until the sensitivity of 'the method of analysis has been increased. Summary 1) . Several species of animals were simultane ously exposed to 17 mg./l. (5,000 p.p.m.) of dichloromethanc. Repeated 7 hour exposures, 5 days a week, for 6 months were tolerated without evi dence of toxic reaction by rats, rabbits and dogs. :In the case of guinea pigs there was an adverse effect upon rate of growth, but the animals appeared in good condition. The mean arterial blood pressure of the dogs remained within normal limits. No significant changes were observed in the blood picture of dogs or rabbits. No evidence of liver damage was given by the following studies on dogs: 1) scrum icterus index, 2) plasma total protein level and A/G ratio, 3) testing for uro bilinogen in the urine, 4) brohnsulfalein excretion tests, 5) pathological examination of the liver, 6) prothrombin level. Repeated clinical examina tions of the urine were negative. Microscopic sections of various organs were normal. 2) . Repeated 4 hour exposures, 5 days a week, for 7i weeks to 10,000 p.p.m. produced no micro scopic evidence of liver damage in monkeys,. rabbits or rats. Two out of 4 dogs showed slight to moderate fatty degeneration of the liver after 6 exposures. Four out of 6 guinea pigs also devel oped liver injury. This concentration of dichloromethanc causes light to moderate narcosis. Several of the animals died, apparently because of acute pulmonary congestion and edema. 3) . Repeated applications of dichloromethane to the cars of albino rabbits lead to hyperemia fol lowed by scaly exfoliation. 4) . Data are presented on the concentration of dichloromethane in the blood and urine of various exposed animals. 5) . These experiments confirm other reports on the low toxicity of dichloromethane compared with other halogenatcd hydrocarbon solvents. No data arc available concerning the effects on human subjects of repeated exposures. From these ani mal experiments, it is tentatively suggested that the maximum allowable concentration for 8 hours daily exposure is 500 p.p.m. f . , \ 16 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY ' [vcl.26, iw.i j REFERENCES . y'' (1) NdCKOLLS, A. H.: The comparative life, fire and (7) Ibid. explosion hazards of common refrigerants. (8) Dudley, H. C.: A portable unit for the determine. Underwriter's Laboratories' Report. Miscel tion of balogenated hydrocarbons. Public laneous Hazards No. 2375,1933. Health Reports, 56:1021,1941. (2) Fluky, F., and Zernik, F.: Schadliche Gase. ' Julius Springer, Berlin, 1931. (3) Lehmann, K. 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