Document 99VmzDJDVQ2YnYDn9B2EYKOz6

._.. ENVIRONMENTAL HEALTH - -PREVENTIVE MEDICINE OCCUPATIONAL MEDICINE AEROSPACZ MEDlClNf VOLUME 12 JAKUART THROUGH J U X E , 1966 AMERICAN MEDICAL ASSOCIATION Publication 11! 1f ' 1; ,! ,1 6 tion, (2) skin application, and ( 3 ) sub- Submitted for oubilutton AUQ 20 1966: accepted Nov 2. ,.. e Medicine and Publlc Health..D_insi1onBroafncInh.duCsatlrviaesltoHn).giene. Tile Unlverslty of uests to Inatirutc ol Ennronmental Heiltb. Tricyclln Alkyl benzenes C, c IO CII Clt CII ._ Omol % 4?mol % 3 inol % 3mol % Omol % OrUol% om01 5% 71x101% 10 mol % 24mol Yo Smol A 1 mol % St. Oklahoma City 73101 (Dr. Nau). 13.5 mol Z nnphthalene. 9.5 mol %?-methyl naphthalene. A r c h Environ Hzalth-Vol 11, March 1966 TES ri of materials dies (Fig 1) used parts: (1) the surge chamber inner chamber hese parts were .heet metal and 1. T h e Venturi ing vapor from with the inlet air-vapor mixye chamber and r plate into the `et mixture of ie exposed anioutside atmosprovided in the This was for re to which the d. The animal `ier were made :loth. A 2 foot -n top of the or the animal removed from `kyl Aromatic C I I - C I IFmctlon B P:392460 F Bmcrf% Omol % 44 mol % 27 mol % 23 mol ?5 ' Omol% 7mot lo LO mol % 24 mol % Smol% l m o l Yo .?ethylnaphthalene. CrC,, FRACTIONS-LVAU ET AL 383 The results obtained when animals were exposed by inhalation to Ce-Clo o r Cll-C12 were compared to results obtained when the Same species of animals were exposed to identical concentrations of benzene or normal decane. Criteria used to detect changes from the normal in exposed animals were: ( 1 ) appearance and behavior of the animal, (2) body weight gains as compared to expected gains, (3) organ weights, (4) hema- tological findings, ( 5 ) bone marrow change^,^ and (6) gross and microscopic pathological changes. The chambers were so operated that there was a constant and known quantity of the test material fed into the vaporizers. Determinations of the concentrations of the vapor in each chamber were made each hour. The air flow into the chamber was measured with a thermo-anemometer and a dry-test meter (Table 2 ) . Inhalation Studies Using Rats.-Benzene: Concentration of Benzene, 1,ooO PPM. Rats exposed 235/2 hours per day, seven days per week, appeared to be in "poor" condition, with a loss of body weight after 183 hours of exposure. They hemorrhaged from the nose and mouth; the TABLE&-Vapor Conccntratiom E m p l o y e d for Inhaiation Material Benzene CI-Ci a Cir-Cla Ndecane Control C+-ClO c 11-cI1 Conccnrntion VarLatLon Ln Marfmum Hours of Days of No. of Exposure Exposure ~ n l m a l s Rat Studle4 1.000ppm 2M) ppm 50ppm 1.000 p p m 616ppm ?00ppm 50ppm mppm ?M)ppm XI ppm 560 ppm No erposure 12?-1.885 i37-749 ?56-733 232-1.43 54-2,;22 `12-739 697-715 Sgl,?35 730-744 722746 36?-1.501 105 92 90 78 150 83 90 51 93 90 91 38 18 23 38 60 IS IS 37 17 18 41 M o n k e y Stud[- ?00ppm Sppm MOppm swm 630 630 630 630 90 . 90 90 80 3 3 3 3 stomach was distended; the gut was empty; and the blood vessels of the lungs, liver, kidneys, intestines, and omental tissues were engorged. The white blood cell count fell significantly; there was a reversal of the polymorphonuclear-lymphocyte ratio, and the deoxyribonucleic acid fell significantly. Microscopic studies of the bone marrow showed an increase in the proportion of erythrocyte precursors (stimulation of erythrocytic activity). .. . I Fig 1.-Vaporizer and exposure chamber. Arch Etizn`ron Hrcllth-Vol 12, March 1966 384 C,-CtI F R A C T I O N S - N A U E T A L Rats exposed to this same concentration 18 hours per day, seven days per week f o r as long as 1,782 hours showed changes similar to the ones described above. Some of the rats were "set aside for observation" for 6y? months after 1,782 hours of exposure. All blood changes returned to normal values with the exception of the deoxyribonucleic acid which remained low. Concentration of Benzene, 200 PPM. Rats exposed eight hours per day, five days per week, showed a fall in WBC after 750 hours of exposure with no change in the polymorphonuclear-lymphocyte ratio or expected weight gain. The microscopic examination of the bone marrow revealed some depression of myelocytic activity and a stimulation of erythrocytic activity. Concentration of Benzene, 50 PPM. Rats exposed eight hours per day, five days per week for as long as 756 hours showed a fall in the white blood cell count' with no other significant findings except that after 600 hours of exposure, 50% o i the rats devel- oped bilateral cataracts. As with 200 ppm exposure, the rats developed lower DNA values and revealed depression in the bone marrow of myelocytic activity and a stimulation of erythrocytic activity. C9-ClO: Concentration of C&o, 1,000 PPM. Rats exposed 18 hours per day, seven days per week, developed congestive changes in the lungs and liver, large spleens, and hemorrhagic kidneys at the end of the first day of exposure. There was a significant lowering of the white blood cell count by the eighth day, as well as a shift in the polymorphonuclear lymphocyte ratio. DNA values were lowered and the expected body weight gains were not observed. There was an increased transparency and fragility of the femur of the exposed rats. Concentration of Ce-Clo, 616 P P M . Rats exposed 18 hours per day, seven days per week, for as long as 2,124 hours showed a significant interference in expected weight gain as well as a significant fall in the total white blood cell count. There was evidence of congestion and hemorrhagic change in the lungs,liver. kidneys, spleen, and omental tissue after only a few days oi exposure. Focal, inflammatory changes in the lungs were noted as well as fatty changes in the liver after 54 hours of exposure. Afte: 400 hours of exposure there were hemor- rhages around the nose and mouth; transitory blood changes occurred in the rats exposed f o r 2,424 hours. Seventy percent of the rats, which were set aside for two months with no additional exposure, devel- oped bilateral cataracts. There were no cataracts observed in unexposed rats of the same age o r in rats exposed to any cancer.- tration of n-decane. The primary histological changes observed in the eye of the exposed rat occurred in the lens where one could observe, in focal areas, accumulations of enlarged epithelial cells with cytoplasm frequently vacuolated. Also, there were areas of proliferation of fibro- blasts. There were no inflammatory cells. These changes in the rat were similar ta those found in the lens of the chick embryo given naphthalene,10 maleic anhydride, or hydro-quinone. There were no significant organ weight changes. Microscopic examination of the bone marrow indicated a stimulation oi myelocytic activity. Rats exposed to 616 ppm of Cg-CI0 231,.:! hours per day for seven days had blood in the urine and hemorrhagic changes in the kidneys, omental, and subcutaneous tissues. Rats exposed continuously for 24, 36, 54, or 72 hours (one single exposure) to the above concentration of Co-Clo showed only transitory blood changes. Rats exposed to 616 ppm o i Ce-Clo on three alternating days for 1 s hours each day and then set aside nine months ior observation did not develop any catarac;;. There were no significant changes iron1 the normal obsemed. Concentration of Cs-CIo, 200 PPbI. Rats exposed eight hours per day, five days per week (90 exposures), showed no persistent o r significant peripheral blood changes. weight gains, bone marrow o r eye lens changes. Concentration of C9-CI,,. 50 PPAI. Ra:' exposed eight hours per day, five days per week. showed no detectable changes froiii the normal after 715 hours of exposure. CrCu FRACTIOLC'S-LVA U ET AL 385 Iiihalaticln Studies UsrJigJlorrkeys.-C,- Jl0: Vapor concentration of Co-ClO, 200 PPJI. Thri:e Rhesus (4Ib; 1.8 kg) monkeys .xposed by inhalation to 200 pprn of Ce-Clo ;even houis per day, five days per week, lor a total of' 90 exposures appeared 'groggy" and sedated while being exposed. rhe white blood cell counts fell and there vas a reversal in the polymorphonuclearynphocyte ratio. During the first week of he exposure the monkeys developed a noiceable tremor which became less proiounced as the exposure was continued. rhere was evidence of skin irritation with a loss of hair and the formation of a dry and "leathery" skin. The monkeys killed after 90 exposures showed no significanl gross or microscopic changes from the nor.mal other than those referred to but had a bone marrow suggestive of depression of both the myelocytic and erythrocytic activity. Vapor Concentration of CO-Clo, 50 PPM. Three monkeys exposed to 50 ppm of CpClo seven hours per day, 5 days per week, for a total of 90 exposures showed no consistent significant changes by gross or microscopic examination except an increase in up -----v .,,,.,,.,,,.I , 6 I I a, *I , I .. . .11 1. I. -e. 0. ,..0.U. --.I. .I. 0.. I" 0,. Fig 2 . - ( A - 0 ) G-CU inhalation studies of rats. Arch Enziron Heolth-Vol 12, hfarch 1966 ,*.on." 111 386 C,=C, F R A C T I O N S - N A U ET A L hematocrit readings and a shift in the polymorphonuclear-lymphoctyte ratio. Idialatiou Stiiciies Usitig Rats.-Cll-Clz: Vapor Concentration of Cl1-CI2, 500 PPM. Rats exposed 1s hours per day, seven days per week, did not tolerate such an exposure. Fifty percent of the rats exposed were dead at the end of the first 18 hours of exposure. Their weight loss was impressive. The organs were all engorged with blood, the spleen was small, and the intestines were filled with a liquid and a gaseous material. IVhen the exposure time was reduced to eight hours per day, seven days per week, the rats deteriorated rapidly (within 1j days) in appearance and weight loss. Postmortem changes noted included a small spleen, hemorrhagic lungs,'and liver, and the intestines filled with a bloody liquid and gaseous material. When the exposure time was reduced to five hours per day, seven days per week, the rats developed a dull and coarse hair (which tended to fall out), emaciation, ant1 vesicles around the eyes, nose, and feet, They appeared narcotized. There was a ! t.-h r / ..,I. D , I. I.,. .. ,, " ,,,I , I I I, 0 , as I. II , 1. I ,,,, I on 111 , I9.I" , 91 , m. 0. '"0.U.e- ,I10U. C.. 0.. Fig 2 . - ( C - D ) G-Cu inhalation studies of rats. Arch Environ Health- Vol 12. hlarck 1966 ? , ., 1 !I 1 week, In 15 Post- small ., ani1 d and ced to week, :hair 1, and feet. vas 7. - -- CrCn, FRACTIONS-NAU ET AL. 1 387 marked fall in the WBC, a reversal of the: polymorphonuclear-lymphocyte ratio, and a. very slight impairment of the expected weizht gains. When these rats were exposed as long as 1,683 hours the WBC and DNA decreased. Expected bOdy weight gains were not observed; the spleens were small and the femurs appeared soft; the bone marrow was "watery." N o cataracts were noted. Rats set aside for four months after the 1,683 hours of exposure appeared to make a good recovery. Vapor Concentration of C I I - C ~ ~2,00 PPM. Rats exposed for eight hours per day, five days per week, for 90 exposures developed a definite and gradual fall in the WBC and the expected body weight gains. "I Fig 2 . - ( E - F ) C.-C, inhalation studies of rats. Arch Enziron Hcalth-Val 1,March 1966 - 388 CrCi1 FRACTIONS- NAU ET AL - TABLE 3.--Gr0~1tll and Hematological Changes in Rats Folloiifing Inhalation of Test Vaporz * hnirnal Croup Preerposure Period Mean No. Value SD End ot 57 Days Mean No. Value SD End o fIO5 Days Mean Sot , Value SD - Control animals Total leukocytes 20 22.760 3.060 20 21,055 1.610 17 20,510 ?.ID Poly leukocytes 20 16 5 20 18 8 17 22 7 Total lymphocytes 20 82 6 20 81 9 17 TI 6 A&dl weight 20 295 33 20 335 94 17 423 23 Benzene anima& Total leukocytes 14 22,650 750 12 4,625 2,045 6 5.4?5 4.140 Poly. leukocytes 14 ?2 9 12 52 16 6 54 13 Total lymphocytes Animal weight 14 14 77 9 12 318 21 12 47 16 6 254 39 6 *:46 13 266 C I-C I B 3nimaIs Total leukocytes 30 25.350 1,6S5 2a 12,6SO 1,190 26 16,920 1,lOJ Poly. leukocytes 30 20 8 28 16 5 26 ?I 7 Total lymphocytes 30 i9 8 28 63 5 26 78 7 Animal weight 30 233 20 18 294 21 26 3?3 20 C ~ I 1C1 animals Tot31 ieukocytes Poly. leukocytes Total lymphocytes 25 25 25 21.790 1.5 84 1.940 10 11 5 16,570 5.m - -5 64 7 5 36 Animals sacrificed becauseof pour hellth. A n h a 1 weight 25 36? 47 5 266 ?6 Endor91 D3ys Decane animsk Total leukocytes 4 3 21,490 i30 31 1i,i65 1.600 18 ??.SI5 1.530 Poly. leukocytes 43 18 S 31 22 E IS 3 14 Total Ipnphocytei 43 91 8 31 76 6 IS i2 14 Animal weight 43 301 40 31 456 26 1s 176 3.; Benzene. 1.ooO ppm; CrC I D , 616 ppm; C I ~ - ICX. 500 ppm; n-decaoe. 540 PPm. One rat out of 17 developed a cataract. Es- Iiihalatioii Stirdies Usirig Moiikegs.-C11- amination of the bone marrow o i the C12: Vapor Concentration of C11-C1ZOo exposed rats revealed stimulation of erythro- PPM. Four Rhesus monkeys exposed seven cytic activity and delayed depression of hours per day, five days per week (90 myelocytic activity. exposures) showed evidence of irritation Vapor Concentration of Cll-C1z, 50 PPM. on the face and in the eyes. Diarrhea was Rats exposed eight hours per day, five days noted on the second or third day of esposi*:'t. per week, for 90 exposures developed some There was no change in the expected b. ._.. very slight interference in the espected body . y i g h t gain; the DNA fell slightly. After ha2v"ing been set aside for observation for weight gain. There was, however, a reversui of the polymorphonuclear-lymphocyte ratio and the bone marrow showed eviclence of a stimulation of erythrocytic activity arid de- four months without further exposure, these pression of myelocytic activity. same changes were observed. Bone marrow Vapor Concentration of Cll-C12,50 PI'JI. changes notcd were the same as those ob- Four monkeys exposed seven hours per day, served in rats exposed to 200 p p i i of five days per week, for 90 exposures devel- C11-C*.'. oped diarrhea on the third day of ~spos:;:? TABLE4.-Sig)iijicance of Growth and Hciirntologicnl Clinitgcs in Rats Froiir Iiiltnlatioin of Test Vnporr * * Blood Element c vs R 57 Day ESpOSUtO PCrlOd c VS A. C ~s.4. C vsD C vs I3 Complete Erposiur Period C vsA. c VSA. Totd leukoc) tes -11s -US -us -1IS -ns - Poly. leol;oc>-tcs +I15 Tot31 I>mphocytes -119 ss +[IS h'8 -KIS N3 +rrs NS -rw -8 Anunnl welght I 8. - - Y 215 xs +RS -11s -a m : POW1 A. C & I D Jlkyl m m n l t c fractlon (filb ppm) -s: PO01 d, C I I - CI ? d k y l aroinxic fracilon (500 ppm) -NS: PO 03 C Cant rol animals + -Increnss In functlon B Benzene exposed nnbiinls (1.mppnr) - -Decreue in luncrlon D N-decuna exposed anuiinL (510 ppro) -11s SS s3 413 A r c h Enziron Hcallh-Vol 12, .\larch 1966 NO Siurtwnp AolmaLs c VI D +us SY NS +I15 I I ,! ! _-Tmi. , S.-.-i!clttc l.mC, br Rats Weighing 150 G7n Each (96 Hours Waiting F't?riod) ~_c_ 'rc3:3i N O o t No. 01 Rats per Exposure PPM Deaths in SIate1ki1 Rats t;Sl!li Each Test Uroup Time (Hr) v;v XO. of ErcIurs Benzeufi c r C10 c-._-_1_1-c1 x 70 70 70 ~ 10 10 10 +. and irritation of the face and eyes. There was a reversal o f the polymorphonuclear- .lymphocyte :ratio, a stimulation of erthro- cytic activit]. of the bone marrow, and a depression o I the myelocytic 3.ctivity. lrzhalatiort :;tictiits Usi~lgh'-Dccarre.-N- Decade: Vapor Concentration of N-Decane, 5.10 ppl1: ]X;lts exposed to this concentra- tion of n-decane by inhalation 18 hours per day, seven days pel- week showed a signifi- cant positive effect on the expected weight gains and a sipificant fall in the total \vBC aiter 57 days o f exposure. After 123 days of exposure there was still 2x1 increase in the expected Lveight gain and also an increase in the total WEC. There was no change' in the polyinorphonuclear-l~mphocyteratio at either of these times nor were there bone marrow changes o r gross or microscopic organ changes of significance. Some rats set asid? without additional exposure for one month showed no changes from the normal. Some of the heinatological findings in rats exposed to the various materials nsmed are shown in Fig 2, A - F , and Tables 3 and 4. It was noted that with benzene all rats which died were found dead at the end of seven hours of exposure (Table 5). With Co-Clo only four were dead at the end of the seven hours of exposure and one more died within 31 hours after the expo- - sure ended. With CII-CU no rats were deah at the end of the seven hours of exposure; one died within 24 hours after the exposure ended, and four more died within 96 hours after =Dosure ended. i 69&9 i o ( i l .7 2iX 4 (71 5 ("1) '. i 713 0L-'( 4 (96) As with rats, it was noted. that with benzene all mice which died were found dead at the end of the 3% hours of exposure (Table 6). With C9-Cl0 four mice were dead at the end of the 3% hours of exposure. Fourteen more died within the five additional mice died within 24 hours after the exposL1re en With Cli-ci3 no mice were dead at the end of the 3% hours of exposure. Xin6. 't. died within the nest four hours after the" .; . exposure ended. Thirteen additional mice , died within 24 houI ended and four additional mice died within four days after the esposure ended. IYith n-decane no mice were dead when the 3% hours of exposure ended. None died within the next 24 hours but four mice , died before the end of the fourth day after the exposure. The data for L C ~ Ofor rats and mice are similar showing the immediate effects o i benzene and the delayed or cumulative ei- fects of Cs-clO and c11-cls(Table 7). .-Recovery of Aromz ' . . -. . of Rats Expose For this study Ce- used in concentratio 2,000 ppm ppm for b for en-- ~C-e--CTI'1 animal \vas iso-octane a the Beckmail uLl A review of tl . ...,conce,n.tration of thes i c i1 f 31 $ Benzene c*c I O CII.CI1 I .N-docaoe it . ;..-,;,- 33 10 PllU 3 % 13.100 50 10 Plus 3 ? j 1.'Joo 52 10 Plus 3 ?5 528 2s 10 plus 3 !Q 30 Benzene Cr Cio c I 1-c11 Rats 1 1.5 5.0 ' Arch Environ Hsaith-Val 12, M a r c h 1966 Mce 1 4.2 14.0 - 390 CrCJs FRACTIONS-AJAU ET A L - very rapidly after the exposure by inhalation ends. It was possible to recover from 90% to 100% of these aromatics in the blood. Skin : Hyperkeratosis Atrophy of epidermis Inflammatory reaction Perikeratosis Ulceration' 31% 24% 417~ 2370 23% Lungs : Focal hemorrhage 12% Skin Application Pigmentation 10% Inflammatory reaction 16% Benzene-C.P., Co-C1o, C11-C12, and n-decane-CP were applied individually to the skin of 20 male CSH mice, using a onefourth inch brush and making one stroke up the middle of the mouse's back from the base of the tail to the base of the neck. About 0.10 to 0.15 gm of material was applied three times each week on alternate Atelectasis 7% Spleen : Amyloidosis 57% Liver : Focal. areas of necrosis 7% Amyloidosis 3.5% Kidney : Cortical scarring 35.5% Sclerosis 27 7% Necrosis of renal papilla 12% 2. Hematology-WBC increased gradually until at termination. it was double the initial count. days. About 150 applications were made per mouse. The mice were observed closely for detection of any gross changes from the normal, and at the end of one year they were killed. A postmortem evaluation was done and all organs and tissues were examined microscopically. T h e ratio of lymphocytes to polymorphonuclear leukocytes was Lariable but there was a relative increase in the number of polymorphonuclear leukocytes. C. C,,-C,. fraction-S5 mice (16.79 ~ aIppIlied per mouse) 1. Pathology ( a ) Gross-skin thick. dry and scaly. Higi: Our findings were as follows: A. Benzene" mice (11.01 g m applied per mouse) 1. Pathology ( a ) Gross-no detectable changes : . ( b ) Microscopic - . Skin : Dermal fibrosis Edema Pi,mentation -10% 20% 20% Hyperplasia 10% i. Kidney: .. .' Lungs : 42. Focal chronic inflammation Bronchopneumonia and bronchitis 5% 5% Spleen : Lymphoid hyperplasia Lymphocytopoiesis Metaplasia Pigmentation red pulp 2. Hematology-no significant changes 10% 5% 20% 5% from the "normal" total W B C differential count were noted. incidence of lobulation of the kidney (less than with CO-C,). ( b ) Microscopic Skin : Hyperkeratosis Perikeratosis Atrophy of epidermis Inflammatory reaction 30% 16% 29% 23% Ulceration Kidney : NO significant findings 6% Lungs : Atelectasis 37 Focal hemorrhage 75. Liver: Pigmentation Inflammation Necrosis 17% 17% 16% Spleen : Amyloidosis 7% 2. Hematology- the IVBC increascrl with in- creasing amounts oi inaterial applied. The "poly" count increased slightly as p3intin.c was started, dropping back to its initial value and remained constant as the painting \vas continued to termination. 3. Expected weight gain-was ilnpairetl. 3. Expected weight gain-no effect B. C-C,, fraction-79 mice (10.62 gm applied per mouse) D. N - d e c a n e 4 5 mice (16.33 grn applied per mouse) 1. Pathology 1. Pathology * ( a ) Gross-skin thick, dry, scaly. Higli in- cidence of lobulation oi kidney. ( a ) Gross-skin thick, dry, scaly. NOother changes. ( b ) Microscopic ( b ) Microscopic Skin: Fibrosis of dernlis -13% 2 ,' Material supplementary to this article (Tahlcs 8.13 and I:i< 3 and 4 ) has been dcpositcd as Document number 8721 with the A D 1 Auxiliary Publications Project. Photodwlicatiori Service, Library of Congress. Washtnnon 25, DC. A copy may be secured hy citing the Docunlent number and by remittha f2.50 for photoprints. or $1.75 lor 35 mm micro- film. Advance payment i s rcquircd. M a k c checks or nrorley orders paynhle to: Chief. Photoduplicattun Service. Library o f Cot1gcss. Kidney : Pigmentation Ulceration Hykerkeratosis Perikeratosis Hemorrhage Pigmeritation Intta~iiitiat ion 29% lS% 7% 7% 33% 33% 1S% Arch Enziron Health- Vol 12, ~Ifarrh1966 - C r Ctt F R.i CTIO.\'S-hTA U ET A L 391 . Spleen : Amyloidosis 10% material by the formation of fibrous tissue. Lungs : Hemorrliage Pigmentation Intianitnation Xecrosis 55% 24% 2870 115'0 2. HetnatologyTNo significant changes from the normal were observed. 3. Expected weight gains-no effect. E, Controls-764 mice (no chemicals applied to the skin) 1. Pat110l0gy (a) Gross-no detectable changes ( b ) Microscopic (757 niiimals read) Skin : Dermal fibrosis Edema Pigmentation Hyperplasia Hyperkeratosis Atrophy oi epidennis Intinmmation Perikeratosis Ulceration 3.170 1.4% 0% 0.7% 1.670 0.9% 0% 070 070 Kidney: Cortical scarring Atelectasis Amyloidosis Focal chronic inflammation 3.8% 0% 0% lS70 Lungs : Bronchopneumonia Bronchitis Focal hemorrhage Pigmentation Inflammatory reaction A te1ectasis Spleen : Lymphoid hyperplasia L>mphocytopoiesis Xetaplasia Pigmentation red pulp Amyloidosis 4.5% 0% 07 0 0% 0% 0% 3.4% 0% 3.5% 3.7% 1.7% Liver: Amyloidosis 0.5% Necrosis 070 2. Hematology- the WBC in nonexposed niicc increased with increasing age (over 3 period - of nine months). During this period there was no change in the differential &tint. 3. Expected weight gain-normal. There may be an increase or a decrease in the total WBC count, or no change. The same effects were noted in rats injected F' subcutaneously with cottonseed oil alone./ .- Thirty male IVistar rats weighing 300 gin each were injected daily subcutaneously in the right and left groin for 14 successive days using an equal mixture of C&~O and coLtonseed oil with each injection (0.3 cc). At the end of the eighth day (tqtal injection being 2.4 cc of the misture) two rats were killed. The IVCC, hematocrit reading, and weight were determined before injec- tions were started and when the aniinals were \. killed. There was a slight increase in the :. .( hematocrit evaluation of the rats killed and some increase in the WCC. This latter may have been the result of the irritation and inflammation. At the end o i the 14th day of the injection (total injection being 4.2 cc of the mixture) five more rats were killed. Again there was an increase in the IYEC and the detection of 17.S ppm of C9-Clo in the rats' blood. -.< Injections were terminated after the 14th day. Rats were killed on the I&h, Z n d , 31st, 37th, and 46th days. The WEC was increased in each case and the femur of the rats, killed on the lSth, 2211~1, and 31st days, was found to be fragile as compared to the femur of control rats. For a period of time the f u r of the experimental rats appeared coarse and discolored. A return to normal was noted as the last rats were killed. I t was also noted that after the second or third day of injection the rats developed a urethral discharge which cleared up after two o r three days. The urine in Subcutaneous Injection of Rats W i t h C8-CI0 the urinary bladder appeared clear and a routine urinalysis of bladder urine revealed T h e subcutaneous injection of 2.5-5.0 cc of Co-Clo suspended in an equal amount of cottonseed oil into rats (Tables 11 and 12)t produced results which were not consistent. Often there was a "slough" formed at the site of the injection. This may have been preceded by estensive inflammatory changes and an effort to "wall ofT" the injected t Tables I t . 12. and 13 have been omitted and n i l 1 k dcpodcd with the AD1 Auxiliary Publications Project. no significant abnormality. Also, the expected weight gain of the injected animals was slightly impaired. T h e amount of CO-Clo found in the rats' blood as injections were continued did not increase. This map have been due to an effective "walling off" as a result of the irritation, or the developnient o i a method of detoxification, or of the metabolism of the injected material. Arch Enairon Hcaltir-Vol 12. Jfarck 1966 p : ~ ~s ]c~l:i,:h:e~ iriji.:cted i-:ia.terial were I .o:~ t div1ie::i E: osi mor :xns \vert lone. h 1ir';:er dost: ctf an q u a l m i s t u r e : of Ca-C c, x > t icoi.tonsc:ed oil \vis t heti injected SmbcLt inwLisIy in 12 male 'iV'star rats weigiiiri;; :!Z~-Z!CI p i . .A tctal of 2 . 5 cc of tlhe nirxture was injected ~daily. Four rats were I.:il't:d 24 hours after 5 cc of the mixture -,v;isinjectec ; four ivere killed 24 hours Lh.ing<bj noted aboie were rrore evident. Stimulation of the myelocj-tic selies was revealed by an intrease in the number of ( a ) "stabs", ( b ) Juveniles, (c) myelocytes, and ( d ) "blast" forms. There was also a more significant reduction in the normoblast. This was comparable to the findings in rats inhaling C D - ~ I O . a f t e r ,7.5 [cc were injected: arid four were killed 21 hours after 10 <:I: o f the mixture were injected. All rats injected showed an increase in the 'iVI3C prior to killing. There ivas a loss in weight of t l x last group of four killed. ontj group of four rats killed showed some increase in the blood level for C&LO. This \.;ilut: fell sipificantly in the blood of the tliirti group ~f four. DXA values f o r these rats were significantly increased as compared to control rats (Table 11). This experiment was repeated using 16 Odor Detection by Humans The determination o i odor detection \vas done in the Laboratory of Industrial Hygiene, Eastman KOdak Company, Rochester, NY, using the followiny method. X total of 1 cc C9-Clowas diluted with 100 cc of methyl alcohol, which dilution was then put into an air blender. The air flow through the blender was varied until a definite odor was perceived by the observer. C9-Clo has a minimum identifiable odor level of 11 nig/cu in. Assuming a molecular weight of 125, this level o i odor has a female \Vistar rats weighing 310 ,sm. I t is concentration of 2 ppm. Similarly, the noted that there \vas in the injected rats: minimum identifiablz concentration o i 1. --Inincrease in the \VBC 2. --Idecrease in hematocrit values 3. Blood levels ior CD-Cl0in the injected animals reached a peak of 11.87 pprn anti then fell off even though injections yere continued. This, as previously suggested, may be due to an effective walling off or an effective method of C11-Ci2was found to be 2.5 mg/liter or 0.5 ppm on the basis of an assumed mo- lecular weight of 150. The minimuni concentration which produced lacrimation and/or irritation of the eyes, skin, and niucuus surfaces in humails was found to be 25 ppm for CD-Cloand 19 ppm for Cll-C12. r;;e'tabolisin (Table 13). Conclusions IYhen rabbits or monkeys were injected subcutaiieously with an equal mixture of cottonseed oil and Cg-Clo (20-SO cc) there was a distinct fall in the IVBC (Table 13). \Lre believe that, under the conditions of our experiments, with the animals used the following conclusions are warranted: 1. The inhalation of Ce-Clo and Cll-C1l' bIicroscopic bone inarrow studies re- alkyl aromatics in n concentration o i 3.2 vealed that: mg/liter (1,000 ppm for benzene, 616 pprn 1. IVitli the smaller ninounts of Cg-Clo for Cg-ClO, and 500 ppm for CI1-Cls) leads injected repeatedly subcutaneously to significant changes from the normal in - there was a stimulation of the myelo- rats (Table 4), but does not produce the cytic series reflected by an increase in myelotoxic effects characteristic of benzene. the tuyelocytes arid "hlast" cells. There 2. The inhalation by monkeys of 50 or - was evidence of a depression of the 200 pprn o f C&lo or CI1-CLffor 90 expo- ; ? erythrocytic series as reflected by a re- sures of seven hours each led to skin e: duced number of normoblasts. irritation. n reversal of the polymorphoriu- 2. \Vith the larger amounts o i CY-CIOin- clear-lyinphocyte ratio, depression oi the jected repeatedly subcutaneously the myelocytic and stimulation o t the erythro- :... .i.. -.~> Cs-Cm FRACTIONS- NAU ET AL 393 cytic activity of the bone. There was a pro- liquid hydrocarbon fractions be carefully nounced early tremor and a fall o f the W B C avoided by the use of protective shields and in those monkeys exposed to Ce-Cio. clothing. 3. T h e inhalation of n-decane in a concen- tration o f 3.2 mg/ liter (560 PPm) leads to significant changes from normal insofar as total J\'BC and expected animal body weight p i n s are concernea. The blood changes are in the opposite direction to those produced by benzene, Co-Clo and C I I - C I ~(Table 4). 4. The ratio of the acute inhalation toxi- S u m m a r y of Findings .. -O n the basis of our studies the alky1 / aromatics present in these catalytic reform- ate fractions do not evert any myelotoxic effects characteristic of benzene. Exposure. to these compounds by inhalation does lead city in rats (LCao) for benzene, CQ'ClO, and to significant hemotologic changes which, Cll-CI3 on the basis of equivalent Exposure times is 1:1.5:5. I n mice this ratio is 1: 4.3:11. 5 . The threshold of odor detection by humans is 2 ppm for C Q - C ~aOnd 0.50 ppm f o r CI1-Cl2, whereas the minimum concen- tration producing irritation to the eyes and mucous surfaces in humans was 25 ppm for Cg-Clo and 19 ppm f o r C11-Cn. 6. These aromatics can be recovered quan- however, are reversible if the exposure is terminated early enough. I n terms of acute vapor exposure, the f ractiori richer in higher molecular weight aromatics is more toxic than the lower molecular weight aromatic 3fraction, but both fractions are more toxic than benzene or normal decane. '3 .e Percutaneous exposure repeatedly to these . aromatic fractions may lead to significant titatively from the blood of rats inhaling systemic changes as well as to .ulceration thern o r into which subcutaneous injections and atrophic changes of the skin. have been made (using a 1:l mixture in cottonseed oil). 7. These materials disappear rapidly from the blood of a rat which had ceased to in- hale them. . 8. Skin application of CQ-ClO,or Cll C12, leads to pathological changes in the skin and Exposure of the eye t o vapors of these fractions is irritating; liquid contact may lead to scar formation. Prolonged exposure may lead to the formation of cataracts. "hi5 study was supported by the Medical and Health Committee of the .%merican Petroleum Institute. internal organs of greater intensity and se- REFERENCES verity than that produced by benzene or normal decane. I 9. T h e results of subcutaneous inoculation of these materials is not consistent with the inhalation toxicity or percutaneous toxicity of the same compounds. 10. It is suggested that a tentativ concen- \tration of 50 ppm for the CB-Clo a k y l aro- matic fraction and 25 ppm for the CI1-CE alkyl aromatic fraction of the approximate composition used in this study be used as T L V values for eight hour inhalation exposures. Naphthas of greater or lesser aromatic content might be assigned correspond- 1. Wolf, 3I.A.. et al: Toxicological Studies of Certain Alkylated Benzenes and Benzene, -4rch I#dirdr Health 14 : 387-398. 1956. 2. Werner. H.W.: Dunn. R.C.. yon Oettingen, W.F.: The Acute Effects of Cumene Vapors in Mice. J Indusfr NSg To.? 26:?61, 1911. 3. JIallery. T.B.; Gall, EA.: m d Brickley, W.J.: Chronic Exposure to Benzene: 111. The Patliolonic Results. J IitduJtr H y g Tor 21:335. 1839. 4. Goidwater. L.J.. and Tweksbury, U.P.: Recovery Following Exposure to Benzene. J I d u s t r H y g Tor 23 :217. 1941. 6. Svirbely. J.L; Dunn. R.C.; and von 0etting.cn. W.F.: The Acute Toxicity of Vapors of Certain Solvents Contain- ing Appreciable .*mounts of Benzene and Toluene, 3 Zmhutr Hyg Tor 2 5 3 6 6 . 1913. 6. Svirbely. J.L; Dunn, R.C.; and van Oettingen. W.F.: The Chronic Toxicity of Jloderate Concentrations of Benzene and oi Mixtures of Benzene and Its Homologues for Rats and Dogs. J InduJtr H y g Tor 26:37-46. 1944. 7. Gerarde. H.W. : Toxicological Studies on Hydrocar- hnns: I. -% Method for the Quantitative Collection of Femoral