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fly AN D APPLIED PIiARMACOL0GY 54, 32.3-331 (198 bs Archie R Unit me Inhalation Toxicology of Benzene: Incidence of Hematopoietic Neoplasms and Hematotoxicity in AKR/J and C57BU6J Mice CARROLL A. SNYDER, BERNARD D. GOLDSTEIN, ARTHUR R. SELLAKUMAR, ISABEL BROMBERG, SIDNEY LASKIN,A' ND ROY E. ALBERT Y York University, In,sritut,e of Envirclnm!entalMedrkine, 550 First Avenue, New York, Nerv Y#ork 10016 ceived Septt?mber25, 1979I. accepted February 26, 1980 The Inhalation Toxicology of Benzene: Incidence of Hematopoietic Neoplasms and Hematotoxicity in AKWJ and C57BU6J Mice. SNYDER, C. A., GOLDSTEIN, B. D., SEL- LAKUMAR, A. R., BROMBERG, I., LASKINS, . , A N D ALBERT, R. E. (1980). Toxicol. Appl. Piiurmacol. 54, 323-331. AKWJ mice and C57BU6J mice were given lifetime exposures i io0 and 300 ppm benzene, respectively. Peripheral blood cell counts were obtained bi- Ncrkly throughout the exposures. Anemia and lymphocytopenia were produced in benzene-exposed AKR mice. Twenty percent of the exposed AKR mice developed bone marrow hypoplasia, compared to 2% for the controls. The benzene exposures did not alter the incidence or induction time of the viral-induced lymphomas commonly seen in AKR mice. In C57BL mice, exposure to benzene produced anemia, lymphocytopenia, and neutrophilia accompanied by a left shift. Thirteen (33%) of the exposed C57BL mice developed bone marrow hyperplasia and in four of these mice, hyperplasia was essentially limited to granule 4 poietic elements. None of the control C57BL mice developed bone marrow hyperplasia. In benzene-exposed C57BL mice there was a significant increase in the incidence of hematopoietic neoplasms including six cases (15%) of thymic lymphoma. Although two control nwe (5%) died with lymphoma neither of these tumors involved the thymus. Thymic iirnphoma is rare in C57BL mice but can be produced by ionizing radiation and chemical carcinogens. I. Human exposure to benzene has been asso- tory (Snyder et al., 1978a) there appear to dated with pancytopenia and its variants be no reports of the experimental produc- [&soy et al., 1971, 1972, 1976; Mallory tion of benzene-induced myelogenous leu- tf a!., 1939; Pollini et al., 1969; Saita and- , kemia. Inhalation studies have, for the most woli, 1954) and acute myelogenous.' 'part, been of short duration and/or have in- b e r n i a and its variants (Aksoy et al. ,' volved acute or subacute exposure levels 1976;Saira and Vigliani, 1962;Tareeffet al., (Boje et al., 1970; Deichmann et al., 1963; The cyto- Jenkins et al., .1970;Nau et al., 1966; Uyeki haveb been et al., 1977). In addition,'a greatideal of reproduced in animals, most often previous work has produced confusing and by Parenteral injection (Gerarde and Ahl- . often contradictory resuits regarding both 1966; Latta and Davis, 19141;Miniai, the :openic andI proliferative effects of elling, 1916; Weiskotten et al., 1916). ben .e .e (Laskin" and- :-Gold- for work- perf,o~ir.lm,l re, tdrt in 61% , tlnis, e l a b 0ra- stei 97 The purpose of our1stu I. Deceased. hematological dose-resp NOTICE: This __ 0041-008X180/0803 copyright 0 1980 by . -SNYDER ET AL. from lifetime, mo Bate-level, in1ialation ex- f o r 2 weeks. Animals were then randomly dist"bubl posures of rodents. Attempts have been into teh and control groups. There were 50 mice made to choose exposure levels that would each of the AKR test and control groups and 40 + .. in each of the C57BL test and control groups, produce significant hematological damage posures were conducted in stainless-steel, dynamicg. but would not 'greatly'shorten survival. In this way, the peripheral blood cell responses of a number of different species and/or strains exposed to benzene vapor could be characterized perhaps leading to a clearer posure chambers (Drew and Laskin, 1973). Test rai amals were exposed for 6 hrlday x 5 daydweek for to either 100 ppm benzene (AKR) or 300 ppm a, zene (C57BL). Sham control animals were to filtered conditioned air in a duplicate ch- during the same time periods as expose: picture of experimental benzene-induced Chamber exposures continued until the last tq hematotoxicity. '. animal died. Any surviving control animals wen The first series of studies involved AKR" then allowed to live out their lives without further& sham exposures. Methods of animal housing mice and Sprague Dawley rats exposed to handling as well as exposure methods includh ~ 300 ppm benzene (Snyder et a l . , 1978b). generation and analysis of test atmospheres WQI This exposure level caused high mortality, as previously described (Snyder et a!., 1978b).Briedy, lymphocytopenia, anemia, neutrophilia, and reticulocytosis in the mice; but only lymphocytopenia in the rats. We now wish to report on AKR mice exposed to 100 ppm and C57BL mice exposed to 300 ppm. The benzene atmospheres were generated by feeding & with entrained benzene vapor into the expo= chamber. Benzene concentrations in chamber air wqc determined every 30 min during the dail! -:xposum using an ultraviolet spectrophotometric teimique. Animals were clinically observed daily for evidew severity of the peripheral blood cell responses generated by exposures of AKR mice to 300 ppm prompted the lifetime study of morbidity. Weight determinations were performed weekly-for the first 4 weeks and biweekly them after. of these mice at 100ppm. The C57BL strain was chpsen to determine if AKR mice were Blood Parameters unique or if another mouse strain would also be more susceptible than rats to the hematotoxic effects of benzene. A further purpose was to study the effect of chronic Venous tail blood from 10 test and 10 control m h of each strain was used for all determinationc. Test ad control animals were bled every other H -::. on th same days within a period of 1.5 hr tc mi- benzene exposure on the lymphoma incidence in these strains. The AKR strain carries a virus that spontaneously produces a high incidence of lymphoma and kills most of the affected mice within 1 year differences caused by handling, circadian rhythms,eY. o d y males were used in the study in order to a v d anomalous white counts due to estrus. In the CT]BL study, as mortality increased, mice from each (either benzene exposure or air exposure) were CY? into the respective groups of mice undergoing the b. (Kahn and Novak, 1973). The C57BL strain carries a virus that has been shown to yield a high incidence of lymphoma following exposure to radiation, carcinogens, and immunosuppressive agents (Kaplan, 1967;Igel weekly blood cell monitoring so that throughout md of the study there were always 10 mice in each o r b test and control groups used for blood cell C O Y Red cell, white cell, and white cell - ! , f f e r c d counts were performed as previously desc- 4(sal. der et al., 1978b). Individual absolute neutroPbJ et al., 1969; Imamura et al., 1973). and lymphocyte counts were determined by multiPb ing the percentage of mature cells of each t Y P 5. the differential count of a given animal by the td METHODS white cell count for that animal. Exposure Pathology Male, &week old, AKWJ and C57BU6J mice (Jackson Laboratories, Bar Harbor, Maine) were quarantined and observed for anomalous behavior Tissues routinely sectioned from animals dying @ad ing the study included lung, bone marrow, as well as liver, spleen, kg all abnormally -.?Pd BENZENE-INDUCED NE(IPLASIA AND TOXICITY 325 V- 5 9 13 17 21 25 29 33 37 Weeks After first Exposure FIG. 1. Lymphocyte counts of AKR mice. An asb k denotes lower by statistically significant dif- b n c e s (22 SE). w s . These sections were fixed in formalin, ema d c d in paraffin, sectioned at 5 pm mounted on slides Od stained with hematoxylin and eosin. All slides were reviewed by the same pathologist. RESULTS AKR Mice at 100 ppm The chronic exposure of these mice ended after 505 days with the death of the last lest animal. There were a total of 337 expo- ws at an integrated mean benzene conCentration of 100 ppm. There were no statisticalij .ignificantdifferences between test &d control mice in median survival (39 and (I weeks, respectively) or in rate of weight Ph. Hematological monitorings were susended after 37 weeks because too few aniU S remained due to advancing mortality. Statistically significant2 lymphocytopenia observed in treated mice vs controls d e r I exposure week and this trend con: hued tl:;oughout the exposure (Fig. 1). Lymphocyte depressions in exposed mice, bowever, were not as severe as those obw e d in AKR mice exposed to 300 ppm et al., 1978b). After the start of exposure red cell levels of treated animals consistently depressed relative to t Z?standard errors ( 2 2 SE) is the criterion used **tatistical significance in the differences between hlood counts of test and control animals. 02d I 5 9 13 17 21 25 29 33 37 Weeks After First Exposure FIG.2. Red blood cells of AKR mice. An asterisk denotes lower by significant differences ( 2 2 SE). controls and these depressions were statistically significant at 9/19 monitoring periods (Fig. 2). Test mice showed some tendency toward neutrophilia throughout the exposure, however, neutrophil levels were elevated by statistically significant amounts in only 3/19 monitoring periods (Fig. 3). Little or no morphological differences were observed in the peripheral blood of test and control animals. Malignant lymphoma was confirmed in 29/49 treated animals3 and in 24/50 controls. Disseminated, lymphocytic lymphoma with thymic involvement was the predominant type of lymphoma found in both exposed and control mice. Of the 29 exposed mice found with lymphoma, 15 exhibited lymphoma cell infiltrates in the bone marrow, One animal was removed from the at risk pool after it was found outside of its living quarters. 10-3 8- 5- 6 - Benze+n,e, , ** . A; ; ;*i0 I3 I7 5; 3; 3!7 WeekAfterFnstExpOw~ FIG.3. Neutrophil counts of AKFt mice. An asterisk denotes lower by statistically significant differences ( 2 2 SE). 326 SNYDER ET AL. ....8 .88 Test .8 .8 Control 8 . Most of the animals in both exposed control groups developed respiratory infR, tions (bronchitis, pneumonia) and sOm showed liver necrosis but it is difficult b determine whether these conditions wen 08 the cause of death in each case. C57BL Mice at 300 p p m Weeks After First Exposure ._ FIG. 4. Comparison of cumulative mortality between C57BL mice exposed to 300 ppm benzene and air controls. Only every other datum point is shown. 7 were found with hypoplastic bone marrows, and 7 were found with essentially normocellular bone marrows. Of the 24 control mice found with lymphoma, 5 exhibited lymphoma cell infiltrates in the bone marrowy l was found with bone marrow hypoplasia, and 18 mice were found with essentially normocellular bone marrows. Data on lymphoma occurrence was analyzed by the log rank method (Peto, 1974; Pet0 and Pike, 1973; Pet0 et af., 1977)which allows for comparison of tumor incidence and tumor induction time between two or more groups corrected for survival. No statistical difference in lymphoma type or occurrence was found between exposed and control AKR mice. Bone marrow hypoplasia was found in a total of 10 treated mice (20%) but in only one control. Bone marrow hypoplasia in these mice was manifested by a diminution of all formed elements, and the appearance of a few clasmatocytes and areas of hemorrhage. The difference in the incidence of bone marrow hypoplasia found in exposed and control mice was significant at the p < 0.05 level (xz = 4.81). The 20% in- cidence found in animals exposed to 100 ppm is substantially less than the 81% incidence of bone marrow hypoplasia found in AKR mice exposed to 300 ppm (Snyder et al., 1978b). The chronic exposure of C57BL mice Wu completed after 488 days with the death o( the last test animal. There were a total 4 331 exposures at an integrated mean COS centration of 300 ppm. After the start exposures treated mice had a median SUI. viva1 of 41 vs 75 weeks for controls (Fig.11 and showed weight gain depression reiatibx to controls throughout the stud: 'Fig. $1. Acquisition of peripheral blood data uxc suspended after 61 weeks because too fer animals were alive in the test group. After the start of exposures, test m i a exhibited a statistically significant degree d lymphocytopenia in 29/30 monitorin4 periods and a statistically significant degm of anemia relative to controls in 30i30 m o 6 toring periods. These differencs beafter l exposure week and . m i n d throughout the course of the study (Figs.6 and 7). Neutrophilia was evident in 1H mice vis-a-vis controls after 17 weeks the degree of neutrophilia was statistic* 160 $1 '8 8 Weeks After First Exposure FIG. 5. Comparison of percentage weigh: =han@* tween C57BL mice exposed to 300 ppm -;Xnide air controls. Only every other datum poifi :' She" BENZENE-INDUCED NEOPLASIA AND TOXICITY 327 *ific :t in 15of the 22 remaining monitoripB periods (Fig. 6). AS exposures progressed, qualitative w g e s were observed in the peripheral cell morphology of test animals. Mjsocytosis appeared after 4 exposure cceks and poikilocytosis began to appear a e r 15 weeks. Hyperlobulated, mature Jrutrophils were observed and a neutroe i c !t -* shift appeared concurrently with ne:. rophilia. The left shift was sigfzled, at first, by the appearance of elevated kvels of metamyelocytes and progressed in &tensity until, by the end of the first year, myelocytes and promyelocytes began to rppear. Giant platelets also appeared by the a d of the first exposure year. Similar changes were not observed in the peripheral blood of control animals. Histc. thological evaluations revealed m increased incidence of hematopoietic mplasms as well as bone marrow and splenic hyperplasia in exposed mice. These Wings are presented in Table 1. The eight exposed animals dying with hematopoietic mplasms were alive for an average of 262 ~ Y afSter the start of exposure (first, 213 bays; last, 344 days). Six of the exposed mice die?, with lymphocytic lymphoma, one died wi... plasmacytoma (myeloma), and I I I I III 20 40 60 80 WeeksAfterFintExpoSwe 6. WBC of C57BL mice. Neut, mature neutro- %lymphs, mature lymphocytes. Only every-other $turnPoint is shown. 2t weeks After Fint Exposure FIG. 7. RBC of C57BL mice. Only every other datum point is shown. All points after start of exposure are different by statistically significant amounts ( 2 2 SE). one died with a leukemia in which the predominant cell type appears to be a hematocytoblast. Two control animals died after 282 and 608 days with lymphocytic lymphoma. There was thymic involvement in all six of the lymphocytic lymphomas found in benzene-treated mice. In contrast, there was no thymic involvement in the two control mice dying with lymphoma. An actuarial plot of the incidence of hematopoietic neoplasms in both groups is given in Fig. 8. The increase in the overall incidence of hematopoietic neoplasms in exposed mice is statistically significant by the log rank method at the p < 0.005 level, 2 = 9.81. When lymphoma incidence is compared in both groups, the increase in ex- posed mice is significant at the p < 0.01 levels, 2 = 7.06. If only,the incidence of thymic lymphoma is compared in both groups, the increase in exposed mice is sig- nificant at t h e p < 0.005 level, 2 = 9.70. . Comparison of those animals in both groups dying without evidence of neoplasia shows highly significant increases ( p < 0.001) of bone marrow hyperplasia (2 = 16.37) and spleen hyperplasia (2= 15.93) in exposed mice. Of the thirteen cases of bone marrow hyperplasia in exposed mice, four involved proliferation of granulocytic cells while the remainder consisted of a general hyperplasia which, although predominantly myeloid in nature, involved all 328 SNYDER ET AL. TABLE 1 HISTOLOGICAL EVALUATIONS OF C57BL MICE EXPOSED TO 300 ppm BENZENE AND OF AIR S HAM CONTROLS Incidence Test Control 1. Hematopoietic neoplasms 2. Bone marrow hyperplasia without evidence of hematopoietic neoplasm 3. Spleen hyperplasia without hematopoietic neoplasm 8/40 13/32 16/32 2/40 0138 2138 formed elements. The splenic hyperplasia was due, predominantly, to ectopic hematopoiesis. Respiratory infections, especially pneumonia, were found in nearly all exposed and control mice, however, it would be difficult to pinpoint the exact cause of death in each case. DISCUSSION The peripheral blood cell effects observed in this study are qualitatively similar to those reported in the previous study (Snyder et al., 1978b). At 300 ppm, some cytopenic effects have been produced in all animals (Sprague-Dawley rats, AKR mice and C57BL mice). At this concentration, all animals exhibited peripheral lymphocytopenia and the mice also exhibited anemia. At 300 ppm, the AKR mice were more sensitive to the peripheral cytopenic effects of the exposure to benzene than the C57BL strain. Sprague-Dawley rats, on the other hand, were less sensitive to the effects of benzene than either mouse strain, exhibiting milder lymphocytopenia and only a trend to anemia. The AKR mice gave evidence of a dose-response effect since the degree of lymphocytopenia and anemia at 100 ppm were not as severe as those observed at 300 ppm. At 300 ppm, the red cell levels of these animals declined to 6 x 106/mm3whereas at 100 ppm, the levels were about 8 x 106/rnrn3.Likew:>e, lym. lmphocyte counts declined to less than mm3 at 300 ppm but only to about 3000 mm3 at 100 ppm. Nevertheless, the cyto. penic effects observed at 100 ppm do repR. sent statistically significant differences from control peripheral blood cell values. Neutrophilia has been produced in AKR and C57BL mice following to 300 ppm benzene. A dose- -2bponx effect is evident in the AKR strain since the neutrophilia was not as marked at 100 ppm as at 300 ppm (Snyder et af.,1978b). In view of the cytopenic effects of the ex. posures on peripheral lymphoid and eq. throid cells, the proliferative effects on the granulocytic cell line represent a sharp cob trast in response. To our knowledge, then has been only one report of bezene-induced myelogenous leukemia 1 - dnimals (Snyder et a / . , 1978a)even though there is a direct association between benzene e x p sure and myeloid leukemia in humam (Laskin and Goldstein, 1977). The production of these granuloproliferative responses in these exposed mice may be meaningful, especially in view of the concurrent lefi shift and myeloid hyperplasia observed in exposed C57BL mice. There is a marked contrast in i.iS effect5 of benzene exposure on the occurrence lymphoma in the AKR and C57BL StrainsAt 300 ppm, the survival of the AKR mice Intervals (days) BENZENE-INDUCED NEOPLASIA AND TOXICITY 329 drrbs enict.faedcetsqoufatbeentozenaleloown an evaluation lymphoma in- &,-tion. Median survival of 300-ppm- treated animals was only 3 months (Snyder 1978b). In the 100-ppm study, median gpivd of treated AKR mice was 39 weeks d i c h is almost identical to our control and rcpofied control values (Kahn and Novak, 1973). No effects on the incidence or type d lymr'?oma or on induction time was d d e n i :owever, in AKR mice exposed to 100 ppm benzene. On the other hand, there was a significant *ease in hematopoietic neoplasms, par- tjcularly thymic lymphomas, in C57BL &e exposed to 300 ppm benzene. This in- qease occurred despite the shorter survival exposed mice compared to controls. Thymic involvement was evident in all six cases c-' :ymphocytic lymphoma found in c.poseG .nice but was not evident in either case of lymphocytic lymphoma in control mice. Thymic lymphoma usually develops kt C57BL mice in association with a virus following exposure to ionizing radiation, chemical carcinogens (Kaplan, 1967) or the hmunosuppressant azathioprine (Imamura 11 al., 1973). In contrast to AKR mice, rpontaneous thymic lymphoma is ap- parentl: --are in C57BL mice (Dunn and &ringer. 1968). The depression in circulating lymphocyte b e l s observed in these studies may in- t h e that benzene is an immunosuppres- a t . The neoplasm incidence and induction h e produced in C57BL mice by chronic aPosure to 300 ppm benzene closely re- vmbles those produced in these ani- as foi'l.wing chronic administration of ne (Imamura et af., 1973). This mY strengthen the argument that a reduc- tion in immune competence may be a re- quirement for the production of lymphomas a these animals. CS7BL mice have been the subject of a uudy by Ward et al. (1975) in which ben- he was administered subcutaneously over a %Week period. The hematopoietic' neo- plasm incidences of control groups in this study differ from the findings of our study. In the Ward et af. study, hematopoietic tumors appeared in both the benzene-injected and control animals in the second year after the start of the experiment and incidences were higher in the control groups (25%, 5/20) than in the test group (17.5%, 14/ 80). In addition, Ward et af. reported no incidences of thymic lymphoma in either the benzene-treated or control groups. Thymic lymphoma was produced in a positive control group receiving nitrosobutylurea. The overall hematopoietic tumor incidence in benzene-treated mice in the Ward et al. study is in good agreement with the incidence observed in the benzene-exposed mice in our study. There is a contrast, however, between the low neoplasm incidence (5%) in the control mice of our study and the relatively high incidence (25%) in the controls of the Ward study. The reported spontaneous lymphoma incidence for C57BL mice appears to be quite low. Igel et al. (1969) found a 6% (4/62) spontaneous incidence at 20 mos; Lieberman and Kaplan (1959) reported a 1.4% (1/74) incidence at 600 d; and Myers et af.(1970) found a 0% (0/370) incidence at 7 months. Since the benzene-exposed, tumor-bearing mice in our study all died within a time frame when these various authors (including Wardet af.) report a very low or zero spontaneous incidence, it appears that the benzene inhalation has increased the incidence of hematopoietic neoplasms and shortened induction time. . The difference in route of administration may explain why no thymic lymphomas were produced by Ward et af. in contrast to our own study. Injury to the bone marrow and spleen as well as the thymus is apparently required to produce thymic lymphoma in these animals (Kaplan, 1967) and a systemic distribution of benzene would seem to be necessary to produce injuries to all of these organs. Subcutaneous injection may not produce the systemic distribu- 330 SNYDER ETAL. . 8 tion of benzene that inhalation exposure would be expected to produce. In summary, the results of our two studies indicate that, for a given exposure level, the two strains of mice tested are more sus- aaccrAND,DINCOL, G. (1972). Details Of blood c., in '3i.patients with pancytopenia associated % long-term exposure to benzene. Brit. J . Ind. M~~ 29, 56-64. s.,AKSOY, M.,ERDEM, AND DINCOL, G.(1976). T~ of leukemia in chronic benzene poisoning. A study ceptible to the cytopenic effects of inhaled benzene than Sprague-Dawley rats, and AKR mice are more sensitive than C57BL mice. These mice also respond to the exposures with a neutrophilia, and, in the C57BL thirty-four patients. Acta Haernatol. 55, 65-72. BOJE, H., BENKEL, W., A ND HEINIGER, H.J. (1- Untersuchungen zur Leukopeose im K n o c h e n a der Ratte nach Chronischen Benzol-inhalation. ~1~ 21, 250-257. DEICHMANN, W. B., MACDONALDV,v E., strain, this is accompanied by a strong left . BERNAL, E. (1%3). The hemopoietic tissue toxjcjv shift and bone marrow hyperplasia. Thus, a. - of benzene vapors. Toxicol. Appl. Pharmacal. proliferative effect of benzene exposure on 201-224. DREW, R. T., A N D LASKINS, . (1973). Environmeu the myeloid cell line has been produced and inhalation chambers. In Methods of Animal this is the cell line that undergoes leukemic mentation (I. Gay, ed.), Vol. 4, pp. 1-41. Academic transformation in most cases of human leukemia associated with benzene exposure (Laskin and Goldstein, 1977). The exposures seem to have little or no effect on the spontaneous lymphoma of the AKR strain Press, New York. DUNN, T. B., ANDDEFUNGEMR., K. (1968).Reticulrup cell neoplasm, Type B, or the "Hodgkin's-like k. sion" of the mouse. J . Nut. Cancer Inst. 40,771821. GERARDE, H. W., A N D AHLSTROM, D. i;. ( 1 % ~ but there appears to be an effect, at 300ppm, on the inducible lymphoma of the C57BL strain leading to an increased incidence of hematopoietic tumors with decreased induction times. The production of thymic Toxicologic studies on hydrocarbons. XI. Influeof dose on the metabolism of mono-n-alkyl deriv, tives of benzene. Toxicol. Appl. Pharmacol. 9, 185-190. IGEL,H. J., HEUBNER, R. J., TURNER, H. C., Konn. P., A N D FALK, H. L. (1969). Mouse leukemia Vina lymphoma in C57BL *miceindicates that in- activation by chemical carcinogens. Science 1 6 haled benzene acts on these animals in a manner similar to other tumorigenic agents. 1624-1626. IMAMURA, N., MIMAKO, N., KAWASE, A., KAWA. *MURA, Y., AND YOHOROK, . (1973). Synr.-cistic Y- tion of N-nitrosobutyi-urea and azathic; in ACKNOWLEDGMENTS duction of leukemia in C57BL mice. Ciunn. 64. 493-498. The authors wish to gratefully acknowledge the following individuals who participated in one or more aspects of these studies: Robert G. Kelly, G. Roger Sparling, and Dorothy J. Natalitio. This investigation was supported by Contract U- 15014(PS-7)from the American Petroleum Institute and by Contract NO1 CP 33260 from the National Cancer Institute and is part of a Center Program supported by Grant ES00260 from the National Institutes of Environmental Health Sciences and Grant CA 13343 from the National Cancer Institute. JENKINS, L. J., JONES, R. A., AND SIEGEL, J . (1QI(R. Long-term inhalation screening studies of benzene. toluene, o-xylene, and cumene on expenmend animals. Toxicol. Appl. Pharrnacol. 16, 818-83. KAHN, R. R., AND NOVAK, D. (1973). VariabiW AKR mouse leukemia m0rtality.J. Nut. Canccrlng. 51, 683-685. KAPLAN, H. S . (1967). On the natural histor! of mu* leukemias. Cancer Res. 27, 1325-1340. LASKIN,S., AND GOLDSTEIN, B. D., ec.-.. (19?' Benzene toxicity: A critical evaluation. J . 7xird Environ. Health, Suppl. 2, November. REFERENCES LATTA,J. S . , AND DAVIES, L. T. (1941). 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