Document ppYYE0QBzq75LXY14jLvGYxyw

T h e e f f e c t of inhaled b e n z e n e o n e m b r y o n a l a n d fetal d e v e l o p m e n t was assessed a n d r a b b i t s CF-1 mice a n d N e w Z e a l a n d w h i t e r a b b i t s w e r e e x p o s e d to 0 or 500 p b e n z e n e for 7 hr p e r d a y f r o m d a y s 6 t h r o u g h 15 (mice)a n d 6 t h r o u g h 18 gestation. Little e v i d e n c e of m a t e r n a l toxicity was seen in e i t h e r species. A l t h o u g h somc signs of e m b r y o n a l toxicity w e r e o b s e r v e d in b o t h mice a n d rabbits, a t e r a t o g e n i c e f f e c was n o t d i s c e r n e d in e i t h e r s p e c i e s i n h a l i n g 500 p p m o f b e n z e n e . --ek h E%.++;>- . . . -u~-+G:+&+" c - * -. ,I 1.' : * %.' - Embryotoxicity of inhaled benzene in mice and rabbits F J MURRAY'. J A JOHN, L W RAMPY. 'R A KUNA and B A SCHWETZ Toxicology Research Laboratory, Health and Environmental Research. Dow Chemical U S A , Midland. Michigan 48640. 'Research and Environmental Heallh Division. E u o n Corporalion, Box 45. Linden. N e w Jersey 07036 ...c- ._-*.- . . . ,-, _I -.: I *. introduction Benzene (benzol, G H s ) is used primarily as a ppm of benzene f o r 6 hr per day from days 6' starting material in the producti0n.d a wide variety of organic chemicals. Benzene is also inherently present in some industrial hydrocarbon solvents and in motor gasoline as a high octane component. Exposure to benzene vapors produces central nervous system depression."' Occupational exposure to high levels of benzene on a chronic basis has been through 15 of gestation; however. increasedembryolethality. was noted a t both levels of i,. .;SI: --. -. - . . A, - exposure. The purpose of the study presented herein wa; *'+A ;.?;.-.y& , .;-. -..1;, - * '-. ...+-*-.-i c . to assess the effect of inhaled benzene on embryonal and fetal development in two additional species, the mouse and the rabbit. associated with aplastic anemia,"'6' a n d chromosomal aberrations."-" Several studies have been conducted in laboratory rats to evaluate the potential teratogenicity of inhaled benzene. In o n e study,"" pregnant rats were exposed too, 10,50, o r 500 p p m of benzene f o r 7 h r per d a y from days 6 through 15 ofgestation; a l o w incidence of fetal malformations (exencephaly, angulated ribs, and non-sequential ossification of forefeet) was observed a m o n g the litters of ratsexposed to 500 ppm. suggesting a possible teratogenic effect at that concentration. No evidence of a teratogenic effect was found by other investigators who exposed rats to 300 to 2200 ppm of benzene for 6 h r per d a y f r o m d a y s 6 t h r o u g h 15 o f gcstation."" In a third study."') a teratogenic clfcct was not seen a m o n g rats inhaling 10 o r 40 materials and methods &_---I- - -Rcnzene (industrial grade), identified a s ID95: .. . ' . 01000-0070, was supplicd for Exxon Chemical Company use in this study by, U.S.A.. Houston,. - +~~~.... : . "m-~+ Texas. However, o n at least t w o days ofi*'-**T"T, a- .i .t - - .:exposure, but n o more than 5 days. the test" ': .. -animals were exposed inadvertently to a sample^- of b e n z e n e o b t a i n e d f r o m M a l l i n c k r o d t - . -..-a. -. ".-..r-'-.<'..-- -. v:; -_. ' ,..! . .Chemical Company; the Mallinckrodt was identified as nanograde quality, . .1043B3. - benzene.-. Lot No.: . _. * * :c-. . . . - ...* .. '. . . ., .. ..4 . . . . . , I . . * . ..-. r .;...,' ". Virgin CF-I mice (Charles River, Portage,, . '. Michigan) and New Zcnland white rabbits . . .' (Langshaws Ihhbitry, Augusta, Michigan) were tiouscd in wire-bottomed cages in temperature- ' and humiclit~-controllcd rooms with a 12 hr light-dark cycle. The animals were maintained: on commercial laboratory chow (Kalston Purina Cornp:inl;, St. Louis. hfissouri) and tap' a. ,. . . . ..' * ' .. 1. ., - . 'Currenl address Svrilcx Corporation. Sranford Induslrial Park. \v;ifer ucl [ i b i f u t t r exccpt while i n t h c e x p o s u r e Palo Alto. California 94304 c1i:tnibcrs. hlicc a n d rabbits were allowed at '.::+.-: . ._. --a Copyright 1979. American Indl1:trial Hygiene Atsoclation American Industrial Hypene Associalion JOUHNAL (40) J //79 993 r . ,. least two a n d three weeks, respectively. for c1eare:l with KOH, stained with alizarin red' acclimatization to the laboratory before use. T h e s,""and exsmined for skeletal alterations. day on which a vaginal plug was observed in At the time of sacrifice, blood samples were mice o r the d a y o n which rabbits were bred obtained through a n incision in t h e neck of naturally was considered d a y zero of gestation. individual rabbit fetuses from five different G r o u p s of 35 a n d 37 bred mice were exposed litters of the control a n d benzene-exposed in chambers to filtered r o o m a i r o r 500 p p m of groups. Hematologic determinations were not benzene, respectively, for 7 hr per day from days conducted o n fctal mice since sufficient 6 through 15 of gestation. Concurrently, groups quantities of blood t o perform the evaluations of 20 bred rabbits each were exposed in t h e s a m e could not be obtained. Additional groups of 8 chambers to filtered room air o r 500 p p m of brcd mice and 4 bred rabbits each were exposcd benzene f o r 7 h r per d a y from days 6 through 18 concurrently wiih the teratology-study animals. of gestation. Blood samples were obtained from mice by Exposures were conducted under dynamic decapitation o n d a y 16 of gestation a n d from airflow conditions in stainless steel a n d glass rabbits by cardiac puncture o n d a y 19 of Rochester-type chambers of4.3 m' volume. T h e g e s t a t i o n . T h e f o l l o w i n g p a r a m e t e r s w e r e atmosphere was generated by metering liquid analyzed in adult a n d fctal blood: packed cell benzene at a controlled rate into a heated volume, percent hemoglobin, red blood cell vaporization flask. T h e vapors were then blown counts, and white blood cell counts. into theairstream being d r a w n into thechamber. T h e Wilcoxon test as modified by Haseman Concentrations in the chambers were analyzed and H o ~ ' 1 'w' a~s~used t o evaluate the incidence of daily t h r o u g h o u t e x p o s u r e by alternately fetal alterations and rcsorptions. Maternal and s a m p l i n g t h e e x p e r i m e n t a l a n d c o n t r o l fetal body weights, food and water consumption c h a m b e r s a t 60 a n d 30 m i n u t e i n t e r v a l s , data.. a n d hematologic data were analyzed r e s p e c t i v e l y , u s i n g a M i r a n 1 i n f r a r e d statistically by a one-way analysis of variance."" spectrophotometer at a wavelength of 3.25 In all cases. the level of significance chosen was p microns. T h e mean ( 5 . D . ) of the daily time- < 0.05. weighted average concentrations of benzene in the exposure chamber was 507+12 ppm. Tlie animals were observed daily from d a y 6 of gestation. and were weighed at several intervals during the e..rperiniental period. Food and water consumptions were recorded during the experimental period at M a y intervals for the micc and at daily intervals for the rabbits. On d a y 18 and 29 of gestation, t h e mice and rabbits, respectively, were sacrificed by carbon dioxide inhalation. T h e number a n d position of live, dead, and resorbed fetuses were noted. All fetuses were \reighed, measured (crown-rump length), sexed, and examined for external alterations and cleft palate. One-third of the fctuscs of each litter, selected at random, was examined immediately for evidence of visceral alterations by dissection undcr a low power stereo microscope."" Heads of mouse fetuses s h i c h were examined f o r visceral alterations were placed in Houin's solution and subsequently examined by the razor-section technique ot' b'ilson.''4' All of the fetuses from each littcr of mice and rabbits were results Exposure of bred miceand rabbits t o 500 p p m of benzene for 7 hr per day had no significant effect on the danis' appearance, demeanor, body weight, o r body iveight gain. Food and water consumptions of pregnant mice were not altered by. inhalation of benzene. The a m o u n t of food and water consumed by pregnant rabbits exposed to benzene was significantly greater t h a n that of the controls o n a few days toward the end of the csposure period. Observations made at the time of sacrifice of t h e d a m s a r e presented-in l a b l c 1. lrihalation of SO0 p p m of benienc: did not significantly affect the incidencc of pregnancy in mice o r rabbits. N o significant effect o n the average n u m b e r of live fetuses or rcsorptions per litter w3s disccrncd in either specics. Mean fct;iI body weight but not crown-rump lcncrh bas decreased sisnificantly a m o n g litters of mice cxposed to benzene. I n rabbits, ictal body mcasurements were not altered signif'icantly by exposuru to benzene. 941 Am Ind tfyg Assac I (40) November. 1979 * .' '< I I ! TABLE 1 Observations at Sacrifice of M i c e and Rabbits Exposed to 5 0 0 P P M of Benzene M i c e__- Control Benzene Rabbits Control Benzene No of bred females % Pregnant No of lifters Implantations/dam' Fetuses/litter' flesorptions/litter* Feral body welghr g' Fetal crown rump length. mm' 35 74 26 12t2 Ilt2 1521 4 101~009 24 1 !1 3 37 81 30 13r2 1 1 +2 1 9L2 1 095+010* 2 3 8'1 1 20 90 18 9f2 8+2 05!06 394'57 97 2 *75 20 95 19 9t2 822 O7tl 0 385'50 95 1 -60 * M e a n of litter values :slandard deviation <"Sqnhcanlly different from the control value, p 0 05 The incidence of fetal malformations among noted in a litter exposed to benzene. Fused litters of mice a n d rabbits is summarized in thoracic vertt'brae acconipanied by fused ribs Table 11. A m o n g the offspring of mice exposed u.ere observed in a n o t h e r benzene-exposed to benzene. no major malformation occurred at litter. Another exposed fctusexhibited fused ribs a n incidence significantly different f r o m that of only. N o major malformations were observed the controls. Cleft palate was observed in single among the control group. Two minor skeletal fetuses in both control a n d benzene-exposed variants, i.e., l u m b a r spurs a n d the proportion of groups of mice; in the latter case, the fetus with fetuses with 13 ribs ( t h e normal n u m b e r is 12 o r clcft palate also had asymmetric vertebrae and 13), occurred significantly less often a m o n g the f u s e d r i b s . S i g n i f i c a n t i n c r e a s e s in t h e litters of rabbits exposed to benzene than among occurrence of several minor skeletal variants the control litters. (not considered to be malformations) were seen in litters of benzene-exposed mice, including Table 111 shows the results of the hematologic delayed ossification of stcrnebrae, delayed evaluation following exposure to benzene. I ossification of skull bones, and unfused occipital Hematologic values of adult bred mice a n d bones of the skull. rabbits exposed to benzene were not In rabbits, exposure to benzene did not significantly different from the control values. s i g n i f i c a n t l y a l t e r t h e o c c u r r c n c e of f e t a l N o significant hematologic chrtngcs were evident malformations, when considered individually or among the fetal offspring of rabbits exposed to _._ .... --- collectively. A single case of gastroschisis H ~ S benzene. Hematologic evaluation of fetal mice TABLE II Fetal Malformations Among Litters of Mice and Rabbits Exposed to 500 P P M of Benzene Mlce Rabbits Control Benzene Control Benzene No Fetuses (Liners) Eaamined External Exainination Visceral Examination Skeletal Examinailon' 2791261 97126) 253(24) 325130) 1 12(30) 286(28l 150(18l SG(18l 1421 171 15211 91 591 19) 152i19) Total Maltormattons N o Fetusas (Litera) Aflected l(11" I(?)' a01 313)" 'Due ruproblenis in fiaation S w e r a l mouse ltllers andone rJhb!t h l 1 w were not ciamined for skCletaI alteratibns 'Cleft palate ' t l e t l palate fused ribs and asymmelrlc vrrtc*brne ~n one fetus "Gdstroschisis fused ribs fused ribs and lhoracc vertebrae I I American Industrial Hygiene Association JOURNAL (40) 11/79 I i . . . . --. ....., . ..,... ..,e . ... **.: -I .. I. -.. . .. . - I , 995 I- .i' TABLE 111 Hematologic Evaluation Following Exposure to Benzene Species Mouse. adult' Benzene (pprn) 0 500 PCV 1%) 50-2 48t2 REC Hgb wec ______-___X I O " / ~ ~ ' g/100 mL x l O ' / r n m ' 86104 163?06 7211 84:02 1 6 0 _ ? 0 6 69tl 6 Mouse, fetal nor determined Rabbsr. adult' 0 40t6 5911 1 133t24 69i29 500 39*.4 50+07 127:l 2 58t07 Rabblt. feral" 0 44tb 3 7 ~ 0 4 12Z+l 4 0 6 5 t O l 3 500 47t4 4 1204 139'1 2 O89t032 *Blood was collected on ddys 16 (mice) and 19 (rabbits) o f geslation %load was collected on day 29 of gestation Values are exnressed as the mean L slandard deviaIion ' I. . .c . a. was not performed since a sufficient quantity of blood could not be obtained. discussion T h e results of the present study, as well as those of earlier teratology studies o n inhaled benzene, a r e summarized in Table 1V. In the present study, benzene was not teratogenic in either mice or rabbits inhaling500 ppm ofthe compound for 7 hr per day during the period of major organogenesis. The few malformations which were observed among the hcnzene-exposed litters were types that have been noted to occur spontaneously at a low incidence in previous control groups. In comparison, a possible teratogenic effect was reported in rats exposcd to 500 p p m of benzene for 7 h r per d a y from days 6 through 15 of gestation."" However, in another study,'"' teratogenicity was not observed in rats inhaling up to 2200 p p m of benzene f o r 6 h r per d a y o n the same days of gestation. Although a teratogenic effect was not found, some evidence of embryotoxicity was noted in mice a n d rabbits exposed to benzene. In mice. increased incidences of delayed ossification of skull bone and sternebrae were observed among the offspring of d a m s exposed t o benzene. This effect was associated with a decrease in the average fetal body weight. In rabbits. the only statistically significant evidence of a n effect o n embryonal or fctal devclopment was a decrease in the occurrence of t w o minor skeletal variants: lumber spurs a n d the proportion of fetuses with 13 ribs. TABLE IV Summarv of Results of Teratoloav Studies on Inhaled Benzene Species Benzene lppml Maternal Weight Gain Feral Body Weight Resorptions Skeletal Variants Malformations CF.1 Mouse N 2 Rabbit S - 0 Rat'"' 500 500 10 50 500 DEC DEC DEC DEC DEC iNC INC INC INC INC' S -D Rat"" S-D R J ~ " ' 100 300 300" 2200 lo 40 DEC DEC _- INC INC INC INC (.I = No slgnqftcant difference c o m p a r d l o t h e c o n t r o l value. DEC - dvirrasrd cvmpared to the control value. I N C = ~ n c r e ~ s ecdompJred 10 Ihe coiilrul value 'Possnble reratogrnic eflect (not srat~sticallvsignificant) ?he 300 pprn concentration was tested twlie 9% Am h d H I P Assoc I (.lo) November. 1919 -- , -., -.--e%- ..- I I In rats, increased frequencies of minor skeletal variants (delayed ossification of sternebrae a n d missing sternebrae) were reported in female but n o t m a l e o f f s p r i n g of d a m s e x p o s e d to benzene.'" ' The present study showed significant increases in the incidences of delayed ossification of sternebrae, delayed ossification of skull bones, and unfused occipital bones of the skull in litters of benzene-exposed mice. As in the above study,"" these observations have been judged minor skeletal variations. not to be considered malformations. Investigators have reported an increased incidence of resorptions among bred rats exposed f o r 6 h r per d a y to 10 o r 40 p p m of benzene."l' But, in two other teratology studies'iw"' on benzene in rats, no significant effect o n t h e occurrence of resorptions was seen at levels of exposure a s high a s 500 a n d 2200 p p m of benzene. I n the present study, inhalation of 500 p p m of benzene did not significantly affect the incidence of resorptions in either mice or rabbits. T h e lack of teratogenicity in mice in the present study contrasts with a n earlier study in which cleft palate was observed in three of fifteen liters of mice given a single subcutaneous injection of 3 rnl- benzene/ kg body weight o n day 13 of gestation."" However, it is pos.sible that the cleft palatcs attributed to benzene wcrc actually produced by the strcss of such a massive subcutaneous injection; unfortunately, a vehicle control group was not included in that study. In thc present study, one fetus with cleft palate was observed in each of the control and exposed groups. T h e concentration of benzene tested in this study, 500 ppm, was markedly higher than the c u r r e n t O S H A s t a n d a r d o f 10 p p m . " " Moreover. the difference in dose between humans and animals may be even greater than that indicated by concentration alone. T h e a m o u n t of a i r inhaled per minute by mice a n d rabbits in proportion to their body weight is approximately I O a n d 3-5 times, respectively, that of hunians.""' Thus. the prcsent findings d o not indicate a teratogenic hazard in hunians exposcd to 10 p p m or less of benzene. -~ . In summary, a tcratogenic effect .was not discerned in eithcr mice o r rabbits inhaling 500 ppm of benzene for 7 hr per day during the p e r i o d of ni;ijor or_cnno_crnesis. T h e o n l y evidence of eiiibryotosicity o r t'rtotoxicity seen in this , t i d y was t h e altercd occurrence of some minor skeletal variations, which in mice was asociated with a decrease in fetal body weight. acknowledgments This work was co-sponsored by Exxon Corporation and T h e [>ow Chemical C o m p a n y . references 1 Browning, E.: Toxicity and Metabolism oflndusfrial Solvents Elsevier Publishing Company, N e w York. N e w York (19651. 2. Aksoy. M.. K. Dincol. S.Erdem, T. A k g u n a n d G . Dincol: Details of Blood Changes in Thirty-Two Patients with Pancytopenia Associated with LonyTerm Exposure to Benzene. Brif J Ind. M e d 29:5664 (1972) 3 Aksoy, M.. S. Erdem, K. Dincol. T. Hepyuksel and G. Dincol: Leukemia and Shoe-Workers Exposed Chronically to Benzene. Blood. 44.837-841 ( 1974). 4 Berlin. M.,J. Gage and E. Johnson: Increased Aromatics in Motor Fuels: A Review of t h e Environmental and Health Effects. Work Envjron. tlealth 11.1-20 ( 1974) 5 Saita. G.: Blood Disorders D u e to Drugs and Other Agents. pp. 1 2 7 -1 4 6 Excerpta Medica. Amsterdam ( 1 9731 6 Aksoy, M.. K. Dincol, S. Erdem and G. Dincol: Acute Leukemia Due to Chronic Exposure to Benzene. A m J Med 52.1GO- 166 ( 1 972) 7 Pollini. G. a n d R . Colornbi: Lymphocyte Chromosome Damage in Benzene Blood Dyscrasia M e d Lavoro 55 241 -255 (1964) 8 Vigliani. E. C. and A. Forni: Benzene, Chromosome Changes. and Leukemia. J. Occup M c d 11.1481 4 9 (1969). 9 Forni. A . M.. A. Cappellini, E. Pacific0 and E. C. Vigliani: Chromosome Chanyes and Their Evolution in Subjec!s with Past Exposure to Benzene Arch. Environ tfealili 23 385-391 11971). 10 Hazelton Laboratories America, Inc.. Vionna. Virginia: (Sqitcmber. 1975). Unpublished data 11 Greene. J. D.. E. K. J. Lcong and S. Laskin: Inhaled Benzene Fetotoxicity in Rais. Toxical Appl. Ptiarmacol 46 9- 18 (1978). 12 Litton Bionetics Incorporated, Kensington. Maryland: (December, 1978) Unpublished data 13 Staples. A . E.: Dctecrion of Visceral Alterations in Mammalian Fetuses Teratology 9 A -3 7 ( 19741 14 Wilson. J. G.: Teratology PriiicipI;*s a n d Tcchmiiucs p p 262-277 T h e Univcrsilyof Chicago Press, Chica(;o. Illinois (1965) 15 Dawson. A. B . : A note on [he Siaining of the Sn.cltaionof Cleared Spccirnms w i t h Allzarin R e d S S! 110 Tech 1 123- 124 ( 1926) .. '. 2 r .. I , . .,.La *. .. a. 9 .. t. . ...... . . .e . . . . '. .- American Industrial Hytiene Associalion JOUfirlAL (10)1 1 179 997 . 16 Haseman. J. K and D. G. Hoel. Table of Gehan's Generalized Wilcoxon Test with Fixed Point Sensoring J Sfarisr Cornput Simul 3 117-135 (1974) 17. Steel. R. G D. and H . ti. Torrie: Pimcples and Procedures of S t a r i s m s M c G r a w - H I I I Book Company, I n c , N e w York. N e w York (1960) 18. Wetmabe. G. and S. Yoshida: The Teratogenic Effect of Benzene in Pregnant Mice. Acta. Medica e! BJO/OylCJ 17 285-291 (1970). 19. 29 C o d e of Federal Regulations: Part 1910.1OOO, Table 2 - 2 . 20. National Academy of Sciences: Handbook of Respirmon W. 8. Saunders Co., Philadelphia. Pennsylvania ( 1 958). Accepted March 20. 1979 .. . -,2 - . . .. . ..' .-* :. . b .._-.. .. .I , * . .. ... . . . 998 Am Ind Hyg Asroc. I (40) Houember. 1979 RECEIVED rm 1978 R. L. DANIEL JUNE 2 2 , . 1978 . .. .. .-. .. . . .