Document M01dz4dKR5OmRdbEMq6bqqE9

Ui Hilt wvf t 14 S/->// O CONFIDENTIAL INFORMATION ffV\I R & D REPORT DOW CHEMICAL U.S.A. pLABORATORY REPORT COOS HET T2.02-193-000-001 DATR ISSUED department March 24* 1983 PROBLEM NO. Health and Environmental Sciences/Toxicology Research Lab, 2,0.8 m ? i TITLE COMPARISON OF CHEMICAL REACTIVITY TO BIOLOGIC ACTIVITY OF THE ALKYL 12 EPOXIDES PAGES IN FULL REPORT author(c) T. R. Fox, R. H. Reitz, and P. G. Watanabe AUTHOR Is ) SI6NATUne(*) /) ,/j) A, L i S3 ?/7 y/fX y reviewer's signature f Thi* report i*: INTERIM |----. UU F,NAL and mainly: Q[] NEW ,---- 1 1___ 1 REVIEW DESCRIPTIVE SUMMARY WITH CONCLUSIONS: The chemical reactivity of ethylene (EO), propylene (PO) and butylene (BO) oxides was determined by reacting with nicotinamide in vitro and comparing their apparent zero order rate constants. The order of reactivity was E0>P0>B0. The ratios of reactivity of EO/PO, E0/B0 and PO/BO were 2.00, 2.49 and 1,25 respectively. Comparison of this sequence of chemical reactivity to the overall general toxicity reported in the literature leads one to the conclusion that toxicity is in a similar order (E0>P0>B0). However, when comparing specific toxicologic effects in complex multicellular systems, differences emerge in these qpalitative biological effects. It is likely that these qualitative differences are a manifestation of differing capacities for detoxification and repair (both cellular and DNA) operating in complex biological systems. Therefore caution must be exercised in the simple extrapolation of biological effects of chemicals from parameters such as chemical reactivity. full RERKT FILED.'--T2.2-193-(1) PHOTO FOR .K-1708-(lOF y K-170M9)* "K-Mges-o)' PK-0317'2251-CO P*-OI75-525S--(0 DO 142637 CONFIDENT! Al.. DISTRIBUTION: ____________ DEPARTMENT FILES R & D ADMINISTRATION CENTRAL REPORT INDEX - 4 COPIES (566 Bldg. - Midland) Form c-joi printed R-to*o Distribution Htx is continued on eneched py. ^^~29^^SS8SiSBSSSSSSSS^B^BS!l COMPARISON OF CHEMICAL REACTIVITY TO BIOLOGIC ACTIVITY OF THE ALKYL EPOXIDES T. R. Fox, R. H. Reitz, and P. G. Watanabe Toxicology Research Laboratory Health and Environmental Sciences, USA Dow Chemical USA Midland, MI DOW CONFIDENTIAL n0 42638 MNFlWNTTM Do j C ONpf 40639 DPwnA( ABSTRACT Comparison of Chemical Reactivity to Biologic Activity of the Alkyl Epoxides. Fox, T. R., Reitz, R. H., and Watanabe, P. G. The chemical reactivity of ethylene (EO), propylene (PO) and butylene (BO) oxides were determined by reaction with nicotinamide in vitro and comparing their apparent zero order rate constants. The order of reactivity was EO>PO>BO, The ratios of reactivity of EO/PO, EO/BO and PO/BO were 2.00, 2.49 and 1.25 respectively. Comparison of this sequence of chemical reactivity to the overall general toxicity reported in the literature leads one to a similar conclusion that toxicity is in a similar order E0>P0>B0, However, of toxicologic significance when comparing specific toxicologic effects such as mammalian mutagenicity, teratogenicity and carcinogenicity marked differences emerge in these qualitative biological effects. It is likely that these qualitative differences are a manifestation of differing capacities for detoxification and repair (both cellular and DNA) operating in complex biological systems. Therefore caution must be exercised in the simple naive extrapolation of similar biological effects to chemicals of closely related structure and chemical reactivity. DOW CONFIDENTIAL OO comf 40 TPMTTAi DO DONF 7^?647 DOW CHEMICAL U.S.A. BIOCHEMICAL RESEARCH LAE. MIDLAND. MICHIGAN 48640 TOX. no. . "------------------ 7 rc.-X -/f3 -Or REQUEST FOR TOXICOLOGICAL STUDIES 1803 BLDG. tiE-f Ti'Oi-MrM>-tfo/ fJAMt OF MOE^IA' TO BE TESTED ___ Sllf/LrJ__ OftQE: JgWlTTED "" ~w (FfiQpYLEnJt OftfCE I,?.-BdTV(AP CXO'E) V* JVDat*! \ ^ J I^BmildtnJ' f j"7Phnt ~ y /hn/: /?<fV I Scl L- -rffjr RVISOR fSiynBiUffc'1 c ^ IPROJECT SAFETY COORDINATOR Jfestimated cost Vf /2- I 1 ACCOUNT j HUMBER 1 DO NOT WRITE BELOW ROUTE OF ADMINISTRATION /A/ 1//77ZD LENGTH OF TREATMENT SPECIES: AX-A. RAT UF rabbit f THIS guinea PIG LINE Dm qf DOG Cm f monkey lJm Ljp HAMSTER Dm f Tn" !tj V /H<n DOSES LOCATION GROUP 1 SECTION PUn(!7I5n !AIj&. PLwf iUFFu i 1 PROBLEM NO. to BE USED BY TOXICOLOGY lab. PROBLEM index no. ... . PROBLEM 1 !i ! , j : COMPLETE ALL INFORMATION CN REVERSE,/ NOTES. CHANCES. ADVERSE EFFECTS NOTED &i(ujUue. Ojidc ; K'liol? " Qo) ?wptykut (Pride', H-/O0I' L*l) 1, Ottit \ IC '(eMM'Cl) CHECK POINT Sample RECEIVED TOXICOLOGY estimated starting time ESTIMATED REPORTED DATE REVISED ESTIm aTE reported REPORT DRAFT ED DATE 10/10fay \ ohc \ t/?0 \ \ "----- ------------| j REPORT ISSUED fate of sample RETURNED DESTROYED DATE 1 REFERENCE SAMPLE STORED REFERENCE SAMPLE TO K STORAGE NOTES 1 confidential date project completed DBm i i AM O^eHATlNG UNIT OB TWf OOW CHEMICAL COWAM' SAMPLE INFORMATION ___ CyMOLECULAR FORMULA OAlpEiffapYligjg, 6[lp, hi SvTYLEil/ dx/l/l 0 W o I 3" MOLECULAR WEIGHT " " I iAMrtf Rt&fRfNCt, S&UNL t AND OR IDENTIFICATION / L-Qf #37/ - gS7 7- OKIk&hJS. STRUCTURAL formula or COMPOSITION /flv AND FER TIES o < Q* * *U XW a- U3J X LU estimated purity of SAMPLE __ >K?. 10BOILING POINT c ^0 mmHG CORROSIVENESS (to common moiolil LC^V CHEMICAL REACTIVITY ftt an STABILITY (o pH chonge, H**i, light) <Hrcf) SAMPLE ANALYSIS j RE5UL TS JZLtfs______________i____ !no____________ 3r%nEXPLOSIVE LIMITS' _ BY [ F^aSH POINT VOLUME* _ IN*|R|| 2-J) F IGNITION TEMP. 53/ * 6AS1 PHYSICAL STATE MfMELTING POINT -c COLOR ODOR (melud* concentration m air) a/oa/ )< 3/VAPOR PRESSURE ^hy ore tomeologicol studies needed? Include $toge or OBveiopmem, numon ewoosure' lj%, MrLfJ* -hrt C^A/uS-^ 25mcG yt\T~ ^hcck-iL ^rurh^j Chteb? /7. 3?> j'Cfcrtt ^ SitCL't/ltTrx Z/VU^7/>vi_ ^ tfpc'Ls/f J PROTOCOL COMPLETED J ANIMALS ORDERED Z] ANIMALS STARTED ON TEST Z NECROPSY DONE 'M C*3?10A PRINT* C* tfSi, irfo DATE | J[___ STATISTICS DONE 1 1 CLINICAL TESTS REPORTED 1 1 HEMATOLOGY TESTS 1___ 1 REPORTED r~l J1 SLIDES STARTED DATE 1 j Q SLIDES TO BE READY DATE j Q SLIDES READ i ; 1----- 1 ___ HISTOPATH REPORTED 1i ----i 1----- 1 WORK ALL DONE, READY | 1 TO WRITE [50 1 42643 OONF T DFNT T Al -1- INTRODUCTION Ethylene oxide (EO) has been reported to increase the frequency of spontaneously occurring tumors in animals (Snellings et al., 1981) and. preliminary information from Europe (Reuzel et al., publication in progress) indicates that propylene oxide (PO) may have a similar effect although PO is less potent than EO in this respect. Butylene oxide (BO) is a structural analogue of EO and PO, but appears to be less toxic (acutely and subchronically) than either EO or PO (Hine et al, 1981; Miller et al., 1981). An inhalation bioassay of BO for carcinogenicity is presently being conducted by the National Toxicology Program (Contract #600253, agent C5527), and the results will probably be reported sometime in 1983-4 (personal communication, Dr. Thomas Cameron, NCI). It appears that the toxicity of the alkyl epoxides may be related to the capacity of these materials to covalently react with nucleophilic sites within biological macromolecules. In order to objectively assess the relative potency of these three epoxides, the relative reactivity of these materials were determined under identical conditions. Nicotinamide is readily alkylated by the alkyl epoxides in an m aqueous solution at physiological pH (Ellis et al., 1982). The Nyalkylnicotinamides formed by such a reaction can be converted to cyclic products which display absorbence maxima around 360 nM. Such a reaction is ideally suited to compare the in vitro reactivity of EO, PO and BO. There are limitations in using in vitro measurements to try to estimate in vivo effects. However, determining the reactivity of EO, PO and BO with nicotinamide should provide an objective quantitative measure of the chemical's potential to react with biological macromole cules . MATERIALS AND METHODS Samples of the alkyl epoxides PO and BO (production sample, 1,2-butvlene oxide. Lot #167) were obtained from the production facil ities of the Dow Chemical Company. A cylinder of EO (Lot #J11-Q517) was purchased from Valley Oxygen (Bay City, MI). Nicotinamide was obtained from the Sigma Chemical Company (St. Louis, MO). All other chemicals DOW CONFIDENTIAL DO 14?644 CONFIDENTIAL -2- used were of standard reagent grade and obtained from commercial suppli ers. The in vitro measurements of the alkylation of nicotinamide by EO, PO and BO were conducted essentially as stated by Ellis et al. (1982). The reactions were carried out in 1.8 ml Teflon* lined screw capped vials in a total reaction volume of 1.0 ml. The composition of the reaction mixture is described below; 700 yl 0.1 M Phosphate buffer (pH 7.4) containing 7.1% ETOH 200 ul Nicotinamide (250 yM/ml) in above buffer 100 yl Ethanolic epoxide solution The ethanolic BO and PO solutions were prepared directly by addition of epoxide to dry ice-chilled ethanol at a concentration of 100 umoles/ml. Because EO is a gas, it was necessary to prepare the stock solution by bubbling 10 ml of gas into 4 ml of dry-ice chilled ethanol to obtain a concentration similar to the BO and ?0 solutions. The samples were Incubated at 50C. The color was then developed by adding 0.5 ml acetone and 0.2 ml 6 H K0H, waiting 5 minutes, and adding 2.0 ml distilled water. After 11 minutes the optical density was read at 358 nM in a Beckman Acta CII1 spectrophotometer. The concentration of the epoxide in the liquid phase of the re action vials was determined analytically by flame ionization gas chromatography using a Varian 3700 gas chromatograph. Separation was achieved on a 3.5 m x 2 mm, 20% Carbowax 20 M on Chromosorb WAW-DMCS (80-100 ml) column. The column temperature was varied between 80-100C isothermal depending on the epoxide analyzed. Nitrogen carrier flow was at 15 ml/min. Injection port and detector temperatures were 120^ and 250C respectively. The extinction coefficients of the N-alky1-nicotinamides were determined by incubating the nicotinamide (6.22 x 10-4M) with a 20 fold excess of epoxide under the conditions described for the rate determinations. The optical density was then measured when the reaction had reached completion. The molar extinction coefficients were calculat ed directly using Beer's law. *Trademark of the DuPont Chemical Company DO 3 42&4<$ 0NFTD BNTTai DOW CONFIDENTIAL RESULTS The three epoxides used in this study have fairly high vapor pressures at the 50C incubation temperature and in an aqueous environ ment might undergo hydrolysis. Therefore it was important to analytical ly determine the epoxide concentration prior to and at the termination of the incubation period. The concentration of the three epoxides as measured by GC analysis is listed in Table 1. At the beginning of the incubation the concentrations were EO (5.1 mM) , PO (8.7 mM), BO (9.2 mM). In a set of replicate vials incubated without nicotinamide it was observed that the EO concentration had decreased by 8 percent and the PO and BO concentration by 3 percent at the end of the 80 minute incu bation. These decreases are not considered significant and did not impact on the experimental results. The reaction rates of EO, PO and BO with nicotinamide were de termined by measuring the initial rate of change in optical density resulting from the production of the nicotinamide adduct. To make this direct comparison of reactivity based on a comparison of the slopes of the molar absorbence versus time graph, the molar extinction coeffi cients must be the same. These coefficients were determined by reacting a 20 fold excess of epoxide with nicotinamide and measuring the optical density when the reaction had reached completion. Using Beer's law, the molar extinction coefficients of the three N-alkylnicotinamide adducts were computed and are essentially identical (Table 2). The relative reactivity of EO, PO and BO can be determined by comparing the apparent zero order rate constants. Figure 1 represents a plot of the molar absorbence (MA) versus time of incubation for the reaction of the three epoxides with nicotinamide. Because the concen tration of the epoxides varied between EO, PO and BO, the optical densities were normalized on a per mole per liter basis. As can be seen in Figure 1, a linear relationship exists between the molar absorbency and time for all three epoxides thereby satisfying the zero order conditions (only 2-5 % of either reactant was consumed). Linear re gression data for the three plots is tabulated in Table 2. The relative reactivity of the three epoxides is determined by computing the ratio of the rate constants derived from the slopes of the linear plots in Figure 1. These ratios, found in Table 3, indicate that DOW CONFIDENTIAL qq l42646 CONFIDENTIAL EO is about 2.5 times more reactive than BO and 2 times more reactive than PO. A comparison of the rate constants between PO and BO showed PO to be slightly more reactive (1.25 times) than BO. DISCUSSION It has been suggested that the toxic, mutagenic and carcinogenic potential of a chemical can be related to chemical reactivity or alkylation capacity. In this study the in vitro reactivity of EO, PO and BO with nicotinamide was determined. The results indicated that EO was about 2.5 and 2.0 times more reactive than BO and PO respectively. A comparison between the reactivity of PO and BO resulted in PO being 1.25 times more reactive than BO. Recently the assessment in the potency of chemical toxicity has been made using a wide spectrum of biological endpoints from simplistic in vitro bacterial systems to more complex animal bioassays. In order to put the reactivity data in perspective with the existing toxicity data it is useful to examine several of these systems in which the epoxides have been tested. Measurements of acute toxicity generally follow the same trend as observed for chemical reactivity. The LC,.q in rats for EO has been determined to be 1460 ppm and the LC^ for PO is approximately 5600 ppm for a 4 hour exposure (Hine et al., 1981). BO is less toxic than PO and appreciably less toxic than EO. Rats can tolerate a single 7 hour exposure to 2600 ppm BO without any mortalities (unpublished data, Dow Chemical Company). Bioassays for EO and P0 have been completed (Snellings et al. , 1980; Reuzel et al., 1982), and a bioassay of BO is underway under the auspices of the NTP (started in December, 1981). Administration of EO was associated with an increase in mononuclear cell leukemia in female F344 rats and peritoneal mesothelioma in male F344 rats, while adminis tration of PO was associated with an increase in benign and malignant mammary tumors in female Wistar rats. These tumors occur at a fairly high spontaneous incidence in the corresponding strains in the absence of chemical treatment, although they appear to occur sooner and with higher multiplicity in the treated animals. In addition, both EO and PO produced a generalized increase in total tumors and total malignant DOW CONFIDENTIAL DO 142647 CONFIDFNTTAl. -5- tumors at the top dose (100 ppm for EO; 300 ppm for PO). Thus EO and P0 appear qualitatively similar in their ability to affect tumor inci dences, although this comparison is complicated by the fact that the bioassays were conducted in different strains of rats. The ability to cause mutation, a manifestation of electrophilic chemicals, has been linked to toxicity and the possible onset of carcinogenicity. The three epoxides used in this study have all been determined to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Embree et al., 1975; Bootman et al., 1979). No qualitative difference in response of the alkyl epoxides is evident in this simple in vitro system. However it is important to note that the bacteria used in this assay have deficient DNA repair mechanisms unlike those found in normal bacteria or other higher mammalian system. However, if one examines several mammalian systems capable of detecting genetic damage, a greater disparity in effects caused by the three epoxides becomes evident. Exposure to EO caused an increase in the number of sister chromatid exchanges in rabbit lymphocytes (Yager et al., 1981), an increase in dominant lethality in rats and mice (Generoso et al., 1980; Embree et al., 1975), and an increased frequency of sex linked recessive lethal mutations in Drosophila (Glaser et al., 1979). While PO was also found to induce mutations in Drosophila the induction of dominant lethality in rats and mice was questionable. In addition, no evidence was found for mutagenic action of PO on mice sperm at 50 and 250 mg/kg/day (Bootman et al., 1979; NTP, 1982). On the other hand, BO failed to produce any of these effects in similar studies (eg. dominant lethality, mouse sperm head morphology, Drosophila sex-linked recessive lethal, unscheduled DNA synthesis, or rat bone marrow cytology (NTP, 1982) . Reproductive and teratogenic effects have also been reported for EO. Significant reduction in fetal body weight and an increase in malformed fetuses were observed at a 150 mg/kg/day dose (LaBorde, 1980). In contrast, BO did not produce any significant embryotoxicity or teratogenicity at either 250 or 1000 ppm exposure in rats or rabbits (Sikov et al., 1980). From the data presented, the quantitative differences in chemical reactivity between the three epoxides are relatively small. Using this DOW CONFIDENTIAL -\ 4'Zk&fc oo -roHN-rr^- CONF -6- data alone one might postulate that there should be very little difference in the qualitative biological effects of the three epoxides. However, in examination of several biological endpoints ranging from simple bacterial mutagenicity to more complex multicellular systems this simplistic hypothesis weakens as differences emerge in the observed qualitative biological effects. It is likely that this difference can be attributed to various biological barriers, the presence of enzymatic detoxification (GSH), and repair mechanisms (cellular and DNA) operating in these more complex systems. Therefore, caution must be exercised in simply extrapolating similar biological effects to molecules of similar structure and chemical reactivity. Written by*. T. "Foxi' M.S. Study Monitor Research Biologist R. H. Reitz, Ph.D. Diplomate, American Board of Toxicology Study Director Research Associate P. G. Watanabe, Ph.D. Diplomate, American Board of Toxicology Director Toxicology Research Laboratory DOW CONFIDENTIAL OO 1,42649 CONFIDENTIAL /- REFERENCES Bootman, J., Lodge, D. C., Whalley, H. E. (1979). Mutagenic activity of propylene oxide in bacterial and mammalian systems, Mutation Research, 67: 101-112. Ellis, H. F., Airy, S. C., and Sinsheimer, J. E. (1982). Comparison of the alkylation of Nicotinamide and A-(p-Nitrobenzyl) pyridine for the Determination of aliphatic epoxides. Anal. Chem., 5A: 213-216. Embree, J. W., and Hine, C. H. (1975). Mutagenicity of Ethylene Oxide (abstract). Toxicol. Appl. Pharmacol., 33: 172-173. Generoso, W. M., Cain, K. T., Krishna, C, W., and Gryder, R. M. (1980). Heritable translocation and dominant-lethal mutation induction with ethylene oxide in Mice. Mutation Research, 73: 133--142. Glaser, 2. R. (1979). Ethylene Oxide: Toxicology Review and Field Study Results of Hospital Use. J. Environmental Pathology and Toxicology. 2: 173-208 Hine, C., Rowe, V. K., White, E. R., Daimer, K. I., Youngblood, G. T. (1981). Patty's Industrial Hygiene and Toxicol (3rd Ed), Vol IIA (Clayton and Clayton, Eds.), John Wiley and Sons, New York, p 2163. LaBorde, J, B., and Kimmel, C. A. (1980). The teratogenicity of ethylene oxide administered intravenously to mice. Toxicol. Appl. Pharmacol., 56: 16-22. Miller, R. R., Ayres, J. A., Caldwell, E. A., and Quast, J. F. (1980). 1,2-Butvlene Oxide 90-day vapor inhalation study with rats and mice. Dow Research Report HET-K~6A969-(5), Health and Environmental Sciences, Dow Chemical Company. NTP Technical Bulletin No. 7: April, 1982, page 9. Reitz, R. H., Fox, T. R. (1983). Fate of 1,2-Butylene Oxide in Male Rats Following Inhalation Exposure. Dow Research Report HET-K-6A969-(8), Health and Environmental Sciences, Dow Chemical Company. DOW CONFIDENTIAL DO 147650 CONFIDENTIAL -8- Reuzel, P.G.J., Kuper, C. F., CIVO Institute, Netherlands, Organization for Applied Scientific Research (Publication in progress, Report No. 82.215/280853). Sikov, M. R., Cannon, W.C., Carr, D. B., Miller, R. A., Montgomery, L. F., and Phelps, D. W. (1980). Teratologic assessment of butylene oxide, styrene oxide and methyl bromide. U.S. Dept, of Health, Education and Welfare. Contract No. 210-78-0025. Snellings, W. M., Weil, C. S,, Maronpot, R. R. (1981). Final Report on Ethylene Oxide Two-year Inhalation Study in Rats, Project Report 44-20, Bushy Run Research Center. Tyler, T. R., McKelvey, J. A. (1980). The Effect of Pre-exopsure on Subsequent Disposition of Ethylene Oxide (E0) in Rats. Fed. Pro ceed^, 39(3), 749. Vooyd, C. E., van der Stel, J. J., Jacobs, J. J. J. A. A. (1981). The Mutagenic Action of Aliphatic Epoxides, Mutation Research, 89, 269-282. Yager, J. W., and Beny, R. D. (1982). Sister chromatid exchanges induced in rabbit lymphocytes by ethylene oxide after inhalation exposure. Environmental Mutagenesis, 4:2, 121. DOW CONFIDENTIAL -CI TABLE 1 INITIAL AND FINAL CONCENTRATION OF ALKYL EPOXIDES Compound EO PO BO Cone. Before Incubation 5.1 .48 mM* 8.7 .12 mM* 9.2 l .26 mM* Cone. After Incubation 4.7 i .23 mM* 8.4 .12 mM* 8.9 .08 mM* % Change -8% -3% -3% *n=3 for all determinations DOW CONFIDENTIAL DO 14265? CONFIDENT! Al -10- TABLE 2 LINEAR REGRESSION ANALYSIS AND MOLAR EXTINCTION COEFFICIENT FOR REACTION OF ALKYL EPOXIDES WITH NICOTINAMIDE Equation Correlation Extinction Compound EO PO BO of Curve M, A.S = 3.74 (t)b-2 .98 M. A. = 1.87 (t) -0 .7 M.A. = 1.5 (t) +1 .98 Coefficient .9973 .9992 .9983 Coefficient 6,226 115* 6,293 t 98* 6,266 72* ^.A. * Molar Absorbence bt * Time in minutes *n=4 for all determinations DOW CONFIDENTIAL DO 142653 OONFTDFNTT Al MOLAR ABSORBANCE -12FIGURE 1 TIME (minutes) -11- TABLE 3 RELATIVE REACTIVITIES OF n-ALKYL EPOXIDES WITH NICOTINAMIDE Ratio of Rate Constants Relative Reactivities EO/BO * 3.74 1.5 2.49 EO/PO - 3.74 1.87 2.00 PO/BO - 1.87 1.5 1.25 DOW CONFIDENTIAL 0 14PSS5 confidential