Document nRBwgxmXr62yvbvmJv3L9Q1R

15 1980KfcCEIVED DEC R.U D/ Bio/tlynamics Inc. Division of Biology and Safety Evaluation PROJECT NO. 79-7342 A OOHINANT-LETHAL INHALATION STUDY * WITH 'BENZENE 4N RATS Final Report Submitted To: Chemical Manufacturers Association Washington, D.C. 20009 Date: December 1, 1980 *' ") ' 1 p DO 067489 CONFIDENTIAL YZ0083052 GO Bio/clynamics Inc PROJECT NO. 79-7342 A DOMINANT-LETHAL INHALATION STUDY WITH BENZENE IN RATS ABSTRACT This study was conducted for the Chemical Manufacturers Association and American Petroleun Institute to assess the mutagenic potential of Benzene in male rats using the dominant-lethal test. Benzene was administered by Inhalation at dose levels of 1. 10, 30 and 300 ppm. Male Charles River CD rats (20 males/group) of proven fertility were exposed 6 hours/day, five days/week for a ten-week treatment period. Dominant-Lethal mutagenicity was evaluated over a two-week post-treatment mating period. On the last day of dosing, each male was caged with two virgin, untreated females for seven consecutive days. At the end of this Interval, these females were removed and replaced with another two untreated females. All females exposed to males were sacrificed 19 days after the first day of exposure to the males. Females were scored for corpora lutea and uterine Implantation data. The latter were distinguished as viable fetuses and dead Implants. Parameters evaluated Include: male mortality, physical in-life observa tions and body weight data (treatment, mating and post-mating periods). Each male was given a gross postmortem examination and testes/epididymides were preserved In formalin, and histological evaluations of these tissues were performed for all control and high-dose males. Mutagenicity was evaluated on the basis of pre-imp!antatlon loss (Implantation efficiency ratio) and post-Implantation loss (mean nunber of dead implants and mutagenic ratio, l.e. no. dead implants/ total Implants). Pregnancy rate data were also evaluated. No treatment-related effects were evident In male mortality, In-llfe phy sical observations or body weight data. Pregnancy rates of females exposed to treated males showed no statistically significant changes at Weeks 1 or 2. Implantation efficiency ratios (Indications of pre-Implantation loss) were comparable between the control and treated groups for both weeks of the mating period. P.O.Box 43 MetttersRoad East Millstone, New Jersey 08873 (201)873-2560 * Telex: 644697 Cable: BIO/DYN DO 067490 CONFIDENTIAL YZ0083053 79-734Z The mean number of dead Implants and mutagenic ratios were comparable between the control and treated groups II, III and IV (1, 10 and 30 ppm, respectively) at Week 1, At Week 2, the mean number of dead implants and mean mutagenic ratio data In these same treated groups were slightly higher than control, but comparable to historical control data for these same parameters. Control group data for this Interval was considered unusually low. In the high dose group (300 ppn), the mean number of dead implants and mean mutagenic ratio values were higher than control for each week of the post-treatment matlnq period. These differences were not statistically significant. The Increases In mean number of dead Implants and mutagenic ratio were largely attributed to data for females exposed to a single highdose male. This male Impregnated both females each week and each female had a high number of dead Implants. Following a 10-week p"ost-mat1ng Interval, males were sacrificed and given a gross postmortem examination. These examinations showed several males In each of the treated groups with hepatic pallor and accentuation of lobular pattern of the liver. Microscopic evaluation of testes/epididymides revealed two high-dose males with testicular lesions. Thus, Benzene exposure at dose levels of 1, 10 and 30 ppm did not effect male reproduction performance or have a demonstrable dominant-lethal mutagenic effect. In the high-dose group (300 ppm) the observed increase in mean nunher of dead Implants and mean mutagenic ratio were primarily related to the performance of a single male. It is unclear if these effects were related to benzene exposure as the Increase In group values for mean dead Implants and mutagenic ratio were not statistically significant. No gross or histopathologic lesions were noted during the evaluations of the testes In this same high-dose male. YZ0083054 Table of Contents 79- 7342 Stir n unite* Ino. I. Introduction.................................................................................................................... 1 II. Materials........................................................................................................................... 1 A. Test Substance......................................................................................................... 1 B. Test Animals............................................................................................................. 1 III. Experimental Outline..................................................................................................... 2 IV. Methods................................................................................................................ 3 A. In-Life....................................................................................................................... 3 B. Postmortem.........................................................................................'....................... 6 C. Statistical Analysis............................................................................................. 8 V. Results and Discussion................................................................................................. 9 A. Mortality.......................................................................................................... 9 B. Body Weight Data................................................ 9 C. Physical In-Life ObservationData..................................................................... 9 D. Male Impregnating IndicesandPregnancy Rates............................................... 10 E. Corpora Lutea, ImplantationandImplantation Efficiency Data....... 10 F. uterine Implantation Data Amplified - Mean Number of Viable Fetuses and Dead Implants................................................................................ 11 G. Mutagenic Ratio Data............................................................................................. 12 K. Gross Postmortem Observations............................................................................ 13 I. Histopathologlcal Evaluations............................................................................ 13 Tables 1. Mean Male Body Weights......................................................................................... 15 2. Male Impregnating Indices and Pregnancy Rates - Post Treatment Mating Period......................................................................................................... 17 3. Corpora Lutea, Implantation and Implantation Efficiency Data Post-Treatment Mating Period.......................................................................... 18 4. Uterine Implantation Data Amplified and Mean f^tagenlc Ratio Values....................................................................................................................... 19 Appendices A. Pregnancy and Implantation Data - Pre-Treatment Mating Period...........A-1 B. Inhalation Report........................................................................................ B-1 C. Individual Male Body Weights................................................................................C-l D. Gross Postmortem Observations - Males........................................................... D-1 E. Pregnancy and Implantation Data - Post-Treatment Mating Period......... E-l F. Certified Feed Information.................................. F-l G. Analysis of Benzene..................................................................................................G-l H. Analysis of Water...................................................................................... H-l I. Statistical Analysis............................................................................................. 1-1 J. Hlstopathology Report.................................................. J-l K. Personnel......................................................................................................................K-l DO 0fc74^Ai CONF10FNT1AI YZ0083055 -1- 79-7342 nio/</*y namlcit Inc. I. INTRODUCTION This study, conducted for the Chemical Manufacturers Association and the American Petroleum Institute, was designed to assess the mutagenic potential on male rats of benzene at dose levels of 1, 10, 30 and 300 ppm. Test sub stance was admlnlsterd via Inhalation for 6 hours per day, 5 days a week for 10 consecutive weeks. Mutagenic potential was evaluated by the dominant-lethal test. Species and strain of test animals, dose levels and route of adminis tration were established by the sponsors. Testing was conducted at Bio/dyna mics, Inc., Mettlers Road, East Millstone, New Jersey 08873. All specimens, raw data and final report are maintained In the Bio/dynamics archives. II. MATERIALS A. Test Substance: Supplier: Descrlptlon: Date Received: Lot No.: Stability: Storage; Benzene Chemical Manufacturers Association Clear colorless liquid with characteristic odor. November 13, 1979 API-CMA Benzene Lot No. 1 (Sample withdrawal date 11/13/79) Benzene was analyzed for purity at four Intervals during the study, l.e. Pre-test, prior to Initiation of Treatment, Week 6 of the Treatment Period and at Termination of treatment. (See Appendix G). Amber glass bottles at room temperature DO 067493 CONFTDFNTTA! YZ0083056 1' ------ 2- 79-7342 lllo/, /dynamics Inc * II. MATERIALS (cont.): B* Test Animals: Species: Rat Strain: CD* (Sprague-Dawley derived) Number of Animals: Purchased: Males: 140 Females: 267 (pre-treatment mating period) 459 (post-treatment mating period) Placed on Test: Males: Pre-Treatment Mating Period-total 123 males Initiation of Treatment - total 100 males (20/group) Females: Pre-treatment mating period: 2 females/male; total 246, Post-treatment mating period: 2 females/ male/week; total 396. Age: Males: 10 weeks at initiation of pre-treatment mating period. Females: Virgin; at least 200 grams at Initiation of mating (pre- and post-treatment periods). Suppller: Charles River Breeding Laboratories Wilmington, Massachusetts 01887 Justification for Animal Selection: Generally recognized standard laboratory rat Dates Received: Males: 29 November 1979 Females: 29 November 1979 (pre-treatment mating period) 6 March 1980 (post-treatment mating period) DO 067494 C0NFTDFNTTA1 YZ0083057 4 74'Mznnmlca Inc. III. EXPERIMENTAL OUTLINE: Group Exposure Treatment Males I Control-Chamber-Exposed 20 II Benzene - 1 ppm 20 III Benzene - 10 ppm 20 IV Benzene - 30 ppm 20 V Benzene - 300 ppm 20 No. of Rats Females (Untreated; t>re-treatment Post-treatment Mating Period Matinq Period 40 80 40 761 40 80 40 80 40 80 treatment schedule - 6 hours/day, 5 days/week for 10 consecutive weeks. ^One Group II male died during the treatment period reducing the nuitber of females exposed to males to 38 per week (2/male; 19 males) for a total of 76 for this group. A sufficient nunber of males were acquired to Insure that enough proven fertile males were available to start on treatment. Male fertility was evaluated during a one week pre-treatment mating period; males were considered fertile If they Impregnated at least one female. Dominant lethal mutagenicity was evaluated on the basis of pre-lmplan- tatlon loss (l.e. Implantation efficiency ratio) and post-Implantation loss (l.e. mean number of dead Implants, mutagenic ratio) during a two week post treatment period. IV. METHODS: A. In-Life: Husbandry: Lights: 12 hours on/12 hours off Temperature: Recorded twice dally. During Exposure Period: Housing: Paired In stainless steel wire mesh cages In chamber DO 067495 CGNFTDENTIAl YZ0083058 Min/d/ yn mica Inc. -4- 79-7342 METHODS (cont.) A. In-L1fe (cont.) Food: Water: Hunldlty: During Non-Exposure Period: Housing: Food: None None Relative hunldlty recorded during the dally exposure Intervals (at least three distinct Intervals dally). Initiated during Week 1 of the treatment period. Individual, except during mating. In sus pended wire mesh cages; during mating housed with two females In these same cages. Purina Rodent Laboratory Chow 5002 (Certified) Mash; ad llbltun. See Appendix F Hunldlty: Water: 40-60% recorded twice dally; morning and afternoon. Humidity was recorded on 231 days for the duration of the study. It was found to be out of range approximately 10% of the time. In chambers humidity was found to be out of range approximately 18%25% of the time. Tap - Elizabeth Water Co. automatic watering system - ad llbltun; analyzed for contaminants on a monthly basis. Special water analyses (Appendix H) were done at two Intervals during the study - during the 10-week treatment period and near the termination of the study. Analysis Included: Heavy metals: Cd, As, Pb, Hg, Se, Be, Sb PCB's Organic Phosphate Pestlcldes-Malathlon Chlorinated hydrocarbons Including lin dane, heptachlor, DDT and Dleldrln Benzene Carbamate pesticide screen do YZ0083059 4 -5- 79-7342 mo//f.lvnamlrs .Inr. i IV. METHODS (cont.): A. In-Life (cont.): Identification of Animals: Each male and female were Identified with a metal ear tag bearing Its animal nunber. This number plus the Bio/dynamics project number comprised a unique Identification number. Assignment to Groups: Males of proven fertility (pre-treatment mating period) were assigned to groups so as to equalize pre-test body weight. Benzene Administra tion: Route: Inhalation Chamber Operation: See Appendix B Frequency: 6 hours/day for Group I and test substancetreated groups. Duration: (Dates of Treatment): Five days (Monday-Fr1day)/week, for 10 consecutive weeks (14 January 1980 - 21 March 1980). Atmospheric Sampllng: See Appendix B Observations for Signs of Pharmacologic or Toxicologic Effects and Mortality: Twice dally (morning, afternoon) Detailed Physical Examination Including Palpation for Tissue Masses: Males - weekly throughout the study. During the treatment period the detailed physical examinations were given following the expo sure period. Body Weights: Males - weekly throughout the study. DO 067497 CONFIDENTIAL YZ0083060 -6- 79-7342 ni,/d dy nnmlcc Inc. METHODS (cont.): A. In-Life (cont.): Mating Design and Mating Period: Pre-treatment Mating Period: Fertility of each male used In this study was determined on the basis of performance during a one week pre-treatment mating perl od. During this Interval each male was caged continuously with two untreated, virgin females for seven consecutive days. At the end of this period females were removed, housed Individually and sacrificed 18 days after the first.day of caging with males. Corpora lutea and uterine Implantation data were recorded. Males were considered of proven fertility If they Impregnated at least one female during the pre-treatment Interval. Corpora lutea and Implantation data for females mated to control and treated group males during the pre-treatment mating period are presented In Appendix A. Duration of the pre-treatment mating period23 December 1979 - 30 December 1979. Post-treatment Mating Period: On the last day of treatment at least two hours after termination of exposure each male was caged with two untreated, virgin females for seven consecutive days. At completion of this Interval these females were removed and replaced with another two untreated, virgin females for a second seven day Interval. 6. Postmortem: Sacrifice: Duration of post-treatment mating period Week 1 - 21 March 1980 - 28 March 1980 Week 2-28 March 1980 4 April 1980 Males - sacrificed at completion of the study following sacrifice of the last females. Gross postmortem examination per formed. Males sacrificed - 12 June 1980 00 YZ0083061 -7* 79-7342 Ml/. IV. METHODS (cont.): B. Postmortem (cont.): Females: Pre-Treatment Mating Period: Sacrificed 18 days after the first day of caging with the males, resulting In a gesta tion period of 11-18 days. Ovaries evalu ated for the number of corpora lutea; uter ine Implantation data recorded, the latter were distinguished as viable fetuses or dead Implants. Date of Sacrflce: January 10, 1980 Post-Treatment Mating Period: Sacrificed 19 days after the first day of caging with males, resulting In a gestation period of 12-19 days. Ovaries and uterus were evaluated: Date of Sacrifice: Week 1, 9 April, 1980. Week 2, 16 April, 1980. Sacrifice Method: Lethal exposure to ether. Uterine Implantation Data (Post-Treatment Mating Period): The abdominal cavity of each female was opened and the uterus with ovaries attached dissected free and removed. Immediately, the uterus was evaluated grossly for implan tation sites (l.e., criteria for pregnancy). Uteri with Implantations were further dis sected along the antImesometrial margin to better reveal the Implants. The number of Implantation sites (both horns) was recorded and each Implant further distinguished as: 1. Viable fetus: the amnion was dissected, the fetus observed and viability was determined on the basis of one of the following criteria: fetal movements; beating heart (observed through the transparent thoracic cavity); or general size and vascular development. DO 067499 CONF TDFNTTAl YZ0083062 B. Postmortem (cont.): Uteri ne Impl antatl on 2. Dead Implants: Dead Implants were Identified either as an Implant that elicited a decidual re sponse but no fetus was evident or a fetus that by size and/or viability criteria (l.e. movement of the head and extremities, beating heart or vasularlty) was not considered viable. Ovaries - the ovaries were dissected free from the uterine horns and evaluated grossly for the presence and number of corpora lutea. The uteri and ovaries from all females (pregnant and non pregnant) were appropriately Identified and preserved Intact in a 10% neutral buffered formalin solution. Male Reproductive System: Tissues Preserved: Testes and Epididymides were saved from all males (testes were fixed for 48 hours In Bouln*s solution, rinsed In tap water and stored In lot neutral buffered formalin): epididymides saved In 10% formalin. Tissues Examined Histopathologically: Testes and Epididymides (all control and hlgh-dose animals). C. Statistical Analysis: See Appendix I. 00 067500 CONFTDFNT TAI YZ0083063 ...-- -- V. RESULTS AND DISCUSSION: -9- 1 79-7342 ------ nio/, /dynamics Inc. A. Mortality: No mortality occurred in control group or treated Groups III, IV or V (10, 30 and 300 ppm) throughout the study. In Group II (1 ppm) male No. 300 died during Week 8 of the treatment period and male No. 316 died during Week 2 of the post-mating Interval. Gross postmortem observations for each of these males are presented In Appendix D. B. Body Weight Data: Individual male body weight data during the treatment, mating and post-mating Intervals are presented In Appendix C. Mean body weight data during the study are summarized In Table 1 (page 15-16). Mean weekly body weights during the treatment period and mean weight gain during this same interval were comparable between the control and treated groups. Likewise, mean body weights during the mating period and 10-week post-mating interval were comparable between all groups. No treatment effect on body weight data was evident. C. Physical In-Life Observation Data: A slight Increase In the incidence of males exhibiting lacrimation was noted in Groups III, IV and V (10, 30 and 300 ppm, respectively) during the first three weeks of the treatment period. During this Interval, lacrimation was noted In 3 control, 3 Group II (Ippm), 7 Group III, 9 Group IV and 13 Group V males; this represents an Incidence of 15%, 15%, 35%, 45%, and 65% of the animals In each group, respectively. For the remainder of the study the incidence of males with lacrimation observations was similar between the control and treated groups. DO 067S01 OONFTDFNTTA1 YZ0083064 -10- ___________________________________________________________________________________________________________ /dy ntmtc* In.'. t During the mating period several control and treated males were noted with a swollen cervical area. This observation Is typical of slalodacryoaden1t1s (SOA) viral Infection. The observation of "swollen cervical area" was transient In nature and occurred In both control and treated males; no treatment effect was indicated. Other observations noted during the physical In-life evaluations were noted with similar frequency between the control and treated groups. D. Male Impregnating Indices and Pregnancy Rates: Male Impregnating Indices and pregnancy rates for each week of the post-treatment mating period are presented In Table 2 (page 17). These Indices are summarized from reproduction data presented In Appendix C. Male impregnating Indices for each week of the post-treatment mating period were comparable between the control and treated group. Every control and treated male impregnated at least one female during the total two-week mating period. Pregnancy rates during Week 1 of the post-treatment mating period were lower than control In Groups II, III and IV (1, 10 and 30 ppm, respec tively); these differences were not statistically significant. Pregnancy rates for the high-dose group (300 ppm) at Week 1 were comparable to control. In the absence of an effect on pregnancy rate at the highest dose level (300 ppm) at Week 1, this decline in pregnancy rates at the lower dose levels was not considered Indicative of a treatment effect. During Week 2 of the post-treatment mating period pregnancy rates were comparable between the control and treated groups. E. Corpora Lutea, Implantation and Implantation Efficiency Data: Corpora lutea and Implantation data and implantation efficiency values (representative of pre-Implantation loss) for individual females during the post-treatment mating period are presented In Appendix E. These data are summarized In Table 3 (page 18). DO 06750? OONFTDFNTTAl __ YZ0083065 -11- 79-7342 ------------- -..... .. . /dyn mici Inc. The mean nunber of corpora lutea were comparable between the control and treated groups for each week of the post-treatment mating period. The mean nunber of Implantation sites at Meek 1 was comparable between the control and Groups II, IV and V (1, 30 and 300 ppm, respectively). In Group III (10 ppn), a slight decrease in mean nunber of implants was noted at Week 1; this difference was not statistically significant and was. In part, attributed to a reduced ovulation rate (i.e. decreases in nunber of corpora lutea) for this group at this same interval. The mean nunber of implantation sites during Week 2 of the post-treatment mating period was comparable between the control and treated groups. Mean Implantation efficiency values (Indicative of pre-implantation loss) were comparable between the control and treated groups for each week of the post-treatment mating period. F. uterine Implantation Data Amplified - Mean Number of Viable Fetuses and Dead Implants: Uterine Implantation data for Individual females during each week of the post-treatment mating period are presented in Appendix E. Mean Implantation data distinguished as to nunber of Implants, viable fetuses and dead Implants are presented in Table 4 (page 19). At Week 1 of the post-treatment mating period, the mean nunber of dead implants was comparable between the control and treated Groups II, III and IV (1, 10 and 30 ppn) and slightly higher than control In Group V (300 ppn); however, this difference between the control and high-dose groups was not statistically significant. During Week 2, the mean nunber of dead Implants was slightly higher than control at each dose level; however, these differences were not statis tically significant. The mean nunber of dead Implants In the control group at Week 2 was considered unusually low. Historically, the mean nunber of dead Implants recorded for control groups In studies of this type (Inhalation _ DO 0f>7503 CONFTDFNT TAl VZ0083066 dominant lethal studies) range -12- .4from 0.3-1 with 79-7342 Bio/, ________ /dynamic's In;. a weekly mean of 0.7 over 22 weekly Intervals (three separate studies). In respect to historical control data and concurrent control data at Week 1, the Increase in mean number of dead Implants for treated Groups II. Ill and IV (1, 10. 30 ppm) at Week 2 was not considered unusually high. The slight Increase In mean number of dead Implants In Group V (300 ppm) for each week of the post-treatment mating period was. In part, attributed to an increase In dead Implants In females mated to a single high-dose male (No. 913). During Week 1, the two females mated to this male had a total of 24 implants of which 16 (66.7%) were dead. In Week 2, the two females mated to male No. 913 had a total of 28 Implants of which 16 (57%) were dead. The mean nunber of viable fetuses was considered comparable between the control and treated groups for each week of the post-treatment mating period. G. Mutagenic Ratio Data: Mutagenic ratio data for Individual females during Weeks 1 and 2 of the post-treatment mating period are presented in Appendix E. Mean mutagenic ratio data are summarized In Table 4 (page 19). During Week 1 of the post-treatment mating period the mean mutagenic ratios were considered comparable between the females mated to control males and the females mated to treated males In Groups II, III and IV (1, 10 and 30 ppm, respectively). The mean mutagenic ratio for the females mated to the high-dose males (Group V, 300 ppm) was higher than control; however, this difference was not statistically significant. Within the high-dose group, the Increase In mean mutagenic ratio was largely attributed to three females. Two of these females (Nos. 1026 A 1027) were mated to a single high-dose male (No. 913); female No. 1026 had 14 Implants of which nine were dead (a mutagenic ratio of 64.3%) and female No. 1027 had 10 Implants DO 067504 CONFTDFNTTAL * YZ0083067 -13- 79-7342 nu>"/dy mmloi Inc. of which seven were dead (a mutagenic ratio of 70.0*). The third female with a high mutagenic ratio resulting from an Increase In dead Implants, was female No. 1024 (mated to high-dose male No. 912); this female had 15 Implants of which nine were dead (a mutagenic ratio of 60.0*). The second female mated to male No. 913 during Week 1 had 12 Implants and all were viable. During Week 2, mean mutagenic ratios for females exposed to the treat ed males were all slightly higher than the control group value. This was attributed to unusually low values for the control during this Interval (l.e. a mean mutagenic ratio of 4.4*). Historically, using this strain of rat a mean mutagenic ratio of 7.0% has been observed (this represents 20 weekly Intervals for three dominant lethal Inhalation studies with a range In weekly values of 4.0-11,0*). The mean mutagenic ratio for treated Groups II, III and IV (1, 10 and 30 ppm, respectively) fall within this historical control range. As was the case with Week 1 of the post-treatment mating period, the mutagenic ratio for the high-dose group (300 ppm) was higher (l.e. 11.7*) than the concurrent control and was just outside the range of values noted histor ically (l.e. 4.0-11.0*). During Week 2, the higher mutagenic ratio was again attributed to the two females (Nos. 1066, 1067) that mated with male No. 913. Female No. 1066 had 16 Implants and eight were dead (a mutagenic ratio of 50.0*); female No. 1067 had 12 Implants and eight were dead (a mutagenic ratio of 66.7*). H. Gross Postmortem Observations (Appendix D): Males were sacrificed after a 10-week post-mating Interval and given a gross postmortem evaluation. These postmortem evaluations revealed an Increased Incidence of males with hepatic pallor and accentuation of the lobular pattern of the liver among the treated group animals. Other morphologic findings occurred sporadically in the treated and/or control animals and were not considered to be related to treatment. 06750^ C0NFrnFNTrAl YZ0083068 TTTpr I. Hlstopathologlcal Evaluations (Appendix E): The testes and epididymides of rats from the control and high-dose' groups were evaluated hlstopathologically. In high-dose male No. 908. the testes revealed moderate tubular degeneration and atrophy characterized by multifocal depletion of spermatogenic epltheliun as well as thickening of the basement membrane. During the post-treatment mating period this male Impregnated both females during Week 1 and one female during Week 2. No effect was Indicated In Implantation efficiency values or mutagenic ratio values for females Impregnated by this male. In high-dose male No. 900, one testis revealed focal disarray and syncltal transformation of spermatogenic epithelium. This male Impregnated both females each week of the post-treatment mating period. During Week 2 one female (No. 1041) had an unusually low Implantation efficiency value (Indicative of pre-implantation loss) and for Week 1 both females (Nos. 1000 and 1001) had high mutagenic ratios. In sunmary, microscopic evaluation of the testes and epididymides of control and high-dose rats revealed two high-dose males with lesions; no such lesions were noted In the control animals. Concerning the lesions noted in the high-dose group, there was no conclusive evidence Indicating a compound-related Injury. T(ayniona t. ^cnroeaer $tu<iy Director DO 1 conf YZ0083069 Table 1 A Dominant-Lethal Inhalation Study With Benzene in Rats 79-7342 Mean Male Body Weights - grams n ov 2T1 O ^o TO1 O\J' 2 yi cs --I o ^M X- ou>o Wt. Treatment Period_________________________________ Gains Group Week: Initial 1 2 3 4 5 6 7 8 9 1o 1-10 ppm I Mean 398 406 411 423 438 455 464 476 487 500 500 0 S.D. 35 31 28 28 30 32 31 34 37 39 40 (Chamber- N 20 20 20 20 20 20 20 20 20 20 20 exposed Control) 94 20 20 II Mean 407 406 412 425 441 460 465 471 477 491 491 88 1 S.O. 27 27 31 35 37 39 39 36 39 39 40 21 N 20 20 20 20 20 20 20 20 19 19 19 19 III Mean 407 409 413 430 444 464 471 481 493 505 505 10 S.D. 38 41 42 44 47 51 52 48 54 56 54 N 20 20 20 20 20 20 20 20 20 20 20 96 18 20 IV Mean 406 409 412 426 440 459 464 476 484 495 498 90 30 S.D. 37 36 37 40 40 43 42 43 41 42 42 20 N 20 20 20 20 20 20 20 20 20 20 20 20 V Mean 409 411 412 424 439 458 468 481 492 502 505 300 S.D. 38 34 34 35 39 40 42 43 45 45 46 N 20 20 20 20 20 20 20 20 20 20 20 95 18 20 No statistically significant differences from control. ^ d y n a m ic * In e . Table 1 (cent.) A Dominant-Lethal Inhalation Study With Benzene in Rats 79-7342 Mean Male Body Weights - grams (cont.) o oO zO i--1 a o rr> Z in N o x> Qu>O Group ppn I 0 (Chamberexposed Control) II 1 III 10 IV 30 V 300 Week: Mean S.D. N Mean S.D. H Mean S.D. N Mean S.D. N Mean S.D. K Post-Treatment Mating Period 12 505 521 40 41 20 20 498 518 34 36 19 19 511 526 54 57 20 20 502 519 45 46 20 20 509 524 48 47 20 20 12 538 550 45 47 20 20 534 551 42 45 19 18 544 558 63 67 20 20 537 551 48 48 20 20 544 559 46 48 20 20 Ho statistically significant differences from control. I Post-Mating Interval 345 5 554 568 575 590 49 51 51 51 20 20 20 20 556 571 581 595 48 48 49 48 18 18 18 18 563 579 586 596 66 71 72 72 20 20 20 20 560 578 581 591 49 49 48 50 20 20 20 20 564 579 587 603 49 48 50 52 20 20 20 20 7..... 8 --nr 599 602 616 52 54 59 20 20 20 608 612 630 49 49 49 18 18 18 608 606 630 71 75 74 20 20 20 606 605 624 53 51 52 20 20 20 611 610 629 50 49 53 20 20 20^ < 3 -16.3 r> OTU --- -17- Table 2 A Dominant-Lethal Inhalation Study With Benzene In Rats yrj ^3H2/<|vniinl|,i| Ini'. Male Impregnating Indices and Pregnancy Rates Post-Treatment Mating Period Group ppm I (0) II 0) in (10) IV (30) V (300) I (0) II (D III (10) IV (30) V (300) Male Impreqnatlnq Index No. % Week 1 20/20 100.0 19/19 100.0 19/20 95.0 19/20 95.0 20/20 20/20 100.0 Week 2 100.0 19/19 100.0 20/20 100.0 19/20 95.0 19/20 95.0 Pregnancy Rateb t 36/40 30/38 30/40 29/40 37/40 90.0 78.9 75.0 72.5 92.5 36/40 32/38 32/40 35/40 36/40 90.0 84.2 80.0 87.5 90.0 No statistically significant differences from control. aMa1e impregnating Index No. of males Impregnating at least one female/No. of males caged with females. DPregnancy rate No. of pregnant females/No. of females exposed to males. 00 067509 CONF" TDFNT TAl YZ0083072 -18Table 3________________ A Dominant-Lethal inhalation Study With Benzene In Rats 79-7342 ntoy^yn mlcs Inc. Corpora Lutea, Implantation and Implantation Efficiency Data Post-Treatment Mating Period Group ppm Mean No ./Pregnant Female Corpora Implantation Lutea Sites Week 1 Mean Implantation Efficiency Values9 % I (0) II (1) III (10) IV (30) A+F 14.6 15.3 13.5 14.6 K+ 13.8 14.3 12.0 13.1 V92.7 92.7 85.7 89.4 V (300) I (0) II (1) hi (10) 14.2 A14.7 14.7 14.2 13.1 Week 2 K13.4 12.7 12.8 91.1 V91.4 85.1 89.0 IV (30) 14.7 14.2 95.2 V (300) 14.9 14.0 93.3 Por explanation of Statist leal symbols (l.e. A+, A-, F, k+, K-, V-) see Appendix 1-5. No statistically significant differences from control. aInd1v1dual female values presented In Appendix C* DO 067510 OONFTDFNTTAl YZ0083073 -19- Table 4 A Dominant-Lethal Inhalation Study ------------wren Benzene in Rats------------ /9-/342 Ulr/id/.ynamic* Inr. Uterine Implantation Data Amplified and Mean Mutagenic Ratio Values Group ppm Mean No./Pregnant Females Implantation Dead Viable Sites Imp! ants Fetuses Mean Mutagenic Ratio* % Week 1 I (0) II 0) hi (10) K+ 13.8 14.3 12.0 K- K+J 1.1 12.7 0.7 13.5 0.7 11.3 V7.8 6.0 7.1 IV (30) 13.1 1.3 11.8 11.6 V (300) 13.1 1.6 Week 2 11.5 14.9 I (0) 11 (1) III (10) K13.4 12.7 12.8 K- K0.4 13.0 1.0 11.7 0.8 12.1 V4.4 10.3 7.3 IV (30) 14.2 1.0 13.2 8.2 V (300) 14.0 1.5 12.6 11.7 For explanation of statistical symbols (l.e. J, K+, K-, V-) see Appendix 1-5. *Mutagen1c ratios for Individual females are presented In Appendix E. Mutagenic Ratio - No. of Dead Implants ; a mutagenic No. of Implantation Sites ratio Is calculated for each pregnant female. See Appendix I. no 067SI 1 CONF T DENT I Al YZ0083074 A-l Appendix A A Dominant-Lethal Inhalation Study With Benzene In Rats 79-7342 Pregnancy, Corpora Lutea and Implantation Data - Pre-treatment Mating Period Corp. Lut. Impl. VFS Preface Corpora Lutea Implantations Viable Fetuses DO 06 7 F, 10 CONF IDFNT TA1 YZ0083075 Appendix A (cant.) A Domlnant-Lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora lutea and Inplantation Data Pre-Treatawnt Hating Period* - Group I - Chanter-exposed Control Male Ho. WO 101 102 103 104 105 106 W7 WB 109 no Penal e Ho. 1308 1310 1331 1332 1333 1334 1335 1336 1389 1390 1408 1410 1419 1420 1423 1424 1453 1454 1466 1469 1480 1481 Corp. Lot. Inpl. W6 16 14 Hot pregnant. 12 12 13 13 16 13 17 16 14 13 10 2 13 12 13 12 18 17 19 19 15 15 14 M 19 16 Hot pregnant. 18 12 21 17 18 15 IS 13 15 13 Nuaber of Dead Implants 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 1 0 0 0 0 VFS 6 14 12 13 13 16 13 2 12 11 15 19 15 14 16 11 17 15 13 13 Hale Ho. Ill 112 113 114 115 116 117 118 119 Female No. 1492 1493 1494 1495 1496 1497 1509 1510 1515 1516 1539 1540 1551 1552 1560 1561 1564 1565 Total: Mean: S.O.: N: Corp. Lut. 15 14 Imol. 15 14 Hot pregnant. 15 14 Not pregnant. 14 14 Not pregnant. 17 15 17 17 Not pregnant. Hot pregnant. 13 13 15 14 Not pregnant. 19 18 Not pregnant. Not pregnant. 13 13 458 15.3 2.7 30 411 13.7 3.3 30 Data not analyzed statistically. 79-7342 Number of Dead Implants 0 1 0 0 0 4 VFS 15 13 14 14 15 13 1 12 0 14 1 17 0 11 0.4 0.9 30 13 400 13.3 3.2 30 A-Z DO 0 6 7 .SI 7 C0 NFTDFNTTA1 00 u> I "I Male Mo. 300 301 302 303 304 305 306 307 306 309 310 Female No. 1326 1327 1341 1342 1343 1344 1347 134B 1377 1378 1385 1386 1391 1395 1402 1403 1404 1405 1406 1407 1415 1416 Corp. lut. Impl. Not pregnant. - 17 16 16 16 16 14 14 14 15 15 18 18 14 13 13 13 17 17 12 12 Not pregnant. 17 7 16 6 19 16 22 15 13 13 16 13 14 14 Not pregnant. Not pregnant. 17 14 V Data not analyzed statistically. o o Appendix A (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy. Corpora lutea and Inplantation Data Pre-Treatment Mating Period* - Group M - (1 ppm) Number of Dead Implants 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 1 0 1 VFS 16 16 14 14 15 18 13 13 14 12 7 6 16 15 13 12 14 13 Male No. 311 312 313 314 315 316 317 318 319 Female No. 1433 1434 1447 1448 1457 1458 1490 1491 1517 1518 1519 1520 1525 1526 1541 1542 1547 154S Total: Mean: S.D.: H: Corp. Lut. Impl. Not pregnant. 15 15 14 14 14 14 13 13 Not pregnant. 15 14 13 10 15 15 14 14 Not pregnant. 17 17 14 12 14 13 12 12 13 12 Not pregnant. 14 14 483 15.1 2.2 32 435 13.6 2.5 32 79-7342 Number of Dead Implants VFS 0 IS 2 12 0 14 76 1 13 0 10 0 15 2 12 1 16 1 11 0 13 1 11 0 12 0 14 20 415 0.6 13.0 1.4 2.8 32 32 A-3 CONF TD FN T1 I Male Mo. 500 501 502 503 504 505 506 507 500 509 510 Female MO. 1320 1322 1355 1356 1361 1362 1363 1364 1383 1381 1387 1388 1398 1399 1400 1401 1413 1414 1417 1418 1449 1450 Corp. Lut. til. 14 14 IS 14 13 13 17 14 18 14 16 14 11 4 10 7 44 13 13 Not pregnant. 15 15 14 13 20 17 Hot pregnant. 18 15 15 15 16 16 16 14 15 15 17 17 17 17 o o v o o cvrj N LFl in oo w '4 00 Data not analyzed statistically. I Appendix A (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy. Corpora Lutea and lugtlantatlon Data Pro-Treatment Mating Period9 - Group III - (10 ppn) Number of Dead Implants 0 0 1 0 1 0 0 2 0 0 0 0 0 0 1 1 0 0 1 0 VFS 14 14 12 14 13 14 4 5 4 13 15 13 17 15 14 15 14 15 16 17 Male NO. 511 512 513 514 515 516 517 518 519 Female No. 1461 1463 1498 1499 1500 1501 1511 1512 1521 1522 1531 1532 1537 1538 1549 1550 1555 1556 Total: Mean: S.D.: N: Corp. Lut, Imol. 92 Not pregnant. 16 16 Not pregnant. 16 13 Not pregnant. 13 13 10 4 14 14 Not pregnant. 11 3 13 13 15 15 Not pregnant. 16 16 14 14 17 17 18 8 476 14.< 3.2 33 413 12.5 4.5 33 79-7342 Nw6er of Dead Implants 0 0 0 1 0 0 0 1 0 0 2 1 5 17 0.5 1.0 33 VFS 2 16 13 12 4 14 3 12 15 16 12 16 3 396 12.0 4.7 33 o OX 5o n - o a o; -n N Z--i --n1 --H ? oe w -4 Male No. 700 701 702 703 704 705 706 707 708 709 710 Female No. 1303 1304 1317 1319 1353 1354 1357 1350 1365 1366 1369 1370 1375' 1376 1379 1380 1381 1362 1421 1422 1438 1439 Corp. Lut, Inpl. 16 16 12 12 Not pregnant. 19 14 14 14 14 14 15 14 15 15 16 16 15 10 Not pregnant. IS 14 13 13 12 12 21 15 17 14 61 Not pregnant. 16 16 Not pregnant. 15 14 18 13 Data not analyzed statistically. i Appendix A (cant.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora Lutea and Implantation Data Pre-Treatment Hating Period* - Group IV - (30 ppm) Number of Dead Implants 0 1 0 0 0 1 0 2 0 4 1 0 2 1 0 0 r 0 i VF5 16 11 14 14 14 13 15 14 10 10 12 12 13 13 1 16 14 12 Male No. 711 712 713 714 715 716 717 718 719 Female No. 1455 1456 1474 1475 1466 1467 1504 1505 1523 1524 1527 1528 1543 1544 1545 1546 1562 1563 Total: Mean: S.O.: N: Corp Lut. Impl. 15 15 Not pregnant. 16 15 17 17 61 18 17 13 13 14 13 15 15 17 H Not pregnant. 14 14 12 12 Not pregnant. 14 14 14 14 16 1$ 15 6 487 14.8 3.0i 33 430 13.0 3.6 33 79-7342 Number of Dead Implants 3 VFS 12 0 15 0 17 01 0 17 0 .13 0 13 0 15 47 1 13 1 11 0 14 1 13 0 16 06 23 0.7 1.1 33 407 12.3 3.8 33 A-5 Cl oo zo n *< N nO O c- T1 Nl Z Ln oe ni w x> 00 Mala No. 900 901 902 903 904 905 906 907 908 909 910 Female No. 1313 1314 1337 1338 1345 1346 1349 1350 1351 1352 1359 1360 1373 1374 1411 1412 1427 1428 1431 1432 1436 1437 Corp. Lut,* IMPU 15 10 Not pregnant. Not pregnant. 10 8 12 10 14 14 17 16 16 16 16 14 Not pregnant. 11 11 15 15 15 11 13 It 17 15 15 15 Not pregnant. 10 10 12 4 12 3 17 15 26 14 Data not analyzed statistically Appendix A (cont.) A Doxn'nant-Lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora lutea and Implantation Data Pre-Treatment Mating Period* - Group * - (300 ppm) Number of Dead Implants 0 1 0 0 1 0 0 1 0 1 0 0 0 2 0 0 0 1 VFS 10 7 10 14 15 16 14 10 15 10 11 15 15 8 4 3 15 13 Male No. 911 912 913 914 915 916 917 918 919 Female No. 1440 1441 1442 1443 1472 1473 1478 1479 1482 1483 1484 1485 1488 1489 1502 1503 1553 1554 Total: Mean: 5.0.: N: Corp. Lut, Ifflpl. 16 16 Not pregnant. Not pregnant. 52 15 15 Not pregnant. 16 15 14 14 16 16 14 14 IB 16 18 . 16 Not pregnant. 14 13 15 14 Not pregnant. 16 16 15 15 455 14.it 3.4 31 394 12.7 3.9 31 79-7342 Number of Dead Implants 1 VFS 15 02 0 IS 0 15 0 14 0 16 1 13 1 15 0 16 0 13 0 14 2 14 0 15 12 382 0.4 12.3 0.6 3.9 31 31 JUG I -1- 79-7342 Appendix B A Dominant-lethal Inhalation Study In Rats with Benzene Inhalation Report SUMMARY Four groups of 20 male rats were exposed to vapors of benzene six hours per day, five days per week for ten consecutive weeks. A sham air exposed control group was also included. Each male, following this exposure interval, was mated with two untreated females per week for two consecutive weeks. Air samples from each chamber were analyzed by Infra-red spectroscopy six times during each exposure. The variable background Interference of the infra-red technique, due to the presence of animals In the exposure chamber, was significant at the two lower levels and In part accounts for the greater fluctuations In chamber concentration at these levels. Mean levels and ranges (as determined analytically) on samples of chamber atmospheres (expressed In ppm) were as follows: rr T.B"{d. 3-3,5) Director, Inhalation Technology Report Preparation: Cecelia A. Bozak, B.A. Study Monitor, Inhalation DO 06751R CONFIDENT TA1 YZ0083081 Bio/ * -79-W42 /<,yn nmtof Inc. Appendix B (cont.) A Dominant-Lethal Inhalation Study in Rats with Benzene Inhalation Report INTRODUCTION This study was designed to assess the mutagenic potential of a test material when administered to male rats by Inhalation for 6 bours/day, 5 days/week for ten consecutive weeks. Males of proven mating and re productive capacity were used for exposure. Each male, following the treat ment period, was mated with two untreated females per week for two con secutive weeks. The females were then scored for pregnancy and total number of implants and fetal deaths. Adverse effects on those parameters were con sidered indications of a dominant-lethal mutagenic effect In the males. In this report, the Infrared calibration curve and nominal and actual concentrations are presented from initiation (January 14, 1980) through termination (March 21, 1980) of the study. EXPERIMENTAL DESIGN Group I II III IV V Exposure Treatment 1 Males (target concentration - ppm) Control - Chamber exposed Benzene - 1 ppm Benzene - 10 ppm Benzene - 30 ppm Benzene - 300 ppm 20 20 20 20 20 No. of Rats Females untreated) Bre-treatment ^ost-treatment Mating Period Mating Period 40 80 40 76 40 80 40 80 40 80 ^Treatment schedule - 6 hours/day; 5 days/week for 10 consecutive weeks. DO 067^ T AD C.ODT YZ0083082 -3- 79-734?,0/dv nnmic* Inc. Appendix B (cont.) A Dominant-Lethal Inhalation Study In Rats with Benzene Inhalation Report Test Material Administration Benzene vapor concentrations were generated Into one cubic meter stainless steel and glass chambers using a flash-evaporation generating system. Liquid benzene was pumped Into a heated 3-neck flask through a 3-1nch length of 1/8-1nch diameter teflon tubing from a glass syringe fitted with a 16 gauge stainless steel needle. Flask temperature was maintained at 50C in Groups II, III, and IV and 80C in Group V. These temperatures were selected as the minimum required to achieve complete volitalIzatlon of the test material. The 3-neck flask was heated using a Glas-Col heating mantle and temperature was measured using a Glas-Col laboratory pyrometer. The benzene was pumped using Sage Instruments Model 220 and 341 syringe pimps. Adjustments In chamber concentration were made by adjusting pump flow rates (ml/hr) or chamber air flow (1pm). Air flow was determined by measuring the change in pressure (AP, cm H2O) over a one Inch orifice plate using a Dwyer magnehellc pressure gauge. Airflows were maintained between 150 and 250 1pm for the duration of the six hour exposures. DO 067520 OONFTDFNTTAl YZ0083083 -4- 79-7342 ------------------- Appendix B (cont.I__________ ____ A Dominant Lethal Inhalation Study in Rats with Benzene Inhalation Report Chamber Operation The stainless steel and. glass exposure chambers In which the animals were exposed had a total volutie of one cubic meter with an effective volune of 760 liters# They were operated dynamically at an air flow between 155 and 245 liters per minute, providing one air change In from 6.5 to 4.1 minutes and a 99% equilibration time (tgg) from 29.9 to 18.9 minutes. Dally Nominal Concentration Nominal exposure concentrations were calculated dally using the fol lowing equation: Nominal Cone, (ppm) Dally nominal concentrations are presented for Groups II-V In Table 4. Chamber Atmospheric Concentration One sample per hour (at about 0.5, 1*5, 2.5, 3.5, 4.5 and 5.5 hours) was drawn from each exposure chamber using a Wilks Instrument Company, Mlran Long Pathlength Infrared Analyzer Model IA (In-house Identification numbers "L" and "4")2. The exposure levels were determined by comparing the observed absorbance of these samples to a previously determined standard curve generated using the test material under the same Instrimental settings. The calibration curves for this test material are presented on pages 9-17 of this appendix. 2M1ran L was used from Day 1 (1/14/80) through Day 59 (3/12/80) of the study. Mlran 4 replaced Mlran L on Day 60 (3/13/80) and was used through termination of the study (Day 68 - 3/21/80). Therefore, calibration curves were generated for each phase of the study, the first set corresponding to Mlran L (1/14/80 - 3/12/80) and the second set corresponding to Mlran 4 (3/13/80 - 3/21/80). YZ0083084 -5- 79-734?,,o/ ---------------------------------------- ------- A^penUix B (cont.)-------------------------------- /<j>n,,m,c. me A Dominant-Lethal Inhalation Study in Rats with Benzene Inhalation Report Calculation of Chamber Concentrations Upon initiation of the study, It was observed that the presence of the animals in the chamber created a background Interference in the infrared monitoring procedure used to analyze the benzene exposure concentration. This interference was negligible for the Group IV (30 ppm) and Group V (300 ppm) exposure groups but if uncorrected, could have resulted in a signifi cant overstatement of the concentration in the Group II (1.0 ppm) and Group III (10 ppm) exposure groups. Using the first few weeks of data a correc tion factor was calculated, based on measurements of the background infrared absorption in the control chamber. An attempt was made to adjust the measurements using the control chamber data on a point-for-point basis. However, since the animals were exposed in a separate room, this approach did not produce a good correla tion; therefore, a correction procedure was developed using the average measured absorbance In the control chamber. This procedure involved the calculation of the average measured interference In the control chamber and then correcting each Individual measurement in the Group II and Group III chambers by subtracting this average background reading from the observed exposure chamber absorbance measurement. Monitoring for this background Interference during the first two weeks of the study in the control chamber led to a correction factor of 0.007 absorbance units for Groups II and III. It was also observed that the first one or two samples of the day had higher absorbances than the remain ing samples in both the control and low level exposure groups. This was attributed to the Increased activity of the animals following loading of the chamber. DO 06757? OONFTOFNTT Ai YZ0083085 -6- 79-7342 Appendix B (cont.) A Dominant-Lethal Inhalation study in Rats with Benzene nto/*yn mic Inc. Inhalation Report Calculation of Chamber Concentrations (cont.) At the conclusion of the study, samples collected at each sampling Interval (ie. 1/2 hr, 1-1/2 hr, 2-1/2 hr, 3-1/2 hr, 4-1/2 hr and 5-1/2 hr) were separately averaged and then statistically compared to one another.3 Where possible, separate calculations were performed for each phase of the study. When It was found that a particular interval possessed a unique cor rection factor, either by being statistically different, or In the case of the smaller sample groups, by appearing to be unique, this factor was applied to all absorbance measurements taken at that sample Interval, for that period. These values have been used to calculate the corrected Individual exposure concentrations. The statistical comparison Is presented In Table 1 and the actual correction factors are presented In Table 2. ^F-test and Student's t-test {modified using Cochran's approximation [t] when variances differ). Snedecor G.S., and W.G. Cochran, Statistical Methods 6th ed., Iowa State University Press, Ames, 1967, pp 104-106, 114-119. DO 0<->7S?3 CONFIDFNTTAL YZ0083086 Time Interval Appendix B (cont.) Table 1 A Dominant Lethal Inhalation Study in Rats with Benzene Inhalation Report Statistical Calculation of Correction Factors Used for Determination of Exposure Concentrations for Groups II and III 79-7342 Exposure Nos. 1/2'hr Mean Background Absorbances (Group I) 1-1/2 hr Sample Intervals: 2-1/2 hr 3-1/2 hr 4-1/2 hr b-1/2 hr 1/14/80 - 3/12/80 1-43 3/13/80 - 3/21/80 44-50 Mean S.D. N 0.0128+ 0.003(1 43 Mean s.n. N 0.0115 0.0042 7 0.008!+ 0.0027 43 0.0090 0.0048 7 0.0068 0.0022 43 0.0088 0.0044 7 0.0066 0.0021 43 0.0087 0.0034 7 0.0061 0.0015 42 0.0084 0.0035 7 0.0061 0.0021 43 0.0085 0.0034 7 "Significantly different from succeeding values (p<0.05). different from succeeding values (p>0.05). " n 2To| o o Z-<? oO' n nj z 01 N T-<> > 00 u> 00 i -7- Time Interval Appendix R (cont.) Table 2 A Dominant lethal Inhalation Study in Rats with Benzene Inhalation Report Correction Factors Applied to Measured Absorbance For Groups II and III 79-7342 Exposure Nos. 3WET 1-1/2 hr Correction Factors Sample Intervals: 2-1/2 hr 3-1/2 hr 4-1/2 hr 5-1/2 hr 1/14/80 - 3/12/80 1-43 0.013 0.008 0.006 0.006 0.006 0.006 3/13/80 - 3/21/80 44-50 0.012 0.009 0.009 0.009 0.009 0.009 n ov z-n o -i o aT| &M z yi -t \) 00 u> o i00 00 -8- -9- 79-7342 Appendix B (cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Infrared Analysis of Benzene CALIBRATION FOR: Molecular Weight: Density: Benzene 78.11 amu 0.878 g/ml MIRAN SETTINGS: Wavelength: Path!ength: Range: Slit Width: Meter Response Time: Coarse Zero: 3.3 u - optimized to 3.28 u 21.75 m (14.0 dial reading) 0.1 l (Groups I, II, III) 1.0 & (Groups IV, V) 1 mm 1 second x1 STANDARD: Measured volunes of benzene were Injected into a closed loop circulation system in order to generate atmospheres Inside the Miran of known concentration levels. The closed loop system, including the Miran, measures 5.64 liters In volune. DO 067578 CONFTDFNTTAl YZ0083089 -10- 79-7342 Appendix R (cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Mlran Jtoblent Air Analyzer Closed Loop Calibrations Mlran L (1/14/80 - 3/12/80) Injection Range Volune Concentration (ul) (PPT>) Al Absorbance Ag A3 0.1 \ 0.01 0.02 0.03 0.05 0.10 0.15 0.19 0.21 0.23 0.30 0.50 0.60 0.5 1.0 1.4 2.4 4.8 7.2 9.1 10.1 11.0 14.4 24.0 28.8 0.001 0.003 0.004 0.007 0.015 0.023 0.029 0.032 0.034 0.045 0.075 0.091 0.001 0.002 0.003 0.006 0.014 0.022 0.029 0.032 0.035 0.046 0.077 0.092 0.001 0.003 0.005 0.007 0.012 0.019 0.024 0.026 0.028 0.038 0.065 0.079 1.0 fc 0.50 0.60 0.63 0,65 0.70 1.00 2.00 3.00 4.00 5.00 6.00 6.20 6.30 6.50 7.00 7.50 8.00 24.0 28.8 30.2 31.1 33.5 47.9 95.8 143.7 191.6 239.6 287.5 297.0 301.8 311.4 335.4 359.3 383.3 0.075 0.09 0.10 0.10 0.11 0.15 0.30 0.44 0.56 0.68 0.79 0.81 0.82 0.84 0.89 0.94 0.99 0.070 0.090 0.095 0.10 0.11 0.15 0.30 0.43 0.56 0.68 0.79 0.81 0.82 0.84 . 0.89 0.94 0.99 0.070 0.080 0.090 0.090 0.10 0.14 0.28 0.41 0.53 0.64 0.74 0.76. 0.77 0.79 0.83 0.87 0.91 A4 0.001 0.002 0.003 0.006 0.014 0.021 0.027 0.030 0.033 0.043 0.074 0.088 0.075 0.090 0.095 0.095 0.10 0.15 0.29 0.43 0.55 0.66 0.76 0.78 0.79 0.81 0.85 0.89 0.94 DO 0675?7 OONPTDFNT TA1 YZ0083090 C.cc-? - e.vcr * cort - c cc-r is s0b C CeY - O.0O& .eci - - <t,OCI no v zn o -o"<Tl oOsi' N z 'J' H Ma> oe T> Appendix B (Cont.) A Dominant Lethal Inhalation Study in Rats with Benzene Inhalation Report Infrared Calibration Curve Hiran L 0/14/80 - 3/12/M) Range: 0-2.9 ppm 79-7342 M-iT 79-2<+2S`,7392 m*Ari:L WvtiJtfiftU i 3.3^ (emntizrtt To 3.x?) wmWTH'.jr.7r^ /Vjp) *rww;c: /.rr* Sur* I m* 7c^- /-9-ro i i*iw isu**$ - - <> --i----(----i-----+- f r "Aff/tn . fT NI *< N oe w PPI /-9-WV vKo* N ou>o so -14- 79-7342 Appendix B (cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Ml ran Ambient Air Analyzer Closed Loop Calibrations Ml ran 04 (3/13/80 - 3/21/80) Injection Range Volune Concentration (ul) (ppm) 'Ti Absorbance A2 A3 -TC' 0.1 A 0.01 0.02 0.03 0.05 0.10 0.15 0.19 0.21 0.23 0.30 0.50 0.60 0.5 1.0 1.4 2.4 4.8 7.2 9.1 10.1 11.0 14.4 24.0 28.8 0.001 0.002 0.002 0.005 0.012 0.018 0.024 0.026 0.029 0.039 0.068 0.081 0.001 0.002 0.004 0.007 0.014 0.022 0.027 0.030 0.034 0.044 0.072 0.086 0.002 0.003 0.005 0.008 0.016 0.023 0.029 0.032 0.036 0.046 0.074 0.088 0.000 0.001 0.002 0.003 0.009 0.015 0.020 0.022 0.025 0.033 0.062 0.074 1.0 % 0.50 0.60 0.63 0.65 0.70 1.00 2.00 3.00 4.00 5.00 6.00 6.20 6.30 6.50 7.00 7.50 8.00 24.0 28.8 30.2 31.1 33.5 47.9 95.8 143.7 191.6 239.6 287.5 297.0 301.8 311.4 335.4 359.3 383.3 0.07 0.09 0.09 0.10 0.10 0.14 0.27 0.38 0.48 0.57 0.65 0.66 0.67 0.68 0.71 0.75 0.77 0.07 0.08 0.09 0.09 0.10 0.14 0.26 0.38 0.48 0.58 0.66 0.68 0.68 0.70 0.73 0.76 0.79 0.07 0.08 0.09 0.09 0.10 0.14 0.26 0.37 0.46 0.55 0.62 0.63 0.64 0.65 0.68 0.71 0.75 0.07 0.08 0.09 0.09 0.10 0.14 0.25 0.37 0.46 0.55 0.62 0.63 0.64 0.65 0.69 0.72 0.75 00 067-S31 CONFTOFNTT Al YZ0083094 Ort? - e.evr - - o-ooi C.Cflf is St $ P.0OH ' O.OC-i - - c.oox - o 0.001 -oo Z-Tl o -H O N o-T( TV'i z vn, oo wo \> T> VtnO Appendix B (Cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Infrared Calibration Curve fifran 4 (3/13/80 - 3/21/801 Range: 0-2.8 ppm 79-7342 flt-iT 79-2VJS-, 7?VS C/H t#1*7/0 N J.J.V re e*T>`LrrJr-'rv t ??z*o*istz Is?* 5i i T"* f n*n /onC-t r o.i 0 iv-.o) ifc L-ty c*/ HiAutsKh*. cin ft-' Appendix 8 (Cont.) 79-7342 *< N VO I N oo Sso I -4 Appendix 0 (Cont.) 79-7342 t t "fs| ! rf -18- 79-7342 Appendix B (cont.) Table 3 A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Cunul atlve Exposure Record Group II - 1 ppm Oate Test Mean Dally Exposure Exposure ConcenNumber tratlon SO Dally Dally Concen- Total Exposure tratlon Exposure Hours Hours Hours Cumulative Inte- Total grated Concen- Mean tratlon ConcenHours tratlon 1/14/80 1/15/80 1/16/80 1/17/80 1/18/80 1/21/80 1/22/80 1/23/80 1/24/80 1/25/80 1/28/80 1/29/80 1/30/80 1/31/80 2/1/80 2/4/80 2/5/80 2/6/80 2/7/80 2/8/80 2/11/80 2/12/80 2/13/80 2/14/80 2/15/80 2/18/80 2/19/80 2/20/80 .1 2 3 4 5 8 9 10 11 12 15 16 17 18 19 22 23 24 25 26 29 30 31 32 33 36 37 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 0.5 0.4 6 3.0 6 3.0 0.3 0.4 6 1.8 12 4.8 0.8 0.6 6 4.8 18 9.6 0.5 0.5 6 3.0 24 12.6 0.5 0.3 6 3.0 30 15.6 1.1 0.7 6 6.6 36 22.2 1.0 0.6 6 6,0 42 28.2 0.9 0.6 6 5.4 48 33.6 1.6 0.5 6 9.6 54 43.2 1.2 0.6 6 7.2 60 50.4 1.2 0.6 6 7.2 66 57.6 1.4 0.4 6 8.4 72 66.0 0.9 0.5 6 5.4 78 71.4 1.3 0.4 6 7.8 84 79.2 1.1 0.4 6 6.6 90 85.8 1.5 0.9 6 9.0 96 94.8 1.2 0.4 6 7.2 102 102.0 0.7 0.5 6 4,2 108 106.2 1.5 0.7 6 9.0 114 115.2 0.7 0.3 6 4.2 120 119.4 0.4 0.4 6 2.4 126 121.8 0.8 0.3 6 4.8 132 126.6 0.9 0.6 6 5.4 138 132.0 0.7 0.9 6 4.2 144 136.2 1.1 1.2 6 6.6 150 142.8 1.5 0.4 6 9.0 156 161.8 0.5 0.6 6 3.0 162 154,8 1.2 0.5 6 7.2 168 162.0 0.5 0.4 0.5 0.5 0.5 0.6 0.7 0.7 0.8 0.8 0.9 0.9 0.9 0.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.9 1.0 1.0 1.0 1.0 DO 067535 OONFTOFNTT Al YZ0083098 -19 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study in Rats with Benzene Inhalation Report Cumulative Exposure Record Group II - 1 ppm (cont.) ........... .. Mean Daily Exposure Test Exposure ConcenDate Day Number tration SD - Cumulative Inte- Daily Total grated DallyConcen- Total Concen- Mean Exposure tration Exposure tration Concen- HoursHours Hours Hours tration 2/21/80 2/22/80 2/25/80 2/26/80 2/27/80 2/28/80 2/29/80 3/3/80 3/4/80 3/5/80 3/6/80 3/7/80 3/10/80 3/11/80 3/12/80 3/13/80 3/14/80 3/17/80 3/18/80 3/19/80 3/20/80 3/21/80 39 40 43 44 45 46 47 50 51 52 53 54 57 58 59 60 61 64 65 66 67 68 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 1.2 0.3 6 0.9 0.3 6 1.0 0.4 6 0.8 0.6 6 0.8 0.7 6 1.1 0.4 6 3.5 4.9 6 1.3 0.7 6 1.0 0.4 6 0.8 0.3 6 1.4 0.3 6 0.9 0.8 6 1.1 0,7 6 1.2 0.5 6 1.0 0.4 6 0.8 1.1 6 0.3 0.4 6 0.3 0.4 6 1.4 0.6 6 0.4 0.4 6 0.4 0.6 6 0.0 0.0 6 7.2 174 169.2 1.0 5.4 180 174.6 1.0 6.0 186 180.6 1.0 4.8 192 185.4 1.0 4.8 198 190.2 1.0 6.6 204 196.8 1.0 21.0 210 217.8 1.0 7.8 216 225.6 1.0 6.0 222 231.6 1.0 4.8 228 236.4 1.0 8.4 234 244.8 1.0 5.4 240 250.2 1.0 6.6 246 256.8 1.0 7.2 252 264.0 1.0 6.0 258 270.0 1.0 4.8 264 274.8 1.0 1.8 270 276.6 1.0 1.8 276 278.4 1.0 8.4 282 286.8 1.0 2.4 288 289.2 1.0 2.4 294 291.6 1.0 0.0 300 291.6 1.0 Standard deviation of Cumulative Integrated Mean concentration for Group II is 0.2 for entire study. c no ONF ^7S36 TOFNT TAI YZ0083099 -20- 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Cumulative Exposure Record Group III - 10 ppm Date Mean Dally Exposure Test 1Exposure ConcenDay Number tratlon SO Cumulative Inte- Dally Total grated Dally Concen- Total Concen- Mean Exposure tratlon Exposure tratlon Concen Hours Hours Hours Hours tration 1/14/80 1 1 1/15/80 2 2 1/16/80 3 3 1/17/80 4 4 1/18/80 5 5 1/21/80 8 6 1/22/80 9 7 1/23/80 10 8 1/24/80 11 9 1/25/80 12 10 1/28/80 15 11 1/29/80 16 12 1/30/80 17 13 1/31/80 18 14 2/1/80 19 15 2/4/80 22 16 2/5/80 23 17 2/6/80 24 18 2/7/80 25 19 2/8/80 26 20 2/11/80 29 21 2/12/80 30 22 2/13/80 31 23 2/14/80 32 24 2/15/80 33 25 8.6 0.8 6 8.7 0.7 6 9.2 0.6 6 7.4 0.4 6 8.6 0.5 6 9.6 0.3 6 8.9 0.4 6 8.5 0.7 6 9.7 0.4 6 11.5 0.7 6 12.4 1.6 6 10.4 1.4 6 10.1 9.8 1.1 2.1 6 6 9.3 1.6 6 10.2 3.4 6 12.7 2.6 6 8.6 0.9 6 9.5 0.8 6 10.0 1.4 6 9.4 1.0 6 8.5 2.9 6 11.3 2.3 6 9.6 1.1 6 10.4 1.6 6 51.6 . 52.2 55.2 44.4 51.6 57.6 53.4 51.0 58.2 69.0 74.4 62.4 60.6 58.8 55.8 61.2 76.2 51.6 57.0 60.0 56.4 51.0 67.8 57.6 62.4 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 - 120 126 132 138 144 150 51.6 103.8 159.0 203.4 255.0 312.6 366.0 417.0 475.2 544.2 618.6 681.0 741.6 800.4 856.2 917.4 993.6 1045.2 1102.2 1162.2 1218.6 1269.6 1337.4 1395.0 1457.4 8.6 8.7 8.8 8.5 8.5 8.7 8.7 8.7 8.8 9.1 9.4 9.5 9.5 9.5 9.5 9.6 9.7 9.7 9.7 9.7 9.7 9.6 9.7 9.7 9.7 00 o YZ0083100 -21- 79- 73 42 Appendix 6 (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Cumulative Exposure Record Group III - 10 ppm (cont.) Date Mean Dally Exposure Test Exposure ConcenDay Number tration SD ' Cumul ati've Inte- Dally Total grated Daily Concen- Total Concen- Mean Exposure tration Exposure tration Concen Hours Hours Hours Hours tration 2/18/80 2/19/80 2/20/80 2/21/80 2/22/80 2/25/80 2/26/80 2/27/80 2/28/80 2/2 9/80 3/3/80 3/4/80 3/5/80 3/6/80 3/7/80 3/10/80 3/11/80 3/12/80 3/13/80 3/14/80 3/17/80 3/18/80 3/19/80 3/20/80 3/21/80 36 37 38 39 40 43 44 45 46 47 50 51 52 53 54 57 58 59 60 61 64 65 66 67 68 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 10.6 1.3 6 8.9 0.6 6 10.5 1.3 6 11.2 1.2 6 9.7 1.3 6 10,1 0.7 6 10.2 1.6 6 10.3 0.7 6 10.4 0.8 6 8.1 4.0 6 10.2 1.2 6 10.4 0.6 6 10.6 1.5 6 10,6 0.7 6 10.4 1.0 6 9.4 0.7 6 11.4 1.4 6 9.6 0.6 6 10.3 1.0 6 9.3 0.9 6 9.1 0.6 6 9.5 0.8 6 9.3 1.2 6 9.3 2.1 6 8.9 1.6 6 63.6 156 1521.0 9.8 53.4 162 15 74.4 9.7 63.0 168 1637.4 9.7 67.2 174 1704.6 9.8 58.2 180 1762.8 9.8 60.6 186 1823.4 9.8 61,2 192 1884.6 9.8 61.8 198 1946.4 9.8 62.4 204 2008.8 9.8 48.6 210 2057.4 9.8 61.2 216 2118.6 9.8 62.4 222 2181.0 9.8 63.6 228 2244.6 9.8 63.6 234 2308.2 9.9 62.4 240 2370.6 9.9 56.4 246 2427.0 9.9 68.4 252 2495.4 9.9 57.6 258 2553.0 9.9 61.8 264 2614.8 9.9 55.8 270 2670.6 9.9 54.6 276 2725.2 9.9 57.0 282 2782.2 9.9 55.8 288 2838.0 9.9 55.8 291 2893.8 9.8 53.4 300 2 947.2 9.8 Standard deviation of Cumulative Integrated Mean concentration for Group III is 0.4 for entire study. 00 067538 OONF T DF.NT T Al YZ0083101 -22- 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation study In Rats with Benzene Inhalation Report Cumulative Exposure Record Group IV - 30 ppm Date Mean Dally Exposure Test Exposure ConcenDay Number tratlon SD tumulstive Inte- Dally Total grated Daily Concen- Total Concen- Mean Exposure tratlon Exposure tratlon Concen Hours Hours Hours Hours tratlon 1/14/80 1/15/80 1/16/80 1/17/80 1/18/80 1/21/80 1/22/80 1/23/80 1/24/80 1/25/80 1/28/80 1/29/80 1/30/80 1/31/80 2/1/80 2/4/80 2/5/80 2/6/80 2/7/80 2/8/80 2/11/80 2/12/80 2/13/80 2/14/80 2/15/80 1 2 3 4 5 8 9 10 11 12 15 16 17 18 19 22 23 24 25 26 29 30 31 32 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 32.7 32.2 28.5 30.6 30,1 29.0 29.3 29.5 29.5 29.5 32.2 32.8 29.0 30.6 32.2 33.3 32.2 31.7 30.1 30.6 29.5 31.1 30.6 29.0 29.5 2.4 2.0 6.8 2.7 2.6 6.7 2.2 2.4 1.3 1.3 2.0 3.2 2.9 4.0 2.0 4.8 0.0 1.3 1.7 1.8 1.3 2.6 3.4 2.1 1.3 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 196.2 193.2 171.0 183.6 180.6 174.0 175.8 177.0 177.0 177.0 193.2 196.8 174.0 183.6 193.2 199.8 193.2 190.2 180.6 183.6 177.0 186.6 183.6 174.0 177.0 6 196.2 12 389.4 18 560.4 24 744.0 30 924 .6 36 1098.6 42 1274.6 48 1451.4 54 1628.4 60 1805.4 66 1998.6 72 2195.4 78 2369.4 84 2553.0 90 2746.2 96 2946.0 102 3139.2 108 3329.4 114 3510.0 120 3693.6 126 3870.6 132 4057.2 138 4240.8 144 4414.8 150 4591.8 32.7 32.5 31.1 31.0 30.8 30.5 30.3 30.2 30.2 30.1 30.3 30.5 30.4 30.4 30.5 30.7 30.8 30.8 30.8 30.8 30.7 30.7 30.7 30.7 30.6 DO 067539 CONF TDFNTT Al YZ0083102 -23- 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Cumulative Exposure Record Group IV - 30 ppm (cont.) Date Kean Oally Exposure Test Exposure ConcenDay Number tratlon SD Cumulative Inte- Daily Total grated Dally Concen- Total Concen- Kean Exposure tratlon Exposure tratlon Concen- Hours Hours Hours Hours tration 2/18/80 2/19/80 2/20/80 2/21/80 2/22/80 2/25/80 2/26/80 2/27/80 2/28/80 2/29/80 3/3/80 3/4/80 3/5/80 3/6/80 3/7/80 3/10/80 3/11/80 3/12/80 3/13/80 3/14/80 3/17/80 3/18/80 3/19/80 3/20/80 3/21/80 36 37 38 39 40 43 44 45 46 47 50 51 52 53 54 57 58 59 60 61 64 65 66 67 68 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 29.5 1.3 6 29.5 1,3 6 29.5 1.3 6 30.1 2.6 6 29.5 1.3 6 29.5 1.3 6 29.5 2.4 6 29.0 2.1 6 30.1 1.7 6 28.5 1.3 6 30.6 2.7 6 30.1 1.7 6 29.5 1.3 6 30.1 1.7 6 29.5 1.3 6 29.0 2.1 6 29.5 1.3 6 30.1 3.3 6 30.1 1.4 6 30.7 2.2 6 33.0 1.8 6 30.7 3.1 6 30.7 2.2 6 31.9 2.8 6 30.2 2.6 6 177.0 177.0 177.0 180.6 177.0 177,0 177.0 174.0 180.6 171.0 183.6 180.6 177.0 180.6 177.0 174.0 177.0 180.6 180.6 184.2 198.0 184.2 184.2 191.4 181.2 156 4768.8 162 4945.8 168 5122.8 174 5303.4 180 5480.4 186 5657.4 192 5834.4 198 6008.4 204 6189.0 210 6360.0 216 6543.6 222 6724.2 228 6901.2 234 7081.8 240 7258.8 246 7432.8 252 7609.8 258 7790.4 264 7971.0 270 8155.2 276 8353.2 282 8537.4 288 8721.6 291 8913.0 300 9094.2 30.6 30.5 30.5 30.5 30.4 30.4 30.4 30.3 30.3 30.3 30.3 30.3 30.3 30.3 30.2 30.2 30.2 30.2 30.2 30.2 30.3 30.3 30.3 30.3 30.3 Standard deviation of Cumulative Integrated Mean concentration for Group IV Is 0.5 for entire study. DO 067540 TDF-MTT AL r.ONF YZ0083103 -24- 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Cumulative Exposure Record Group V - 300 ppm Date Mean Dally Exposure Test Exposure ConcenDay Number tratlon SD ' Cumulative Inte- Dally Total grated Dally Concen- Total Concen- Mean Exposure tratlon Exposure tratlon Concen- Hours Hours Hours Hours tratlon 1/14/80 1/15/80 1/16/80 1/17/80 1/18/80 1/21/80 1/22/80 1/23/80 1/24/80 1/25/80 1/28/80 1/29/80 1/30/80 1/31/80 2/1/80 2/4/80 2/5/80 2/6/80 2/7/80 2/8/80 2/11/80 2/12/80 2/13/80 2/14/80 2/15/80 ) 2 3 4 5 8 9 10 11 12 15 16 17 18 19 22 23 24 25 26 29 30 31 32 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 292 25 275 3 300 22 291 22 293 15 296 8 296 14 284 12 285 12 305 3 295 7 303 6 303 16 304 19 306 5 303 29 302 3 299 13 303 4 304 6 299 11 300 5 300 12 300 3 301 12 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 1752 6 1,752 292 1650 12 3,402 284 1800 18 5,202 289 1764 24 6,966 290 1758 30 8,724 291 1776 36 10,500 292 1776 42 12,276 292 1704 48 13,980 291 1710 54 15,690 291 1830 60 17,520 292 1770 66 19,290 292 1818 72 21,108 293 1818 78 22,926 294 1824 84 24,750 295 1836 90 26,586 295 1818 96 28,404 296 1812 102 30,216 296 1794 108 32,010 296 1818 114 33,828 297 1824 120 35,652 297 1794 126 37,446 297 1800 132 39,246 297 1800 138 41,046 297 1800 144 42,846 298 1806 150 44,652 298 DO 067541 CONFIDFNTTAl YZ0083104 -25 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study in Rats with Benzene Inhalation Report Cumulative Exposure Record Group V - 300 ppm (cont.) Date Mean Dally Exposure Test Exposure Concen Day Number tration SD Cumulative Inte- Daily Total grated Daily Concen- Total Concen- Mean Exposure tratlon Exposure tration Concen Hours Hours Hours Hours tration 2/18/80 2/19/80 2/20/80 2/21/80 2/22/80 2/25/80 2/26/80 2/27/80 2/28/80 2/2 9/80 3/3/80 3/4/80 3/5/30 3/6/80 3/7/80 3/10/80 3/11/80 3/12/80 3/13/80 3/14/80 3/17/80 3/18/80 3/19/80 3/20/80 3/21/80 36 37 38 39 40 43 44 45 46 47 50 51 52 53 54 57 58 59 60 61 64 65 66 67 68 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 301 6 299 5 301 8 296 7 301 8 299 4 299 17 302 12 303 4 291 11 311 42 301 5 299 11 300 14 300 5 303 20 310 22 297 25 291 18 300 32 233 18 302 32 303 31 319 51 302 23 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 1806 156 46.458 298 1794 162 48,252 298 1806 168 50,058 298 1788 174 51,846 298 1806 180 53,652 298 1794 186 55,446 298 1794 192 57,240 298 1812 198 59,052 298 1818 204 60,870 298 1764 210 62,634 298 1866 216 64,500 299 1806 222 66,306 299 1794 228 68,100 299 1800 234 69,900 299 1800 240 71,700 299 1818 246 73,518 299 1860 252 75,378 299 1782 258 77,160 299 1746 264 78,906 299 1800 270 80,706 299 1758 276 82,464 299 1812 282 84,276 299 1818 288 86,094 299 1914 294 88,008 299 1812 300 89,820 299 Standard deviation of Cumulative Integrated Mean concentration for Group V is 4.0 for entire study. 00 06754? C0NpT Of-NTTAl YZ0083105 Date Mlran L 1/14/80 1/15/80 1/16/80 1/17/80 1/18/80 1/21/80 1/22/80 1/23/80 1/24/80 1/25/80 1/28/80 1/2 9/80 1/30/80 1/31/80 2/1/80 2/4/80 2/5/80 2/6/80 2/7/80 2/8/80 2/11/80 2/12/80 2/13/80 2/14/80 2/15/80 26- 79-7342 Appendix B (cont.) Table 4 A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Dally Nominal Concentrations Test Exposure Day Number Nominal wncentratlon '.PPm, Group Group Group Group II III IV V 11 22 33 44 55 86 97 10 8 11 9 12 10 15 11 16 12 17 13 18 14 19 15 22 16 23 17 24 18 25 19 26 20 29 21 30 22 31 23 32 24 33 25 0.7 8.1 0.7 8.1 1.0 8.5 0.9 8.4 1.3 8.2 1.3 8.2 1.4 8.2 1.4 8.0 1.5 8.8 1.4 10.9 1.5 6.1 1.3 10.6 0.7 10.7 1.6 10.4 1.4 10.2 1.4 5.1 1.3 9.9 1.5 8.5 1.8 9.0 2.0 11.5 aa 1.0 9.1 0.8 9.1 1.9 12.7 0.8 10.4 29.8 30.7 29.0 28.5 30.6 28.9 27.9 28.2 30.7 31.4 25.9 32.7 31.3 31.3 29.5 25.2 30.2 29.8 30.2 30.6 23.5 30.3 31.1 30.9 28.6 316 305 337 302 307 321 310 290 300 300 299 290 317 312 317 302 312 320 324 319 308 319 314 329 321 Nominal concentration suspect due to possible weighing error, not Included in mean calculation. 00 067S43 CONFIDENTIAL YZ0083106 -27- 79-7342 Appendix B (cont.) Table 4 (cont.) A Dominant Lethal Inhalation Study in Rats with Benzene Inhalation Report Daily Nominal Concentrations (cont.) Date Test Exposure Day Number Nominal concentration Ippm) Group Group Group Group II III IV V Mi ran L (cont.) 2/18/80 2/19/80 2/20/80 2/21/80 2/22/80 2/25/80 2/26/80 2/27/80 2/28/80 2/29/80 3/3/80 3/4/80 3/5/80 3/6/80 3/7/80 3/10/80 3/11/80 3/12/80 36 37 38 39 40 43 44 45 46 47 50 51 52 53 54 57 58 59 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 1.2 11.3 1.3 10.3 1.1 10.1 1.1 9.5 1.1 10.6 1.0 10.5 a 10.0 1.1 12.1 1.9 10.4 1.3 8.3 1.4 10.3 1.2 10.8 1.2 10.2 1.4 10.9 1.2 10.4 1.3 11.3 1.0 10.5 1.3 10.7 30.0 29.7 28.7 28.3 29.1 29.3 30.1 30.5 30.3 29.4 32.5 31.0 30.4 30.3 30.4 30.6 32.4 31.3 325 317 326 315 320 318 324 325 319 314 318 321 337 338 343 344 361 338 Mi ran 04 3/13/80 3/14/80 3/17/80 3/18/80 3/19/80 3/20/80 3/21/80 60 61 64 65 66 67 68 44 45 46 47 48 49 50 1.2 11.9 1.5 11.0 1.9 6.9 0.8 9.9 0.9 10.3 1.0 11.3 1.0 10.4 35.9 32.9 32.0 34.5 34.7 32.4 34.4 352 362 321 375 355 343 363 Mean S.O. N 1.3 0.3 48 9.8 1.5 49 30.4 2.2 50 323 19 50 aNom1nal concentration suspect due to possible weighing error, not Included in mean calculation. 00 067544 CONF TDFNTI At YZ0083107 *. . -28- 79-7342 Appendix B (cont.) Table 5 A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Mean Daily Nominal Concentration VS. Mean Dally Actual (IR) Concentrations' Group II III IV V Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Nominal Cone. Ippro) 1.3 0.3 48 9.8 1.5 49 30.4 2.2 50 323 19 SO Actual Cone. t ppm) 1.0** 0.5 50 9.8 1.0 50 30.3 1.2 50 299** 7 50 Significantly different from nominal concentration: **[><0.01. Mean dally actual (IR) concentrations were compared t'o "mean daily nominal concentrations by the F-test and Students' t-test. When variances differed significantly (F-test), Students' t-test was appropriately modified using Cochran's approximation (t`). Snedecor, G. S., and W.G. Cochran. Statistical Methods. 6th ed., Iowa State University Press, Anes, 1967, pp. 104-106, 114-115. DO 067545 CONFTDFNT TAl YZ0083108 n oo ZTl o o o 3" z---si t*Or' Appendix C A OoMfnant-Lethal Inhalation Study With Benzene in Bats Individual Male Body Weights (grans) Group I (Chamber-Exposed Control - 0 ppm) 79-7 M2 An. No. 100 101 102 103 104 105 105 107 108 109 no in 112 113 114 115 1w 117 118 119 Initial A416 364 402 423 371 389 485 386 321 409 412 440 408 357 414 364 410 385 385 424 Week 1 A420 364 403 419 377 384 497 386 432 410 411 437 412 362 418 370 417 389 390 422 Week 2 A433 368 404 417 386 386 487 396 437 412 416 443 423 370 398 368 428 402 394 433 Week 3 A448 383 420 426 403 396 492 406 447 418 431 454 441 383 408 391 440 420 403 446 Treatment Period Week 4 Week 5 Meek 6 Week 7 A- A- A- A465 480 485 489 400 410 422 431 436 458 461 472 438 464 479 512 420 425 440 455 402 417 431 442 519 531 541 557 423 432 446 457 468 484 498 507 424 441 449 460 448 464 472 487 471 498 506 517 449 466 477 489 398 417 426 4M 432 452 462 472 398 412 418 423 455 469 480 494 440 473 481 494 423 430 436 445 460 467 471 482 Ueek 8 A504 435 486 519 460 456 580 466 526 473 97 532 502 438 477 439 506 513 451 486 Week 9 A515 446 491 538 476 464 597 483 539 480 506 546 519 451 494 446 526 528 461 486 Week 10 520 47 500 538 477 453 598 466 548 477 501 540 508 455 488 444 521 534 466 489 Change In Body Wt. 1-10 A100 83 97 119 100 69 101 100 116 67 90 103 96 93 70 74 104 145 76 67 Mean S.D. N 398 406 411 423 438 455 464 476 487 500 35 31 ze 28 30 32 31 M 37 39 20 20 20 20 20 20 20 20 20 20 500 40 20 94 20 20 Tor explanation of statistical symbol: A-; see Appendix I C-l N 0u>0 *i Appendix C (coot.) A Dominant-lethal Inhalation Study With Benzene in Rats Individual Male Body Weights (grans) Group II (J ppm) 79-7342 rtfla W 300* 301 302 303 301 305 306 307 309 309 310 311 312 313 314 31S 316 317 313 319 initial 455 419 393 455 417 411 432 415 415 401 410 351 396 371 305 379 394 391 445 399 452 416 395 463 405 399 425 415 399 391 407 345 397 370 396 396 390 402 451 400 week z 471 426 401 469 402 401 428 436 424 38$ 409 342 406 370 402 407 404 403 463 393 week 3 493 447 414 491 412 419 445 457 435 388 428 357 418 370 408 410 421 418 473 404 Treatment Period Week a Week 5 Week 6 week 7 TleeE B~""Week 9 "Week Iff 506 532 539 46B 461 479 473 486 419 454 430 442 452 459 465 485 510 533 535 541 548 567 434 452 445 467 489 499 439 465 454 466 484 496 449 472 482 502 514 527 476 495 503 5)5 530 533 449 466 464 473 481 483 393 409 . 409 421 437 443 44$ 474 481 484 454 498 373 386 400 404 416 425 434 459 466 472 482 499 380 394 391 398 407 419 421 446 451 465 479 488 434 444 453 467 473 488 436 456 459 474 492 501 431 454 465 476 484 494 498 516 521 533 533 555 423 444 447 458 465 476 494 474 569 487 495 538 546 481 451 481 433 504 410 493 493 456 500 548 471 Change In Body Wt. M0 7B 79 106 82 gg 113 131 83 60 74 88 107 40 98 97 66 98 97 71 Mean 5.0. K 407 27 20 406 27 20 412 31 20 425 35 20 441 37 20 460 39 20 465 471 39 36 20 20 477 39 19 491 39 19 491 40 19 88 21 19 Mo statistically significant differences from control. Aninal found dead during Week 8 of the treatment period, C-2 YZ0083111 ro> o zo -n H o O O' T! sj Z-t U>i m a> T> Appendix C (cent.) A Dominant-Lethal Inhalation Study Kith Benzene in Rats Individual Hale Body Heights (grans) Group III (19 ppm) 79.7342 An. Ho. 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 Mean S.D. N Initial 406 409 440 416 349 415 455 381 414 415 379 404 378 404 379 404 525 354 415 391 407 38 20 Ueet l 398 419 451 428 340 408 466 385 414 420 375 403 392 403 384 400 534 354 418 384 409 41 20 week z 400 420 445 430 351 412 480 397 400 431 368 410 396 412 397 406 540 357 426 391 413 42 20 Week 3 411 436 462 451 365 434 501 414 430 449 377 417 407 423 416 419 561 369 443 405 430 44 20 Treatment Period_________ ifeelc 4 Meet b beck t> weeFT 423 462 463 478 451 467 478 482 479 500 517 527 471 482 488 505 380 386 393 396 446 470 471 480 522 545 550 568 427 445 456 466 449 476 492 498 465 485 496 504 388 403 413 429 431 457 457 471 417 437 452 463 428 44Q 462 466 425 444 451 467 437 469 476 485 582 609 616 597 379 392 387 400 461 484 488 503 414 418 429 443 444 464 471 481 47 51 52 48 20 20 20 20 Meek 8 49e 487 536 522 410 494 578 471 511 509 441 478 475 474 480 491 646 408 510 449 Keek 9 505 507 548 526 419 503 587 . 481 533 524 444 486 484 486 488 513 664 416 525 455 493 505 54 56 20 20 vieek 10 504 498 556 527 421 513 587 487 533 527 438 471 482 490 499 510 647 419 522 463 50$ 54 20 Change in 106 79 105 99 81 105 121 102 119 107 63 68 90 87 115 110 113 65 104 79 96 18 20 Ho statistically significant differences from control. ( ! qO zo -i o OT1 ON'l Z-H L>fi I> N ou>e I Appendix C (cont.) A Dominant-Lethal Inhalation Study With Benzene in Rats 79-7342 Individual Nile Body Weights (grans) Croup IV (30 ppn) An. No. 700 701 702 703 704 706 706 707 708 709 710 711 712 713 714 715 716 717 71B 719 initial 3B1 428 419 343 362 419 365 sas 369 421 430 486 379 400 445 365 437 401 454 430 week l 388 428 . 420 352 363 414 363 391 372 420 419 482 372 412 453 377 442 410 456 440 Week z 388 427 406 362 367 408 366 397 381 422 413 497 377 419 456 382 444 414 477 428 week 3 393 431 421 371 381 423 373 404 388 443 435 508 401 446 474 397 457 422 499 462 Treatment Period Week 4 Week 5 Week 6 Week 7 391 408 412 416 435 457 457 473 433 450 464 480 386 405 417 431 393 414 417 431 449 474 479 494 392 401 407 420 418 448 452 457 414 418 424 442 449 474 483 494 438 460 461 470 526 545 552 563 415 431 444 461 455 477 480 497 496 520 524 540 409 418 426 433 472 488 494 503 437 459 461 468 511 536 536 552 471 489 492 502 Meek 8 421 483 496 437 442 504 436 457 455 510 480 566 482 475 525 444 513 476 571 501 Week 9 431 497 514 447 454 519 442 463 459 S21 491 572 487 487 562 461 522 479 567 516 Week 10 432 503 615 454 446 524 440 461 479 518 488 565 503 502 568 461 526 493 575 514 Change in Body Wt 1-10 44 75 95 102 83 110 77 70 107 98 69 83 131 90 115 84 84 83 119 74 Mean S.D. N 406 409 412 426 440 459 464 476 484 496 37 36 37 40 40 43 42 43 41 42 20 20 20 20 20 20 20 20 20 20 498 42 20 90 20 20 No statistically significant differences frc control. C-4 Appendix C (cont.) A Dominant-Lethal Inhalation study tilth Benzene in Rats 79-7342 Individual Kale Body Weights (grams) Group V (100 ppm) An. No. 900 901 902 903 904 90S 906 907 90S 909 910 911 912 913 914 915 916 917 918 919 Initial 494 436 431 425 374 376 414 413 394 445 381 353 444 409 389 360 391 401 481 371 week I 477 430 434 420 377 388 412 410 407 438 380 363 451 407 400 360 409 3SB 482 375 Week 2 452 438 436 432 386 389 417 405 416 450 386 366 448 411 398 340 412 391 485 378 Wert 3 469 453 461 441 396 399 434 416 427 471 40B 375 448 416 415 352 426 401 491 387 Treatment Period Week 4 Week 5 Week 6 Week 7 489 524 531 548 472 492 494 506 475 483 496 511 462 475 493 513 401 437 437 438 424 441 453 467 439 462 468 480 427 446 454 472 440 459 465 478 489 509 521 528 416 439 444 465 387 403 411 424 470 473 499 513 433 442 455 461 420 436 450 463 357 367 373 387 440 454 462 477 420 448 458 466 516 542 556 569 405 427 435 446 Week 8 561 516 514 522 453 481 491 462 479 536 474 432 541 47B 478 391 481 483 587 454 Week 9 "Weet 10 568 569 518 512 525 533 539 549 466 462 491 495 496 500 499 511 495 493 557 561 477 478 442 450 544 559 485 491 487 481 401 403 502 513 491 493 597 590 459 46t Change in Body Wt 1-10 92 82 99 129 65 107 88 101 86 123 98 87 108 84 81 43 104 100 108 86 Mean S.O. N 409 411 412 424 439 458 468 481 492 502 38 34 34 35 39 40 42 43 45 45 20 20 20 20 20 20 20 20 20 20 505 46 20 95 18 20 Ko statistically significant differences from control C-5 T- 'J' io N 00 U> I Appendix C (coot.) A Dominant-Lethal Inhalation Study With Benzene in Rats 79-7342 Individual Male Body Weights (grams) Group I (Charter-Exposed Control - 0 ppm) Animal Mo. 100 101 102 103 104 105 106 107 108 109 no 111 112 M3 114 115 116 117 118 119 Mean S.D. N Mating Period Week 1 Week 7 A- A* 512 636 447 461 516 541 542 551 476 486 456 468 600 614 489 509 556 574 474 485 519 526 545 574 522 537 460 473 497 523 459 480 521 535 539 547 465 472 496 522 505 521 40 41 20 20 Week I "Week 2 A- A572 590 477 482 562 579 574 595 503 516 485 500 641 656 527 534 592 603 498 512 538 542 595 607 554 575 486 498 539 554 490 503 544 552 561 574 481 486 536 546 538 550 45 47 20 20 Week 3 A597 486 581 588 521 502 663 530 619 520 538 619 586 500 547 496 564 577 490 553 5S4 49 20 Post--Mating Period Week 4 Week 5 Week 6 A- A- A604 612 616 497 511 529 599 608 614 615 622 643 537 537 553 5l6 528 547 692 700 722 546 555 566 630 640 654 543 552 568 562 574 588 629 632 644 597 599 607 511 515 538 547 573 583 512 511 527 573 575 586 566 598 619 502 507 522 563 557 568 568 575 590 51 51 51 20 20 20 For explanation of statistical symbol: A-; see Appendix I. SeeFT" Week 8 A- A634 637 538 533 637 628 656 668 557 557 551 553 723 739 581 585 671 674 574 583 593 597 652 641 616 614 539 543 601 602 537 541 594 599 624 626 532 534 575 580 599 602 52 54 20 20 Week 10 A653 549 641 690 569 557 773 591 699 599 612 666 626 557 619 557 599 634 540 597 616 59 20 DO 0 6 7 5 5 1 CONFTOFNT IA I N QO U> i no o z~n o o om O'd' Z on --vHi ^\) Append 5* C (cit.) A Oouinant-Lethal Inhalation Study With Benzene In Rats 79-73*2 Individual Hale Body Heights (grans) Croup II (1 ppa) AnInal Ho. 300* 301 302 303 304 30S 3ft 307 308 309 310 311 312 313 314 315 316* 317 318 319 Hean S.D. K Hating Period HeeFl Weft 2 523 551 475 495 537 581 491 509 511 525 525 540 554 566 504 529 455 476 495 525 438 460 518 541 415 431 504 516 504 512 490 524 503 509 531 553 484 496 498 518 34 36 19 19 UeeFl . 576 511 600 529 544 569 593 546 482 546 466 555 442 536 529 511 512 577 516 "KeeT? ' . 592 527 616 555 557 592 608 571 497 556 476 584 455 552 535 530 594 529 Week 3 588 533 621 557 557 601 615 579 508 570 481 588 438 548 542 . 538 60S 530 534 551 556 42 45 48 19 18 18 Post-Hating Period Keek 4 Week 5 ... 608 615 637 550 555 576 641 652 665 568 582 593 591 591 609 615 633 642 636 649 663 593 601 621 526 539 556 579 593 611 492 502 513 606 617 629 455 469 490 565 579 588 563 566 576 - 547 555 572 60S 620 620 537 538 556 ' KeekT" "Ifeek fl" 651 654 585 583 679 676 607 597 624 637 659 667 679 686 630 630 564 565 612 617 521 534 642 651 504 509 603 609 587 602 -- 589 591 639 640 563 561 571 581 595 608 612 48 49 48 49 49 10 18 18 18 18 Week 10 676 604 696 614 648 682 705 642 591 621 553 670 525 625 618 608 673 581 630 49 18 No statistically significant differences froa control. JAnlaal found dead during Meek 8 of the treatment period. bAnInal found deed Week 2 of the post-nating period. C-7 YZ0083115 J oo o zo -rt -aT(t oS>i *< Z-h cyni N i> oo 0u>0 i ON Appendix C (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats 79-7342 Individual Kale Body Heights (grams) Group III (10 ppm) Animal No. 500 SOI S02 503 504 505 506 507 500 509 510 511 512 513 514 515 516 517 518 519 Mean S.D. H Hating Period week i Week Z 513 523 502 513 554 575 537 $52 431 439 520 527 599 606 496 517 540 551 528 542 446 461 476 491 486 489 496 519 514 536 509 522 657 692 429 451 529 551 456 471 511 526 54 57 20 20 Meek 1 541 542 602 570 454 538 627 538 566 553 473 507 497 537 547 534 736 466 575 484 544 63 20 Meek Z 566 563 619 580 468 557 639 544 584 575 480 S09 509 5*2 555 544 758 474 593 492 558 67 20 Meek S4 570 576 620 589 475 560 642 538 591 576 485 519 518 545 569 555 764 479 591 492 563 66 20 Post-Mating Period Week 4 Meek 5 Week 6 ` Week 7 Tsrr- Seek 10 585 602 591 619 623 589 605 617 626 634 639 647 652 659 669 602 613 634 650 652 493 497 510 514 524 576 583 596 616 616 655 665 664 697 700 556 567 582 593 590 613 626 626 649 638 591 589 592 611 603 498 498 520 516 528 534 550 556 567 560 523 519 540 554 556 556 550 570 583 521 583 594 603 617 624 574 578 591 597 590 802 806 830 811 826 490 498 506 512 515 611 619 621 643 632 508 517 519 528 527 647 661 712 671 526 638 726 612 677 621 539 583 579 597 654 607 823 525 656 543 579 586 596 608 606 71 72 72 71 75 20 20 20 20 20 630 74 20 Ho statistically significant differences from control C-8 Appendix C (cont.) A Dominant-Lethal inhalation Study With Benzene In Rats 79-7312 Individual fele Body Weights (grams) Group IV (30 ppm) Animal !* 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 715 7)7 718 719 Mean S-D. N Hating Period feet l Wear? 440 449 515 535 520 536 455 476 435 450 534 551 444 463 455 489 498 505 526 540 491 524 579 602 496 505 514 542 580 586 470 479 495 518 488 514 584 609 514 499 502 519 45 46 20 20 Week 1 455 548 568 491 463 555 472 516 521 565 551 617 528 561 611 486 541 531 633 524 537 48 20 Ueek 7---- WeTT 483 492 559 564 590 600 507 514 480 487 568 575 450 501 528 529 527 539 575 595 558 569 631 638 546 556 577 577 633 639 495 502 553 559 548 548 643 656 537 550 551 560 48 49 20 20 Post-Mating Period Meek 4 MT Ueek S 506 512 518 577 590 597 614 621 631 524 529 548 502 501 512 596 594 597 517 517 528 553 555 569 562 564 571 616 626 632' 597 601 600 664 655 672 578 SSI 606 589 594 589 659 658 677 523 527 541 581 580 581 572 S7t 585 656 662 682 567 576 591 578 581 591 49 48 50 20 20 20 Ueek 7 ~Ueek~5 "UeeTTD 531 537 610 613 644 648 561 567 524 518 60S 607 536 540 573 568 584 580 658 654 622 621 691 684 62$ 623 611 611 690 691 544 550 602 608 592 598 701 689 616 607 555 616 656 592 530 625 556 574 615 674 648 709 656 630 703 566 630 609 710 629 606 605 53 51 20 20 624 52 20 Bo statistically significant differences from control. DO 0 0 7 5 5 4 CONFIDFNIT TAl O u0>0 I -3 ro> v z~n o v-1 O O O' "n ni z O' -A O' 3 'Ji 0u>0 < Appendix C (coflt.) A Dominant-Lethal Inhalation Study Kith Benzene In Rats 79-7342 Individual Hale Body Weights (grans) Group V (300 ppm) Animal Ho. 900 901 902 903 904 905 906 907 90B 909 910 911 912 913 914 915 916 917 918 919 Mean 8 Mating Period MM 1 week z 575 605 508 532 532 547 549 560 474 489 497 515 512 502 513 522 504 524 568 589 472 482 415 449 555 575 502 517 489 517 419 436 $16 526 502 5)6 606 608 463 469 509 524 48 47 20 20 Week 1 616 548 573 580 509 541 518 549 536 603 509 475 606 539 524 455 551 536 622 483 544 46 20 Week 2 637 560 591 602 520 558 530 569 558 617 530 491 631 549 529 468 565 554 628 496 559 48 20 Week 3 640 563 599 616 518 576 534 575 562 625 531 491 617 559 538 476 575 551 646 496 564 49 20 Ho statistically significant differences from control Post-Mating Period Week 4 Week 5 Week 6 649 667 683 581 586 607 620 625 634 621 637 641 531 531 540 588 595 613 553 557 573 587 604 615 576 579 597 644 645 671 549 562 577 508 512 515 643 651 683 570 583 590 543 549 568 498 507 525 583 593 623 578 588 600 651 662 668 511 515 531 579 587 603 48 50 52 20 20 20 Week 7 701 614 643 664 544 621 591 626 600 676 585 535 664 609 574 533 613 604 681 539 611 50 20 Week 8 681 614 650 673 551 611 592 631 601 680 565 543 658 610 . 573 539 608 608 681 539 610 49 20 Week 10 717 633 657 686 671 649 610 634 624 697 590 561 709 619 589 544 624 618 698 555 629 53 20 C-10 D-l Appendix D A Dominant-Lethal Inhalation Study With Benzene In Rats 79-7342 Gross Postmortem Observations - Hales Preface Hepatic pallor and an Increased incidence of accentuation of the lobular pattern of the liver were seen In the treated animals* Other morphologic findings occurred sporadically In the treated and/orxontroljsnimals and are not considered to be compound-related. \\ DO 067556 CONFTDFNTt A! YZ0083119 Group ppm 0-2 79-7342 Appendix D (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males Animal " Yype of Number Death8 Observation s)b 100 T 101 T 102 T 103 T 104 T 105 T 106 T 107 T 108 T 109 T no T 111 T 112 T 113 T 114 T 115 T 116 T N.O.A. Lungs: few scattered red foci on all surfaces of all lobes. N.O.A. Lungs: few scattered tan foci on all lobes Lungs: dark red areas (0.3 cm. in dlam.) on left lobe. Liver: accentuated lobular pattern. N.O.A. N.O.A. N.O.A. N.O.A. N.O.A. N.O.A. N.O.A. Lungs: scattered red foci on all lobes and "surfaces. Ear (Right): scab at eartag (0.2 cm.). Ear (Right): scab. N.O.A. N.O.A. N.O.A. 8T Terminal. "N.O.A. No observable abnormalities. 00 067557 CONFTDFNT TAL YZ0083120 Group ppm Animal Number I 0 (cont.) 117 118 119 11 300 1 301 302 303 304 305 306 307 0*3 79-7342 Appendix D (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males Type of Death* Observation s)b T Lungs: scattered tan foci on all lobes and surfaces. Ear (Right): scab (0.2 cm. in diam.) T N.O.A. T N.O.A. S.D. Liver: accentuated lobular pattern of all 1obes and surfaces; slightly enlarged. Lungs: all lobes and surfaces not collapsed; multiple white to tan areas (0.1 - 0.4 cm.); areas confluent; white areas extend beneath surface; found throughout tissue. Brain: cerebrun highly vascularized. T Lungs: left lobe adhered to thoracic wall. Raised, Irregular tan areas (0.1 - 0.3 cm.) with gray centers on all lobes. T Lungs: multiple scattered gray foci on all lobes. T Kidneys (Both): pale tan. Lungs: red focus on right posterior lobe. T N.O.A. T N.O.A. T Lungs: scattered tan and gray foci on all 1obes and surfaces. T Kidneys (Both): pale brown. Liver: all lobes pale brown with extremely accentuated lobular pattern. Lungs: multiple scattered red and gray foci on all lobes. *T * Terminal; S.O* * Spontaneous Death. N.0.A. * No observable abnormalities* DO 0675BB CONFTDFNTT At- YZ0083121 Group ppn Animal Number II 308 1 (cont.) 309 310 311 312 313 314 315 316 0-4 79-7342 Appendix D (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males Type of Death* Observation s)b T Lungs multiple scattered gray foci on all lobes. T Liver all lobes pale brown. lungs all lobes mottled pink and tan. "slight gray color at left lung and bronchi ole junction. Nodules embedded In edge of posterior lobe. Proliferation of 0.1 cm., light green-tan, solid nodules. T Ear (Right): torn with scab at eartaq. Kidneys (Both) scattered tan foci, liver all lobes pale tan. riihgs: all lobes dark red with scattered gray, tan and red foci. T Lungs: few scattered gray foci on all lobes. T KldneysfBoth): mottled red. Lungs: few scattered red foci on all lobes. T N.O.A. T N.O.A. T Lungs scattered red foci on all lobes and s'ur faces. S.D. Seminal vesicles: 2-3 x larger than normal with surrounding edema (clear). Urinary bladder large (3.2 x 1.8 cm.); red. tan, firm, filled with extreme amount of thin red fluid and calculi (0.1 - 0.3 cm. In dlam.). Kidneys (Both) pale brown with distended renal pelvis (0.8 x 0.4 cm.). Surrounding adipose tissue containing moderate amount of clear edema. Thoracic cavity: moderate amount of thin, red hydra-pleura. *T Terminali S.D. - Spontaneous Death. bN.0.A. - No observable abnormalities* CD.OONF 06TD7F5N5T9TAl YZ0083122 D-5 79-7342 Appendix D (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males Group ppm Animal Number Type of Death II 1 (cont.) 316 (cont.) S.D. Observation $)b Lungs: all lobes adhered to thoracic wall ana diaphragm with dark red and tan mottling, scattered with white areas (0.1 - 0.4 cm. In dlam.). 317 T Kidneys (Both) multiple clustered tan foci. Liver: all lobes pale tan; accentuated lobular pattern. Lungs: few scattered tan foci on all lobes. 318 T N.O.A. 319 T N.O.A. Ill 500 10 T Liver: accentuated lobular patterns of all lobes. 501 T N.O.A. 502 T Lungs: scattered red foci on all lobes. 503 T Lungs; few scattered tan foci on all lobes ana surfaces. Liver: all lobes and surfaces slightly pale tan; accentuated lobular pattern. 504 T N.O.A. 505 T Ear (R): torn at eartaa. Liver: all lobes pale tan; moderately accentuated lobular pattern. 506 T Liver accentuated lobular pattern of all lobes. 507 T N.O.A. 508 T N.O.A. *T Terminal; S.D. Spontaneous Death. <>N.0.A. No observable abnormalities. DO 067560 CONFTDFNTTAI YZ0083123 Group ppm Animal Number III 10 (cont.) 509 510 511 512 513 514 515 516 517 518 519 IV 700 30 701 702 D-6 79-7342 Appendix D (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males Type of Death Observation s)b T Lungs: scattered red foci on all lobes and surfaces. T Liver: accentuated lobular pattern of all lobes. Lungs: all lobes slightly red; white dis coloration (0.4 cm. In dlam.) on dorsal surface of right lobe. T Incisors (both: upper): missing. T N.O.A. T N.O.A. T N.O.A. T Liver: all lobes and surfaces slightly pale: accentuated lobular pattern. Ear (Right): scab (0.3 cm.). T N.O.A. T N.O.A. T Kidneys (Both): few scattered tan foci. Lungs :"Tew scattered tan foci on an lobes. T Testes(Both): slightly reddened. Kidneys (Both): mottled red. lunqs: red focus on right median lobe. T N.O.A. T N.O.A. T Liver: all lobes and surfaces slightly pale tan; accentuated lobular pattern. *T Terminal. "N.O.A. - No observable abnormalities. no of>75f> CONFtOFNTT YZ0083124 Group ppm Animal Number IV 30 (cont.) 703 704 705 706 707 708 709 710 711 712 , 713 714 0-7 79-7342 Appendix D (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males Type of Death* Observation! s)b T Testis (Right): not developed; small; 1/8 normal size. T Lungs: few scattered tan foci on all lobes. T N.O.A. T Liver: slightly pale red-brown; extremely accentuated lobular pattern. T N.O.A. T Liver: slightly accentuated lobular pattern of all lobes Forellmbs (Both): moderate hair loss. T . N.O.A. T N.O.A. T Liver: accentuated lobular pattern of all lobes and surfaces. Lungs: few scattered dark red foci on all "lobes and surfaces. T Liver: all lobes pale tan; extremely accen tuated lobular pattern. Lungs: few scattered tan foci. T Lungs: multiple clustered, raised tan areas (up to 0.3 cm. In dlam.) on all lobes. Ear (Right): slight scab. T Liver: all lobes and surfaces slightly pale " tan"; accentuated. Lungs: scattered red foci on all lobes and "surfaces. Kidneys (Both): few depressions (<0.2 cm.). *T Terminal. bN.0.A. * No observable abnormalities. 00 06756? CNFTDFNTTA! YZ0083125 Group ppm Arvfnial" Number IV 715 30 (cont.) 716 717 718 719 V 900 300 901 902 903 904 905 906 0-8 79-7342 Appendix 0 (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males type of Death8 " " ' ' " -"T Observation! s)b T Liver: slightly accentuated lobular pattern 'of all lobes. T Incisors (upper): maloccluded. Lungs: scattered gray foci on all lobes. T N.O.A. T Liver: all lobes pale tan; extremely accen tuated lobular pattern. Kidneys (Both!: extremely pale tan. Lungs: few scattered tan foci on all lobes. T N.O.A. T Lungs: moderate scattered gray foci on all lobes and surfaces. T Lungs: few scattered red foci on all lobes. T Liver: all lobes and surfaces slightly pale tan; accentuated lobular pattern. Lunas: scattered tan and red foci on all lobes and surfaces. T Kidney (Right): embedded cyst (0.4 cm. In Vlam.) near nil us; containing clear, thin fluid. Kidneys (Both); pale brown. T N.O.A. T N.O.A. T Testis (Left): slightly reddened. Lunas: red focus on ventral surface of left lobe. Liver: slightly accentuated lobular pattern of all lobes. T Terminal. "N.O.A. No observable abnormalities. DO 067563 rONFTOFNTTAl YZ0083126 Group ppm Animal Number V 300 (cont.) 907 908 909 910 911 912 913 914 915 916 917 918 0-9 79-7342 Appendix 0 (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Males Type of Death* Observation s)b liver: all lobes pale tan; extremely accen tuated lobular pattern. Lungs: few tan scattered foci on all lobes. T N.O.A. T Lungs: scattered tan foci on all lobes and surfaces. Liver: accentuated lobular pattern of all lobes and surfaces. T N.O.A. T Liver: slightly accentuated lobular pattern of all lobes. T Lungs: scattered tan and green foci on all lobes and surfaces. T N.O.A. T N.O.A. T N.O.A. T Kidneys (Both): mottled red: depressed area (left) : (fl.4 x 1.0 cm.). iver: slightly accentuated lobular pattern of all lobes. Lungs: red area (0.2 cm.) on dorsal surface of right median lobe; red focus of right an terior lobe. Ear (Right): scab at eartag. T N.O.A. T Liver: red areas (0.4 - 0.8 cm. In dlam.) over surfaces of all lobes; moderately accen tuated lobular pattern. *T Terminal. "N.O.A. No observable abnormalities. DO 067564 CONFTDFNTTAl YZ0083127 Group ppn V 300 Animal Number 919 D-10 79-7342 Appendix D (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations * Males Type of' Death Observations)*** T Kidney (Right): renal calculi. Lungs: scattered gray foci on all lobes. Eye (Left): 20% opacity. *T Terminal. bN.0.A. No observable abnormalities. 00 0675A5 CONFTDFNTTAl YZ0083128 E-1 Appendix E A Dominant-Lethal Inhalation Study With Benzene In Rats 79-7342 Pregnancy and Implantation Data - Post-Treatment Hating Period Corp. Lut. Impl. VFS M.R. I;E. Preface 9 9 m. m u Corpora Lutea Implantations Viable Fetuses Mutagenic Ratio (for definition see Methodology and Reference; Appendix I) Implantation Efficiency (for definition see Methodology and Reference; Appendix I) YZ0083129 Appendix E (cont.) A DonInant-Lethal Inhalation Study tilth Benzene In Rats Pregnancy, Corpora Littea and Implantation Data 79-7342 Male Female COrp. Ho. Ho. Lot. Impl. A*F %* 100 200 14 13 201 20 19 101 202 203 1 15 15 15 102 204 205 18 17 Hot pregnant. 103 206 207 13 20 13 15 104 206 209 Not pregnant. 15 14 105 210 211 Hot pregnant. 15 14 10S 212 2131 19 13 17 14 107 214 215 15 12 15 12 10S 216 217 15 15 14 IS 109 218 219 12 16 11 16 110 220 22Ia 14 17 14 19 I.E. X 10-2 V92.9 95.0 93.8 98.3 94.4 98.1 75.0 93.3 93.3 89.5 - 98.3 97.9 93.3 98.3 91.7 96.4 98.2 - Post-Treatment Mating Period {Meek 1) - Group I - Chamber-Exposed Control dumber of Dead Implants It0 2 1 0 N.R. VFS X 10-2 K+J V13 1.9 17 10.5 14 6.7 15 1.7 Male Female Corp. I.E. Ho. -"? Lut. Impl. X 10-2 Ill 222 223 112 224 225 16 12 13 13 15 12 13 13 93.8 97.9 98.1 98.1 Number of Dead Implants 1 1 2 1 M.R. VFS X 10-2 14 6.7 11 8.3 11 15.4 12 7.7 0 17 1.5 113 226 9 8 88.9 227 13 9 69.2 1 0 7 12.5 9 2.8 0 13 1.9 114 228 13 13 98.1 0 13 1.9 1 14 6.7 229 15 14 93.3 1 13 7.1 115 230 Not pregnant. 2 12 14.3 231 14 14 98.2 2 12 14.3 116 232 12 4 33.3 0 4 6.3 1 13 7.1 233 14 14 98.2 0 14 1.8 3 14 17.6 117 234 13 13 98.1 1 12 7.7 1 13 7.1 235 M 14 98.2 1 13 7.1 1 14 6.7 118 236 18 18 98.6 1 17 5.6 0 12 2.1 237 16 15 93.8 0 15 1.7 0 14 1.8 119 238 13 13 98.1 0 13 1.9 1 14 6.7 239 12 12 97.9 0 12 2.1 0 tl 2.3 3 13 18.8 0 / 14 1.8 10 9 52.6 Total: Mean: S.0.: N: -- J95------ 5151.5----------------55-------------' 14.6 13.8 92.7 l.i 2.4 2.8 12.3 1.8 36 36 34 36 458" ' -mrr 12.7 7.8 2.6 9.1 36 36 YZ0083130 For explanation of statistical symbols: W, K+, K-, K+J, V-; see Appendix I. Total nuaber of Implants greater than total mmfoer of corpora lutea; data excluded from calculations of Implantation efficiency. no o z-n( o o OT1 OS!' z yi H-I>H ONJ' t-Z Appendix E (eont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora Lutea and Implantation Data 79-7342 e 1 fe. c o n f id e n t ia l Male Wo. 300* 301 302 303 304 JOS 306 307 306 309 310 Female No. 400 401 402 403 401 405 406 407 400 409 410 411 412 413 414 415 416 417 418 419 420 421 Corp. Lut. Inpl. -- 16 16 17 16 14 13 19 14 18 18 t2 12 IS 15 15 IS 17 15 13 12 Not pregnant. 15 15 14 14 16 16 15 13 Not pregnant. 16 16 16 15 13 10 13 13 I.E. X 10-2 - 98.4 94.1 92.9 73.7 98.6 97.9 98.3 98.3 88.2 92.3 98.3 98.2 98.4 86.7 98.4 93.8 76.9 98.1 Post-Treatment Hating Period {Week 1) - Group II - (1 ppm) Number oT '' Dead Implants H.R. ffS It 10-2 Hale Female Corp. Hu. No. Lut. ' I.E. Imp!, X 10-2 - 311 422 Hot pregnant. - 423 16 12 75.0 1 15 6.3 312 424 15 15 98.3 0 16 1.6 425 17 17 98.5 1 12 7.7 313 426 13 13 98.1 1 13 7.? 427 14 14 98.2 1 17 5.6 314 428 Not pregnant. 0 12 2.1 429, 17 15 88.2 0 IS 1.7 315 430 18 17 94.4 2 13 13.3 431 Not pregnant. 0 15 1.7 316 432 Not 0 12 2.1 433 18 13 72.2 317 434 Not pregnant. 0 15 1.7 435 15 15 98.3 0 14 1.8 318 436 13 12 92.3 1 15 6.3 437 14 14 98.2 0 13 1.9 319 438 15 13 86.7 439 Not pregnant. 1 0 15 6.3 15 1.7 Total: 155" 428 "2770-- Kean: is.:1 14.3 92.7 S.D.: l.B 1.8 8.2 0 10 2.5 N: 30 30 30 0 13 1.9 Number of Dead Implants - 1 2 0 4 2 0 3 1 1 0 0 0 22 0.7 1.0 30 *ta1e Do. 300 found dead during Week 8 of treatment period. oa TNLoOn'i' H.R. YFS X 10-2 - 11 8.3 13 13.3 17 1.5 9 30.8 12 14.3 15 1.7 14 17.6 12 7.7 14 6.7 12 2.1 14 1.8 13 1.9 4o4 toi.o 13,5 6.0 1.9 6.S 30 30 E-3 Appendix (cont.) A Dominant-lethal Inhalation Study With Benzene in Rats Pregnancy, Corpora Lutea ami Implantation Data 79-7342 Male Ho. 500 $01 502 503 504 505 506 $07 508 509 510 female Carp. No. Lut. I-E. Imp!. xio-z 600 13 601 13 13 98.1 12 92.3 602 12 12 97.9 603 Not pregnant. 604 Not pregnant. 605 10 9 90.0 606* 607 14 10 15 6 60.0 608 15 609 14 14 93.3 14 98.2 610* 611 16 17 Not pregnant. 612 Not pregnant. 613 Not pregnant. 614 Not pregnant. 615 18 18 98.6 616 12 617 16 12 97.9 16 98.4 618 12 619 14 12 97.9 12 85.7 620 12 1 621 Not pregnant. 8.3 Post-Treatment Hating Period (Week 1) - Group 111 - (10 ppm) Number of Oead Implants M.R. VFS xlO-2 Male Female Corp Wo. No. tut. Number of I.E, . Dead Implants x10-2 VFS M.R. xlO- 1 12 7.7 511 622 13 3 23.1 0 3 8.3 2 10 16.7 623 11 6 54.5 0 6 4.2 1 11 8.3 512 624 11 11 97.7 1 10 9.1 625 12 12 97.9 0 12 2.1 513 626 16 14 87.5 0 14 1.8 0 9 2.8 627 Not pregnant. 1 14 6.7 514 628 15 14 93.3 0 14 1.8 2 4 33.3 629 13 11 84.6 1 10 9.1 0 14 1.8 515 630 15 13 86.7 2 11 15.4 1 13 7.1 631 Not pregnant. 1 16 5.9 516 632 13 13 98.1 0 13 1S 633 17 17 98.5 0 17 1.5 517 634 635 Not pregnant. 17 13 76.5 0 13 1.9 518 636 11 10 90.9 0 10 2.5 2 16 11.1 637 15 15 98.3 1 14 6*7 0 12 2.1 519 638 12 12 97.9 2 10 16.7 1 15 6.3 639 13 13 98.1 0 13 1.9 0 12 2.1 Total: "TOT"---------- 3TO-- 24tw:r ---------- ZO---------- 340 ` Turr 1 11 8.3 Mean: 13.5 12.0 85.7 0.7 11.3 7.1 S.D.: 2.1 3.9 22.8 0.8 3.8 6.B 0 1 (25.0) N: 30 30 28 30 30 29 rO N F T D F N T *Total manher of implants greater than total nwber of corpora lutea; data excluded from calculation of implantation efficiency. Oo o O' --i O'O' > E-4 Mate Female Corp. Ho. No. let. Impl. 700 800 601 14 12 14 to 701 802 603 14 13 Not pregnant. 702 804 605 Not pregnant. 14 10 703 606 807 13 12 Not pregnant. 704 806 809 12 12 10 12 70S 810 811 14 16 5 15 706 812 813 13 14 13 12 707 614 615 Not pregnant. 15 13 706 816 817 17 17 16 10 709 610 819 16 16 16 13 710 820 621 15 15 15 14 I.E, xlO-z 96.2 83.3 92.9 71.4 92.3 83.3 97.9 35.7 93.8 98.1 85.7 86.7 94.1 58.8 98.4 81.3 98.3 93.3 n oo ZO ~n o a^ 2 cn H NJ N -* o J> 2S CUe>l i Appendix E (cont.) A Dominant-Lethal Inhalation Study Mith Benzene in Bats Pregnancy, Corpora Ltitea and Implantation Data 79-7342 Post-treatment Mating Period (Meek 1) - Droop IV - (30 ppm) Number of Dead Implants K.ft. VF5 nlO-2 Male Female Corp. Wo. Mo. iut, . i'.c* Impl. xlO-2 1 13 7.1 711 822 Not pregnant. 3 7 30.0 823 Not pregnant. 1 12 7.7 712 824 16 825 11 15 93.8 11 97.7 713 826 14 14 98.2 3 7 30.0 827 Not pregnant. 0 12 2.1 714 828 14 14 9B.2 829 Not pregnant. 1 9 10.0 715 830 14 1 11 8.3 831 14 14 98.2 13 92.9 1 4 20.0 716 832 16 2 13 13.3 833 16 16 98.4 14 87.5 0 13 1.9 717 834 14 14 98.2 1 11 8.3 835 Not preghant. 718 836 Not pregnant. 1 12 7.7 837 18 16 88.9 8 8 50.0 719 B38 17 17 98.5 5 5 50.0 839 Not pregnant. i 15 6.3 l 12 7.7 0 15 1.7 i 13 7.1 lotal: -T23---------- 351 2694.(1 Mean: 14.6 13.1 89.4 S.O.: 1.7 2.5 13.8 N: 29 29 29 Hunter o? Dead Imol ants M.S. Vts Klo-Z 1 14 6.7 2 9 18.2 0 14 1.8 0 14 1.8 0 14 1.8 0 13 1.9 2 14 12.5 1 13 7.1 0 14 1.8 2 14 12.5 0 17 1.5 " 39 34Z "335nr 1.3 11.8 11.6 1.7 3.1 13.1 29 29 29 E-5 Appendix E (cent.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora Lotea and Implantation Data 79-7342 Male Female Corp. No. No. let. ft1. 900 1000 1001 15 11 14 10 901 10024 19 1003 18 20 17 902 1004 1005 13 12 12 12 903 1006 1007 17 16 17 14 904 looe 1009 16 12 16 12 905 ion 1011 11 13 B 13 906 1012 n 12 12 Not prefleant. 907 ni4 WT5 13 14 13 13 900 1016 1017 14 13 - 14 13 909 nn 1019 13 10 11 2 910 1020 1021 11 14 11 M K10-" 93.3 90.9 94.4 92.3 97.9 96.5 87.5 96.4 97.9 72.7 98.1 97.9 98.1 92.9 96.2 96.1 64.6 20.0 97.7 98.2 Post-Treatment Hating Period (Week 1) - Group V - [300 ppm) Number of Dead Imolants M.R, Hale Female Corp. VFS xlO-* No. No. Lut. Imp!, xio-* dumber of1 Dead Imolants N.R. WS KlO-< 3 11 21.4 911 1022 12 11 91.7 0 11 2.3 2 6 20.0 1023 13 13 98.1 1 12 7.7 1 19 5.0 912 1024 15 15 98.3 9 6 60.0 0 17 1.5 1025 12 12 97.9 0 12 2.1 0 12 2.1 913 1026 15 14 93.3 9 5 64.3 0 12 2.1 1027 11 10 90.9 7 3 70.0 0 17 1.5 914 1026 Not pregnant. 0 14 1.8 1029 16 16 98.4 1 15 6.3 2 14 12.5 915 1030 M 13 92.9 2 11 15.4 4 8 33.3 1031 Not pregnant. 1 7 12.5 916 1032 K 15 93.8 0 15 1.7 0 13 1.9 1033 15 14 93.3 0 14 1.8 1 11 8.3 917 1034 18 15 83.3 0 15 1.7 1035 23 11 47.8 0 11 2.3 1 12 7.7 918 1036 14 14 98.2 3 11 21.4 0 13 1.9 1037 12 12 97.9 3 9 25.0 0 14 1.8 919 1038 17 17 98.5 1 16 5.9 0 13 1.9 1039 14 14 98.2 4 10 28.6 0 11 2.3 Total: 521-------...W" 175071-- 58 `""476 557.9 2 0 87.5 Mean: 14.2 13.1 91.1 1.6 11.5 14.9 S.D.: 2.7 3.0 15.5 2.4 3.9 21.6 0 11 2.3 N: 37 37 36 37 37 37 1 13 7.1 CONFTDFNTTAl Total matter of Implants greater than total nurtter of copora lutea; data excluded from calculation of Implantation efficiency. zo> D 7N-i iNnj I E-6 Appendix E (cont.) A Dominant-Lethal Inhalation Study tilth Benzene In Rats Pregnancy, Corpora lutea and Implantation Data 79-7342 Hale Female Corp. Ho. No. Lut, Impl. 100 240 241 A13 17 K13 16 101 242 243 14 20 14 14 102 244 24S 12 12 Not pregnant. 103 246 247 11 15 11 13 104 248 249 IS 13 14 11 105 2S0 261 14 14 14 14 106 262 263 15 15 15 15 107 264 266 15 13 Not pregnant. 108 266 257 Not pregnant. 18 14 109 268 269 13 14 13 14 110 260 261 14 IS 14 15 I* Ei KlO-Z V98.1 94.1 98.2 70.0 97.9 97.7 86.7 93.3 84.6 98.2 98.2 98.3 98.3 86.7 77.8 98.1 98.2 98.2 98.3 Post-Treatment Hating Period (Meek 2) - Group I - Chamber-Exposed Control Number of Dead Implants H.R. Kale Female Corp. VF$ xlfl-g No. No. lut. t.E* Impl. xlO-g Number of Oead Implants H.R, VFS xIO-* K- K- V- 0 13 1.9 Ill 262 15 12 80.0 1 IS 6.3 263 11 11 97.7 0 12 2.1 0 11 2.3 2 12 14.3 112 264 19 16 84.2 1 13 7.1 265 16 13 81.3 2 14 12.5 0 13 1.9 1 11 8.3 113 266 8 7 87.5 267 16 16 98.4 0 7 3.6 1 15 6.3 0 11 2.3 114 268 14 13 92.9 0 13 1.9 269 16 14 87.5 0 13 1.9 0 14 l.B 0 14 1.8 115 270 Not pregnant. 0 11 2.3 271 22 15 68.2 0 15 1.7 0 14 1.8 116 272 12 12 97.9 0 14 1.8 273 16 16 98.4 0 12 2.1 a 16 1.6 0 15 1.7 117 274 14 14 98.2 1 14 6.7 275 12 12 97.9 0 14 1.8 2 10 16.7 0 13 1.9 118 276 21 14 66.7 277 16 15 93.8 i 13 7.1 i 14 6.7 1 0 1 01 119 278 13 13 7.1 279 12 13 98.1 11 91.7 0 13 1.9 a 11 2.3 13 1.9 13 7.1 14 l.B 14 6.7 Total: "53B---------W3--"3250"------------ IS----------- Hean: 14.7 13.4 91.4 0.4 S.O.: 2.8 1.8 9.4 0.7 N: 36 36 36 36 597T 13.0 4.4 1.7 3.8 36 36 DO 0 b 7 F ,7 ? OONF TD FN T I At For explanation of statistical symbols: A-, K-, V-; see Appendix I Appendix E (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora Lotea and Implantation Data 79-7342 Male Female Corp. No. No. Lut. 300b 440 441 - IsL. - 301 442 443 12 11 Not pregnant. 302 444 445 15 15 Not pregnant. 303 446 447 Not pregnant. 16 15 304 448 449 14 13 12 12 305 450 451 18 12 16 2 o b-r oo n o C-TJ1 OX'l 306 % 307 308 309 4S2 453 454 455 456 457 458 459 14 13 16 16 24 15 14 13 16 14 15 15 Not pregnant. 14 14 sa) 310 460 > 461 IS 18 7 16 !(* xlO-Z 91.7 98.3 93.8 85.7 92.3 88.9 16.7 92.9 98.4 62.5 92.9 87.5 98.3 90.2 46.7 88.9 Post-Treatment Hating Period (Week 2) - Group II - (1 ppm) Humber of Dead lamlants - M.R, YES xlQ-? _ - Ha ale Female Corp. No. No. Lut, 311 462 15 463 13 Inpl. 14 13 i.e. xlO-" 93.3 98.1 Number of Dead Implants ' VFS 0 14 1 12 N.R. xlO* 1.8 7.7 1 10 9.1 312 464 11 11 97.7 465 16 15 93.6 0 11 2.3 1 14 6.7 0 15 1.7 313 466 19 15 78.9 467 12 12 97.9 5 10 33.3 5 7 41.7 314 468 10 10 97.5 1 14 6.7 469 15 15 98.3 0 10 2.5 2 13 13.3 0 12 2.1 315 470 11 5 45.5 1 11 8.3 471 11 1 9.1 0 5 5.0 I 0 (75.0) 0 16 1.6 316 472 16 15 93.8 2 0 87.5 473 Not pregnant. 0 15 1,7 0 13 1.9 317 474 16 15 93.8 3 13 18.8 475 14 14 98.2 0 15 1.7 0 14 1.6 1 14 6.7 318 476 Not pregnant. 1 12 7.7 477 13 13 98.1 Q 13 1.9 0 14 1.8 319 478 17 17 98.5 2 13 13.3 479 14 14 98.2 1 16 . 5.9 1 13 7.1 2 /12 14.3 0 7 3.6 0 16 1.6 TotlTms9-- Mean: 14.7 S.O.: 2.8 N: 32 (K im.i 12.7 85.1 3.9 23.3 32 32 -------------- 31----------1.0 1.3 32 373-- 11.7 4.0 32 37TT" 10.3 17.0 31 t*Ha1e No. 300 found dead during Week 8 of treatment. is s E-8 ro> o zo ->r-\4 O OT1 3N"i z-) yNiJ o> Male Female Gorp. Ho. No. Let, 500 640 641 12 17 t.f. JSL. KtO-Z 12 97.9 17 98.5 $01 642 643 14 13 Hot pregIftAHU 92.9 502 644 645 17 16 Not pregnant. 94.1 503 646 647 11 13 9 81.8 5 38.5 $04 648 649 11 11 11 97.7 11 97.7 505 660 651 16 16 Not pregnant. 98.4 506 652 653 Not pregnant. 14 14 98.2 507 654 655 Not pregnant. 13 12 92.3 500 656 657 17 12 17 98.5 12 97.9 509 658 659 11 15 6 54.5 14 93.3 510 660 661 13 14 13 98.1 14 98.2 Appendix E (cont.) A Dominant-Lethal Inhalation Study Kith Benzene In Rats Pregnancy, Corpora Lutea and Implantation Data 79-7342 Post-Treatment Hating Period (Nee* - Group III - (TO ppm) Humber of1 Dead Implants 0 1 H.R. VFS x10-2 12 2.1 16 5.9 Kale Female Ho. Ho. Sit 662 663 Corp. Lut. 17 10 i.f. ISBLl xlO-2 4 23.5 5 50.0 Humber of dead Implants 0 0 1 12 7.7 512 664 19 17 89.5 0 665 15 15 98.3 0 1 15 6.3 513 666 16 15 93.8 1 667 Hot pregnant. 4 5 44.4 514 668 Not pregnant. 1 4 20.0 669 14 14 98.2 0 1 10 9.1 515 670 13 13 98.1 0 1 10 9.1 671 16 16 98.4 0 1 15 6.3 $16 672 Not pregnant. 673 13 13 98.1 1 517 674 14 14 98.2 0 0 14 1.8 67S 15 14 93.3 1 518 676 15 15 98.3 1 0 12 2.1 677 20 17 85.0 1 2 15 11.8 519 678 13 13 98.1 0 0 12 2.1 679 14 14 98.2 1 0 6 4.2 2 12 14.3 1 12 7.7 2 12 14.3 Total: T55----- Nean: M.2 5.0.: 2.4 H: 32 411 iwrr -------- a--------- 12.8 89,0 0.8 3.6 19.1 0.9 32 32 32 N.8. VfS xl0~z 4 6.3 5 5.0 17 1.5 15 1.7 14 6.7 14 1.8 13 1.9 16 1.6 12 7.7 14 1.8 13 7.1 14 6.7 16 5.9 13 1.9 13 7.1 35Tw 12 .1 7.3 3.6 8.1 32 32 E-9 O (w--t u> I <1 Appendix (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora Lutea and Implantation Data 79-7342 oo a z-n o a'^Tl o SJ Z i/i H XI .-I Ln 3> Male Female Corp. No. No. tut. Impl. 700 840 841 18 13 18 13 701 842 843 15 14 14 13 702 844* 845 15 14 16 12 703 848 847 14 14 Not pregnant. 704 848 849 15 15 15 14 705 850 851 11 15 11 15 700 852 853 14 14 14 14 707 854 855 13 12 9 12 70S 856 857 15 15 14 15 709 858 659 18 18 18 18 710 860 861 Not pregnant. Not pregnant. I.E. xlO-z 98.6 98.1 93.3 92.9 85.7 98.2 98.3 93.3 97.7 98.3 98.2 98.2 69.2 97.9 93.3 98.3 98.6 98.6 Post-Treatment Mating Period (Week 2} - Group IV - (30 ppm) Number ot Oead Implants N.R. VFS xlO-2 Male Female Corp. No. No. Let. I.E* Impl. xlO-< 1 17 5.6 711 862 14 13 92.9 1 12 7.7 863 15 15 98.3 2 12 14.3 712 864 13 13 98.1 0 13 1.9 865 13 13 98.1 2 14 12.5 713 866 13 13 98.1 0 12 2.1 867 15 14 93.3 1 13 7.1 714 868 14 14 98.2 669 13 13 98.1 0 15 1.7 715 870 16 14 87.5 0 14 1.8 871 16 15 93.8 0 11 2.3 716 872 14 14 98.2 0 15 1.7 873 Not pregnant. 1 13 7.1 717 874 13 13 98.1 0 14 1.8 875 15 13 86.7 5 4 55.6 718 876 Not pregnant. 1 11 6.3 877 w 19 98.7 0 14 1.8 719 878 17 16 94.1 0 15 1.7 879 15 15 98.3 1 17 5.6 2 16 11.1 total: Mean: S.O.: N: 513 14.7 1.8 35 49b 3237.2 14.2 95.2 2.0 5.9 35 34 Nunfcer ot Dead Indents 0 0 1 1 0 5 1 0 1 3 0 2 1 1 1 1 35 1.0 1.3 35 *T tal nwater of Implants greater then total nunber of corpora tutea; data excluded from calculation of Implantation efficiency. N.R. VFS xlO- 13 1.9 15 1.7 12 7.7 12 7.7 13 1.9 9 35.7 13 7.1 13 1.9 13 7.1 12 20.0 14 1.8 11 15.4 12 7.7 18 5.3 15 6.3 14 6.7 461 Ztt/mb 13.2 8.2 2.5 10.6 35 35 E--10 N 0tt--*0l w i 00 Appendix E (cent.) A Dominant-Lethal Inhalation Study With Benzene In Rats Pregnancy. Corpora tutea and Implantation Data 79-73(2 r> oo zo ~n -H o O O' 'll SJ z yi H si M O' x> Hale Female Corp. Ho. Ho. Lut. lt*l. xI0!-2. *00 1040 1041 16 16 16 98.4 B 50.0 901 1042 1043 Hot pregnant. Not pregnant. 902 1044 1045 IS 16 13 06.7 16 98.4 903 1046 1047 13 13 13 90.1 13 98.1 904 1040 1049 14 20 14 90.2 16 80.0 905 1050 1051 16 15 16 98.4 14 93.3 906 1052 1053 14 15 14 98.2 15 98.3 907 1054 1055 13 15 13 98.1 15 98.3 900 1056 1057 16 16 Not pregnant. 98.4 909 1056 1059 14 14 14 98.2 14 98.2 910 1060 1061 1$ 17 IS 96.3 17 98.5 Post-Treatment Mating Period (Meek 2) - Group - (300 ppm) Humber of Dead Implants K.R. 1 VFS xlO-2 Kale Female Corp. Ho. Ho. lut. I.E, Impl, xio-2 1 15 6.3 911 1062 17 17 98.5 0 8 3.1 1063 13 12 92.3 912 1064 IS 15 98.3 1065 Not pregnant. 1 12 7.7 913 1066 16 16 98.4 3 13 18.8 1067 13 12 92.3 0 13 1.9 914 1068 15 14 93.3 0 13 1.9 1069 12 12 97.9 0 14 1.8 915 1070 15 15 98.3 0 16 1.6 1071 20 8 40.0 0 16 1.6 916 1072 16 16 98.4 3 11 21.4 1073 17 16 94.1 0 14 1.8 917 1074 15 15 98.3 1 14 6.7 1075 13 13 98.1 0 13 1.9 918 10764 12 13 2 13 13.3 1077 11 11 97.7 1 15 6.3 919 1078 W 16 98.4 1079 14 12 85.7 2 12 14.3 0 14 1.8 1 14 6.7 2 15 11.8 Total: "537 "' '' Kean: 14.9 S.D.: 1.9 R: 36 505 mi 14.0 93.3 2.2 12.9 36 35 Humber of Dead Implants ~ VFS 4 13 1 11 1 14 H.R. xlO* 23.5 8.3 6.7 8 a 0 2 2 5 0 I 1 1 1 0 0 1 W 1.5 2.0 36 8 50.0 4 66.7 14 1.8 10 16.7 13 13.3 3 62.5 16 1.6 15 6.3 14 6.7 12 7.7 12 7.7 n 2.3 16 1.6 11 8.3 452 T777T 12.6 11.7 3.0 16.0 36 36 *Total tnmber of Implants greater than total nber of corpora lutea; data excluded from calculation of Implantation efficiency. N g u> i V E-ll F-l Appendix F A Dominant-Lethal Inhalation Study With Benzene in Rats 79-7342 Certified Feed Information Week of Study 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Interval AcclImatlon Acclimation Pre-Treatment Mating Period Pre-Treatment Mating Period Female Sacrifices (Pre-Treatment) Treatment Period Treatment Period Treatment Period Treatment Period Treatment Period Treatment Period Treatment Period Treatment Period Treatment Period Treatment Period Post-Treatment Mating Period-Week 1 Post-Treatment Mating Period-Week 2 Female Sacrifice (Week 1) Female Sacrifice (Week 2) Post-Mating Interval Post-Mating Interval Post-Mating Interval Post-Mating Interval Post-Mating Interval Post-Mating Interval Post-Mating Interval Post-Mating Interval Date 12/11/79 12/18/79 12/24/79 12/31/79 1/8/80 1/15/80 1/22/80 1/29/80 2/5/80 2/12/80 2/19/80 2/26/80 3/4/80 3/11/80 3/18/80 3/25/80 4/1/80 4/8/80 4/15/80 4/22/80 4/29/80 5/6/80 5/13/80 5/20/B0 5/27/80 6/3/80 6/10/80 Lot. No. Oct 03792B Oct 03792B Oct 22791D Oct 227910 Oct 22791C Oct 22791C Dec 6791A Dec 06791A Dec 06791A Dec 20791C Dec 20791C Dec 06791E Dec 27792C Dec 27792C Jan 04801C Feb 1480 IE Jan 31801B Feb 14801C Feb 14801C Feb 14801C Feb 28801F Feb 14801A Feb 14801A Mar 06801E Mar 2680IE Mar 19802F Mar 19802F aPurtna Laboratory Chow 5002 (Certified Feed) mash; ad libitum. DO 067577 OONFTDFNTT Al YZ0083140 G-l Appendix G A Dominant-Lethal Inhalation Study with Benzene In Rats 79-7342 Analysis of Benzene________ Pre-test Study Interval Prior to Week 6 of the Initiation Treatment of treatment Period Completion of Treatment Date Collected: 12/3/1979 1/14/1980 2/18/1980 3/21/1980 Freezing Point (C) Purity {I) 5.5064 99.96 5.5084 99.97 5.5188 99.98 5.5291 100.00 Specific Gravity 0.8845 0.8845 0.8843 0.8844 Total Sulfur by Microcou- lometry <1.0 <1.0 <1.0 <1.0 -ppm Sats ppm 42 42 28 45 Toluene ppm 28 28 5 26 Acetone ppm 2 2 0.3 - Analyses were performed at the Exxon Research and Engineering Company Linden, N.J. DO 067578 CONFTDFNT TAl YZ0083141 H-l Appendix K A Dominant-Lethal Inhalation Study With benzene In Rats DETERMINATION OF POLLUTANTS IN DRINKING WATER GIVEN TO ANIMALS FOR STUDIES NO. 79-2425 AND 79-7342 Report No.: Sponsor: Prepared by: Issued by: 80023 Chemical Manufacturers' Association and American Petroleum Institute Washington, D.C. James C. Eschbach Date: September 3, 1980 Blo/dynamlcs Inc. Department of Metabolism and Analytical Environmental Sciences P.0. Box 43 Mettlers Road East Millstone, New Jersey 08873 Chemistry SUMMARY The drinking water given to rats for Studies No. 79-2425 and 79-7342 was sampled and assayed for residues which could interfere with the studies. The compounds assayed Included benzene, trace metals, pesticides and PCBs. The samples were assayed according to E.P.A. proposed and established pesti cide procedures using mass spectrometry, atomic absorption spectrometry and gas chromatography. The results obtained Illustrate that < 1.0 ppb of benzene, < 10 ppb trace metals assayed, <1.0 ppb chlorinated pesticides, < 4.0 ppb carbamates and < 2.0 ppb of PCBs were deter mined In the drinking water samples collected from the animal rooms. Joseph S. O'Srodnlck, Ph.D. Do O67579 Director confidfnttai Department of Metabolism and Analytical Chemistry YZ0083142 H-2 Appendix H (cont.) A Dominant-Lethal Inhalation Study With Benzene in Rats 80023 P 2 Of 4 1.0 INTRODUCTION Drinking water samples were collected at two Intervals from the rooms In which the animals for Studies No. 79-242S and 79-7342 were housed. These samples were assayed for compounds which could have adverse effects on the studies If they were present In the drinking water. The samples were assayed for benzene, trace metals, pesticides and PCBs at the beginning and end of the studies. 2.0 EXPERIMENTAL Water samples were collected and composited since they were from the same water source. An aliquot was taken for benzene for assay according to Method 624 for volatile organic compounds by purge and trap prior to g.c./mass spectrometry. This sample was collected In an air-tight container to prevent volatilization prior to assay. Additional aliquots were collected for the ana lyses of pesticides, metals and PCBs. The assays were performed according to proposed or approved E.P.A. methodology. 3.0 RESULTS The data obtained Illustrate that < 1.0 ppb of benzene, < 10 ppb trace metals, <1.0 ppb of chlorinated and <4.0 ppb carbamate pesticides and <2.0 ppb of PCBs were determined In the water samples assayed. Table I summarizes the data obtained In this study. DO 067580 CONFTOFNTTAl YZ0083143 Appendix H (cont.) A Dominant-Lethal Inhalation Stuuy With Benzene In Rats 80023 P 3 of 4 4.0 ACKNOWLEDGEMENT This study was supported by both the Chemical Manufacturers' Association and the American Petroleum Institute. oo OoNF 06?58] rFNTTAl YZ0083144 H-4 Appendix H (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats TABLE I. RESULTS OF DRINKING WATER ASSAYS FOR STUDIES NO. 79-2425 XND 79-7342 METALS Antimony Beryllium Cadmium Arsenic Lead Mercury Selenium Assayed 3/17/80 < 1.3 <1.0 < 1.0 < 0.5* <1.0 < 0.5 < 1.0 Assayed 6/12/80 ppp < 6.7b < 1.0 < 1.0 < 1.0 1.2 < 0.5 < 8.3 80023 P 4 of 4 PESTICIDES Lindane Heptachlor DDT Dleldrin Malathlon Carbamate PCB BENZENE <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 < 1.0 < 1.0 <1.0 <1.0 <1.0 <1.0 < 2.0 <1.0 a. Level of detection high because graphite method of analysis used Instead of hydride method of analysis. b. Level of detection higher because of possible loss In lamp efficiency. on o^7sa? confidfn^al YZ0083145 1-1 Appendix I A Dominant-Lethal Inhalation Study with Benzene In Rats 79-7342 Methodology and References Parameter Body Weight (Pre-mating Treatment Mating and Post mating periods) Method Torbal Torsion Balance. Statistical Analysis of Data Results of analyses by the indicated statistical procedures were reported for the following: Parameter___________________ _________ Method 1. Male Body Weights - Treatment Period, Mating Period and PostMating Period. 2. Male Body Weight Gain - Treatment Period. A A Corpora Lutea and Implantation Data -Post-Treatment Mating Period 1. Number of corpora lutea 2. Number of Implantations 3. Number of dead Implants 4. Total mmber of dead Implants 5. viable fetuses 6. Implantation efficiency (pre-Implantation loss) 7. Mutagenic ratio (post-Implantation loss) 8. Pregnancy rate 9. Male Impregnating Index A A A A A C C B B YZ0083146 1-2 Appendix I (cont.) A Dominant-Lethal Inhalation Study with Benzene In Rats 79-7342 Methodology and References (cont.) Statistical Analysis of Data (cont.) Method Aj: Statistical evaluation of equality of means was made by the appropriate one-way analysis of variance technique, followed by a multiple comparison pro cedure if needed. First, Bartlett's test was performed to determine if groups had equal variances. If the variances were equal, parametric procedures were used; If not, nonparametric procedures were used. The parametric procedures were the standard one-way ANOVA using the F-dlstribution to assess significance. If significant differences among the means were Indicated, Dunnett's test was used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the KruskalWallis test was used, and if differences were Indicated a summed rank test (Dunn) was used to determine which treatments differed from control. A statistical test for trend In the dose level was also performed. In the parametric case (i.e. equal variance), standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case, Jonckheere's test for monotonic trend was used. The test for equal variance (Bartlett's) was conducted at the 1%, two-sided risk level. All other statistical tests were conducted at the 5% and 1% twosided risk levels. aSnedecor, G.W., and Cochran, W.G., Statistical Methods. 6th ed.,Iowa State Univ. Press (1967), Hollander and Wolfe, Nonparametric Statistical Methods. John Wiley and Sons, New York (1973), Dunne'tt, C.W..J. Am. Sta. Assn., Vol. 50 (1955) and Biometrics, Vol . 20 (1964). Bartletts's Test ANOVA Dunnett's Test Kruskal-Wallls Summed Rank Test (Dunn) Regression Analysis Trend Lack of Fit Jonckheere's Statistic pp. 296-298 pp. 277-279 pp. 1096-1121, 482 pp. 114-116 pp. 131 pp. 149-152 pp. 160-164 pp. 120-123 S&C SAC D H&W HAW S&C S&C K&W 00 067584 OONFTDFNT TAi YZ0083147 1-3 Appendix I (cont.) A Dominant-Lethal Inhalation Study with Benzene in Rats 79-7342 Methodology and References (cont.) Statistical Analysis of Data (cont.) Method Bb: Statistical analysis of incidence data was performed using contingency tables. First, a standard chi-square analysis was performed to determine if the proportion of incidences differed between the groups tested. In keeping with standard statistical practice, if any one cell had an expected value of less than 5, this step was not reported. Next, each treatment group was compared to the control group using a 2x 2 Fisher Exact test and the significance level was corrected via the Bonferronl inequality to assure An overall test of the stated significance level. Thirdly, Armitage's test for linear trend in the dosage groups was performed. ^Chi-square: Snedecor, G.W., and Cochran, W.G., Statistical Methods, 6th ed., Iowa State Univ. Press. Ames, Iowa, 1971, pp. 250-253. Fisher Exact Test: Bradley, O.V., Distribution Free Statistical Tests. Prentice Hall Englewood Cliffs, N.J., 1968, pp. 195-203. Bonferronl Inequality: Miller. R.G.. Jr.. Simultaneous Statistical Inference McGraw-Hill Book CoTT'H.Y.' 1966, p. 15. Armitage's Test: Armltage, P., "Tests for Linear Trends In Proportions and Frequencies". Biometrics, Sept. 1955, pp. 375-386. DO OONF TDFNT T A1 YZ0083148 1-4 Appendix I A Dominant-Lethal Inhalation Study with Fluorocarbon 142b In Rats 79-7288 Statistical Analysis Method C Analysis for dominant-lethal studies based on corpora lutea and implantation characteristics will be as follows. The nunber of corpora lutea, implants (total) and dead implants will be determined for each female in the study, females with fewer than two implants will be excluded from the analysis. The mutagenic ratio or ratio of the number of dead Implants to the total nunber of Implants will be computed. The implantation efficiency or ratio of the nunber of implants to the nunber of corpora lutea will be computed (If the result Is greater than unity, the ratio for that female will be Ignored). The mutagenic ratio and implantation efficiency will be modified by Bartlett's transformation (Snedecor and Cochran, page 328), then trans formed by the inverse sine transformation (Snedecor and Cochran, page 327). The two ratios will be analyzed using a standard nested design with the females nested within males nested within treatments (Gill, pages 201-210). Statistical significance of the treatment differences will be based on the F ratio of "treatment mean square" to "male mean square" In the nested design. If the treatment differences are statistically different (p<.05, or p<.01), statistical significance among individual transformed means will be determined by the least significant differences technique (Snedecor and Cochran, page 272). Untrans-formed mean ratios will be presented. All tests were reported at the 5% and 1% level of significance. References: Snedecor, G.W. and Cochran, W., Statistical Methods, 6th edition, Iowa State University Press, Ames, Iowa, 1971. Gill, J.L. Design and Analysis of Experiments In the Animal and Medical Sciences, Vol. I., Iowa State University Press* Ames, Iowa, 1978. DO 067586 OONFTDFNTTA! YZ0083149 1-5 Appendix I (cont.) A Dominant-Lethal Inhalation Study with Benzene In Rats 79-7342 Methodology and References (cont.) Statistical Analysis of Data (cont.) Statistical Notations Symbol Statistical Statement No Sig p<0.05 p<0.01 Absolute Oata A- No statistical differences among the means (parametric ANOVA). A+ The means differ significantly (parametric ANOVA). * ** Significantly different from control (Dunnett*s). K- No statistical differences among the means (Kruskal-Wallls, nonparametrlc). K+ . The means differ significantly (Kruskal-Wallls, nonparametric). L The response is linearly related to the dose levels. J There is an ordered response to dosage. F Lack of fit. v- F-test Incidence Data + ++ Significantly different from control (Fisher Exact Test). DO 067587 CONFTDFNT TAI YZ0083150 J-l Appendix J A Dominant Lethal Inhalation Study in Rats with Benzene Histopathology Report Bio/Dynamics Project Number M15, 79-7342 Submitted tot Dr. A. L. Kn zevich Director, Pathology Depi Bio/Dynamics Inc. Cast Millstone, N. J. DO 067588 OONFTDFNTTAl YZ0083151 J-2 79-7342 A Dominant Lethal Inhalation Study in Rats with Benzene Histopathology Report Table of Contents 1. Introduction 2* Materials and Methods 3. Results and Discussion 4. Summary Table I - Microscopic Findings Control Rats Table II - Microscopic Findings Exposed Rats Table III - Summary of Microscopic Findings DO 067F89 CONFTOFNTTAl YZ0083152 J-3 A Dominant Lethal Inhalation Study in Rata with Benzene Histopathology Report 79-7342 1. Introduction A study was designed and performed to assess the muta genic potential of a test compound administered by inhalation to Charles River rats. This report denotes and discusses the results obtained from microscopic evaluation of testes and epididymides of rats in the high dose group. 2. Materials and Methods The experimental design is detailed in the protocol (Bi / Dynamics Project No. M-15, 79-7342). Five groups, each con sisting of twenty male rats were exposed for $ hours a day, 5 days a week, for 10 consecutive weeks. Group 1 animals re ceived unmedicated chamber air. Groups II, III, IV and V animals were administered Benzene by inhalation at doses of 1 ppm 10 ppm, 30 ppm and 300 ppm respectively. At the end of the study all animals were sacrificed and necropsied. The testes and epididymides were collected and preserv d for 48 hours in Bouin's fixative and transferred into 10% neu tral buffered formalin. After fixation, the specimens from Groups I and V were embedded in paraffin, sectioned at a thickness of approximately 6 microns and stained with hema toxylin and eosin for evaluation by light microscopy. DO 067590 CONFTDFNT TAl YZ0083153 J-4 79-7342 . 3. Results and Discussion The microscopic findings are presented in Tables I and II and summarized in Table III. Microscopic findings were denoted as follows: N * Within normal histologic limits 2 * Mild degree of indicated change 3 Moderate degree of indicated change * " Tissue absent U * Tissue inadequate (fragmented, etc.) In animal 908 (Group V, High Dose) the testes revealed moderate tubular degeneration and atrophy characterized by multifocal depletion of spermatogenic epithelium as well as thickening of the basement membrane, in animal 900 (Group V, High Dose) one testis revealed focal disarray and syncitial trans formation of spermatogenic epithelium. The changes thus de scribed occurred only in testes of treated animals. However the single occurrences are judged to be much too sporadic to be associated with the experimental inhalation administration of Benzene. oo YZ0083154 J-5 79-7342 A Dominant Lethal Inhalation Study in Rats with Benzene Histopathology Report Summary A study was designed and performed to assess the muta genic potential of Benzene administered by inhalation to Charles River male rats. Microscopic evaluation of the testes and epididymides f control and treated rats (High Dose Croup) revealed few lesions in treated rats. However there was no conclusive evidence indicating compound related injury. DO 067592 OONFTDFNTT Al YZ0083155 3707 3708 101 3709 102 3710 103 3711 104 3712 3713 106 3714 107 3715 3716 109 J-6 79-7342 Table I A Dominant Lethal Inhalation Study in Rats with Benzene Control Rats Microscopic Findings Animal Number Sex Pathology Number 80- Group Testes Epididymides SOT SOT 001 M MM M M M M MM M IIIIXXXIXX NNNNNNNNNN NNN *NNNNMN Ml YZ0083156 79-7342 J-7 Table I A Dominant Lethal Inhalation Study in Rats with Benzene Control Rats Microscopic Findings 3723 116 115 3721 114 3720 113 3719 112 3718 111 3717 110 Animal Number Sex Pathology Number 80- Group Testes Epididymides MMMMM MMMMM I I II II I II1 N NNN N N NN NN * N N N N N N N NN 1 3722 ! 3724 117 3725 118 3726 119 DO 067594 OONFTDFNTIAL YZ0083157 3727 3728 901 3729 902 3730 903 3731 904 3732 3733 3734 907 3735 3736 606 806 006 79-7342 J-8 Table II A Dominant Lethal Inhalation Study in Rats with Benzene Exposed Rats Microscopic Findings Animal Number Sex Pathology Number Group 80- ion VOoDs M M M M M M MM MM VVVVVVVVVV Testes - seminiferous tubules, degeneration - focal syncitial cell formation Epididymides - globular/syncitial cell formation NNNNNNN N 3 2 NNN*NNN*N 2 DO 067595 OONF TDFNT T At YZ0083158 79-7342 J-9 Table II A Dominant Lethal Inhalation Study in Rats with Benzene Exposed Rats Microscopic Findings Animal Number Sex Pathology Number Group 00o ( 73 910 911 912 913 914 916 917 918 6T6 irt pH MMMMMMMMMM r- CO OS m tm- rro- o W pH r* NV f* r> rro- w m hn V VVV VV V V VV Testes Epididymides u N NN N N N U NN NNNNN *NN *N D0 067596 oonftdfntiai YZ0083159 J-10 Table III A Dominant Lethal Inhalation Study in Rats with Benzene Summary of Microscopic Findings Organ/Microscopic Finding Group Testes - seminiferous tubules, degeneration - focal syncitial cell formation Epididymides - globular/syncitial cell formation I 79-7342 `. V 1/18 1/18 1/16 DO 067597 OONFTOFNTTAl YZ0083160 K-l Appendix K A Dominant-Lethal Inhalation Study with Benzene In Rats PERSONNEL DEPARTMENT/TITLE NAME STUDY DIRECTOR Raymond E, Schroeder STUDY MONITOR/REPORT WRITER Loretta Bshasso SMALL ANIMAL: REPRODUCTION A TERATOLOGY SECTION MANAGER Raymond E. Schroeder SUPERVISOR Maureen Kenny TECHNICIAN-IN-CHARGE Kathryn E. Sloan VETERINARIAN Fred A. Hines DIRECTOR INHALATION TECHNOLOGY George M. Rusch SUPERVISOR Gary Hoffman TECHNICIAN-IN-CHARGE Kim Nltahara STUDY MONITOR/REPORT WRITER Cecelia A. Bozak PATHOLOGY PATHOLOGIST Henry F* Bolte 79-7342 DEGREE M.S. B.A. M.S. B.A. B.S. D.V.M. Ph.D. B.A. B.A. B.A. D.V.M., Ph.D. Do &759S CONF TDfrHTTAl YZ0083161 K-2 Appendix K (cont.) A Dominant-Lethal Inhalation Study with Benzene In Rats PERSONNEL DEPARTMENT/TITLE NAME DATA LOGISTICS SUPERVISOR B.J. Guth QUALITY ASSURANCE SUPERVISOR Craig M. Lamb 79-7342 DEGREE B.A. B.A. OO O67599 oonftofNTTA( YZ0083162 R-l Appendix R An Inhalation Female Fertility Study with Benzene in Rats PERSONNEL DEPARTMENT/TITLE } NAME STUOY DIRECTOR Raymond E. Schroeder STUDY MONITOR/REPORT WRITER Loretta Bshasso SMALL ANIMAL: REPRODUCTION & TERATOLOGY SECTION MANAGER Raymond E, Schroeder SUPERVISOR Maureen Kenny TECHNICIAN-IN-CHARGE Barbara Bernard VETERINARIAN Fred A* Hines DIRECTOR INHALATION TECHNOLOGY George M, Rusch SUPERVISOR Gary Hoffman TECHNICIAN-IN-CHARGE Kira Nltahara STUDY MONITOR/REPORT WRITER Cecelia A, Bozak PATHOLOGY PATHOLOGIST DATA LOGISTICS SUPERVISOR Henry F. Bolte B.O. Guth QUALITY ASSURANCE SUPERVISOR Craig M. Lamb 79-2425 DEGREE M.S. B.A. M.S. B.A. D.V.M. Ph.D. B.A. B.A. B.A. D.V.M., Ph.D. B.A. B.A. 00 067600 CONFIDENTIAL YZ0083163 BIO/DYNAMICS INC. METTLERS ROAD EAST MILLSTONE, N.O. OB873 QUALITY ASSURANCE STATEMENT ************ Stucly # 79-7342 Test Article/Substance AP1-CMA Benzene--,,----------- -- Sponsor American Petroleum Institute and Chemical Manufacturers Asspclajign Listed below ere dates that this `tudy was tnspeeJ"urance Unit and the dates findings were reported to the Study Director ana to Management, Datefsj of Inspection Datets) findings reported to Study Director Date(si findings reported to Management 1/10/80 1/23/80 4/02/80 6/12/80 7/14-17/80 8/13-14/80 8/18/80 8/29-9/2/80 1/11/80 1/23/80 4/10/80 6/17/80 9/26/80 1/08/81 8/25/80 9/16/80 1/11/80 1/24/80 4/10/80 4/14/80 6/18/80 6/19/80 9/26/80 10/09/80 8/28/80 1/26/81 8/26/80 9/03/80 12/02/80 12/08/80 date Quality Assurance DO 067601 CONFTDFNTTAL YZ0083164 BIO/DYNAMICS INC. METTLERS ROAD EAST MILLSTONE* N.J. 08873 QUALITY ASSURANCE STATEMENT Study # 79-2426 xest Article/Substance API-CHA Benzene Sponsor American Petroleum Institute and Chemical Manufacturers Association Listed below are dates that this study was Inspected by the Quality Assurance Unit and the dates findings were reported to the Study Director and to Datefsj of Inspection Date(s) findings reported to Study Director Date(s) flndlnqs reported to Manaoement 1/23/00 3/05/80 3/19/80 4/10/80 7/24-8/4/80 8/13-14/80 8/29-9/14/80 1/24/80 3/06/80 3/21/80 4/10/80 1/12/81 1/08/81 10/03/80 1/24/80 3/06/80 3/21/80 3/25/80 4/10/80 1/23/81 1/28/81 8/28/80 1/26/81 12/02/80 12/08/80 date YZ0083165 t