Document NGy5o2gQNpvDKNmMQg54kwrD8

KfcCElVED 0EC15 1980 ftMMUIn. Bio/clynamics Inc. Division of Biology and Safety Evaluation PROJECT NO. 79-7342 A OMINANT*LTHAL INHALATION STUDY * WITH BENZENE4N RATS Final Report Submitted To: Chemical Manufacturers Association Washington, D.C. 20009 Date: December 1, 1980 DO 067489 CONFTDFNTTAL. YZ0083052 Bio/aynamics Inc. PROJECT HO. 79-7342 A DOMINANT-LETHAL INHALATION STUDY WITH BENZENE IN RATS ABSTRACT This study was conducted for the Chemical Manufacturers Association and American Petroleon 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 CO 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-Implantation loss (Implantation efficiency ratio) and post-Implantation loss (mean mmber 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-life 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. ft .Box43 MautoraRoad EastMlllatono,NewJartay06873 (201)873-2360 T4x:844507 CaMa:BKVDYN DO 067490 CONFIDENTIAL YZ0083053 79-7342 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 ppm), the mean number of dead Inplants and mean mutagenic ratio values were higher than control for each week of the post-treatment matlnq period. These differences were not statistically significant, T/ie 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 post-mating 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 number of dead Implants and mean mutagenic ratio were primarily related to the performance of a slnqle 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. do 0 CONpl YZ0083054 Table of Contents 79-7342 nwmlcH inr. r.' Introducln..7Tr. ............. 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 Observation Data................................................................ 9 D. Male Impregnating Indices andPregnancy Rates.............................. 10 E. Corpora Lutea, Implantation andImplantationEfficiency Data.............. . 10 F. Uterine Implantation Data Amplified - Mean Number of Viable Fetuses and Dead Implants............................................................................. 11 G. Mutagenic Ratio Data................................................................................ 12 H. 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 tfctagenlc Ratio Values............................................................................... ............................... 19 Appendices A. Pregnancy and Implantation Data -Pre-Treatment Mating Period............ A-1 B. Inhalation Report.................................................................................... B-l C. Individual Male Body Weights............................................................................ C-l D. Gross Postmortem Observations -Males........................................ D-l E. Pregnancy and Implantation Data-Post-Treatment Mating Period............ E-1 F. Certified Feed Information.................................. ..........................................F-l G. Analysis of Benzene.......................................................................................... G-l H. Analysis of Water.............................................................................................. H-1 I. Statistical Analysis......................................................................................... 1-1 J. HI stopathology Report.............................................. ........................................J-1 K. Personnel......................................................................... K-l DO 0674<^2 confidential YZ0083055 -1- 79-7342 X nioA m. /tfymio lne. - - -.1* INTROOUCJlOfk^. - 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 Blo/dyna- mlcs, Inc., Mettlers Road, East Millstone, New Jersey 08873. All specimens, raw data and final report are maintained In the Blo/dynamlcs archives. II. MATERIALS A. Test Substance: Suppller: Description: Date Received: Lot No.: Stability: Storage: Benzene Chemical Manufacturers Association Clear colorless liquid with characteristic odor. Novenber 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 CONFTDFNTIAI YZ0083056 -2- 79-7342 ,,,0/day tismloit inc. MATERIALS (cont.): ZestAnlmdtyr Species: Rat Strain; CD (Sprague-Dawley derived) Number of Animals: Purchased: Males: 140 9 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 CONFIDFNTTAt YZ0083057 L * namloi Inc. HI. -. f* PRIEMTAL**tJTL TNE: Group Exposure Treatment^ No. of Hats HaTeS------ Females lOltITTSJT'llHBNl Pre-treatment Post-treatment Mating Period Mating Period 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 40 40 40 40 40 80 76l 80 80 80 Treatment schedule - 6 hours/day. 5 days/week for 10 consecutive weeks. lone Group II male died during the treatment period reducing the nunber of females exposed to males to 38 per week (2/male; 19 males) for a total of 76 for this group. A sufficient number 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-Implan tation 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 CONFIDENTIAL YZ0083058 nknilM Inc. -4- 79-7342 IV. METHODS (cont.) A. In-Life (cont.) Food: Water: Hunldlty: s 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 Humidity: 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 hunldlty was found to be out of range approximately 18%25% of the time. Tap - Elizabeth Water Co. automatic watering system - ad libitum 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, DOT and Dleldrln Benzene Carbamate pesticide screen DO 0674-^ [DENIAL YZ0083059 79-7342 II to <Jy mnilca |nr. IV. METHODS (cont.): 'Ar tn-tlfe (cont.): Identification of Animals: Each male and female were Identified with a metal ear tag bearing Its animal number. This number plus the Blo/dynamlcs project number comprised a unique Identification number. Assignment to Groups : / Benzene Administra tion: Males of proven fertility (pre-treatment mating period) were assigned to groups so as to equalize pre-test body weight. Route: Inhalation Chamber Operation: See Appendix B Frequency: 6 hours/day for Group I and test substancetreated groups. Duration: (Dates of Treatment): Five deys (Mondey-Fr1day)/week for 10 consecutive weeks (14 January 1980 - 21 March 1980). Atmospherlc Sampl1ng: 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 06.7497 CONFIDENTIAL YZ0083060 -6- 79-7342 IV. METHODS (cont.): 'Ar -in-tffe (cont.): Mating Design and Mating Period: Pre-treatment Mating Period: Fertility of each male used In this $tu<1y was determined on the basis of performance during a one week pre-treatment mating peri 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 YZ0083#61 -7- 79-7342 Hio/ /dynamic* Inc. IV. METHODS (cont.): " '-B.-Poatmort'SlTfcont.): 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, 1960. 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 antlmesometrlal margin to better reveal the Implants. The nunber 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 CONFTDFNTTAt YZ0083062 ----------------nr:--mrrrouy (coni.)!----------------- -8- 79-7342 nio/. B. Postmortem (cont.): Uterine Impl antatlon 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 s1*e 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 mmber of corpora lutea. The uteri and ovaries from all females (pregnant and non pregnant) were appropriately Identified and preserved Intact In a 10X 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 10% neutral buffered formalin): epididymides saved In 10% formalin. Tissues Examined Histopathologically: C. Statistical Analysis: Testes and Epididymides (all control and high-dose animals). See Appendix I. DO 067500 CONFTDFNTIAt, YZ0083063 .......... i--i----ii-- - V. RESULTS AND DISCUSSION: -9- '-- .................-- 79-7342 llio/ /dynimlc* Inr. A. Mortality: No mortal 1 tjToccurred fn 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 bodty 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 lacrlmatlon 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, lacrlmatlon was noted In 3 control, 3 Group I! (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 lacrlmatlon observations was similar between the control and treated groups. DO 067501 CONFTDFNTTAt YZ0083064 -10- ni a! /dynamics In,-. * During the mating period several control and treated males were noted with'-e-swol WTcerVlcal area. This observation Is typical of slalodacryoaden- Itls (SDA) 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-llfe evaluations were noted with similar frequency between the control and treated groups. D. Mai4 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 summarised 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 sunmarired In Table 3 (page 18). DO 067-SO? CONFIDENT I Al __ YZ0083065 -11- 79-7342 ------------------------------------------------------------------------------------------------------- ------------------------------------------------------------ * /"> namlcs ine. The mean nunber of corpora lutea were comparable between the control -v- --------- - and treated groups JEgr each week of the post-treatment mating period. The mean nunber of Implantation sites at week 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 (l.e* decreases In nimber of corpora lutea) for this group at this,same Interval, The mean nimber 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-lmplantation 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 ppm) 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 067S03 CONFIDENT T.At. YZ0083066 -12- 79-7342 II lo/, ______ /Hynimlc* Inj. # dominant lethal studies) range from 0,3-1.4 with a weekly mean of 0,7 over 22 weekly Intervals (three separate studies). In respect to historical control data add concurrent control data at Week 1, the Increase In mean number of dead Implants for treated Groups II, III 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 Meeks 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). Durlng Week 1 of the post-treatment matlng perlod the mean mutagenlc 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 ppn, 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 fmales (Nos. 1026 & 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 OO 067504 CONFIDENTIAL * YZ0083067 -13- 79-7342 nj*>/ /dynamloi 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 . ^^ ""*-1 " ' 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. Ill and IV (1. 10 and 30 ppn. 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 0): 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. 0fe7505 OoNFjOFn tal YZ0083068 ^nrr . -- I. -H*I*sto*p* a tholeglcal-Evaluations (Appendix E)j The testes and epididymides of rats from the control and high-dose' groups were evaluated hlstopathologlcally. In high-dose male No. 908* the testes revealed moderate tubular degeneration and atrophy characterized by multifocal depletion of spermatogenlc eplthellun as well as thickening of the basement membrane. During the post-treatment,mat1ng 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 spermatogenlc 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 stmmary, 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. Raymorra E.Schroeder Study Director wwi > i vj Ke ni/'jaiig Vice President of Toxicology ofc7*0^, . -r r\C NT ^ YZ0083069 \ no o 2O -mO< Oovj' 2 yi N *HI->< NOJ owo Group ppm I 0 (Chambereiqjosed Control) II 1 III 10 IV 30 V 300 Week: Initial Mean S.D. N 398 35 20 Mean S.D. N Mean S.D. N Mean S.O. N Mean S.O. N 407 27 20 407 38 20 406 37 20 409 38 20 Table 1 A Dominant-Lethal Inhalation Study With Benzene In Rats 79-7342 j -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------1-- Mean Male Body Weights - grams v 'r1 let 2 / wt. Treatment Period ____________________________ 'Gains _T'~jr~ 5 --jz~~~JZ~ 6 "3" 10 1-10 406 411 423 438 455 464 476 487 500 500 31 28 28 30 32 3! 34 37 39 40 20 20 20 20 20 20 20 20 20 20 94 20 20 406 412 425 441 460 465 471 477 491 491 27 31 35 37 39 39 36 39 39 40 20 20 20 20 20 20 20 19 19 19 409 413 430 444 464 471 481 493 505 505 41 42 44 47 51 52 48 54 56 54 20 20 20 20 20 20 20 20 20 20 409 412 426 440 459 464 476 484 495 498 36 37 40 40 43 42 43 41 42 42 20 20 20 20 20 20 20 20 20 20 411 412 424 439 458 468 481 492 502 505 34 34 35 39 40 42 43 45 45 46 20 20 20 20 20 20 20 20 20 20 88 21 19 96 ' 18 20 90 20 20 95 18 20 No statistically significant differences from control. 3U] a a tiu vu A p . 1 no o zTt o >- o *< N Om 0\^j ZHi--I OO00' I> o(*o> Group PP Week: Table 1 (cont.) A Dominant-lethal Inhalation Study With Benzene in Rats Mean Hale Body Weights - grams (cont.) . 79-7342 -----r Post-Treatment Katinq Period iz --f- Post-Katinq Interval . t Z 3 X 5 _6_ ~"T" a --W I 0 (Chamberexposed Control) Mean S.O. N 505 521 40 41 20 20 538 550 554 568 575 590 599 602 616 45 47 49 51 51 51 52 54 59 20 20 20 20 20 20 20 20 20 II Mean 496 516 534 551 556 571 581 595 608 612 630 1 S.D. 34 36 42 45 48 48 49 48 49 49 49 N 19 19 19 18 18 18 18 18 18 18. 18 III Mean 511 526 10 S.D. 54 57 N 20 20 544 558 563 579 586 596 608 606 630 63 67 66 71 72 72 71 75 74 20 20 20 20 20 20 20 20 20 IV Mean 502 519 537 551 560 578 581 591 606 605 624 30 S.D. 45 46 46 48 49 49 48 50 53 51 52 N 20 20 20 20 20 20 20 20 20 20 20 Olli V Mean 509 524 300 S.D. 46 47 N 20 20 544 559 564 579 587 603 611 610 629 46 48 49 48 50 52 50 49 53 20 20 20 20 20 20 20 20 20's 3 No statistically significant differences from control. . * *10- i 93 -- nI/" ---- -- Table 2 A Dominant-Lethal Inhalation Study With Benzene In Bats 79 73m^//<|v"*l"ll>` inr. 1 " ' 11 Male Impregnating Indices and Pregnancy Rates Post-Treatment Mating Period Group ppm I (0) t II 0) III (10) IV (30) V (300) I (0) II 0) in (10) IV (30) V (300) Male Impreonatlnq Index* w. t 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 Preanancv Rateb --r 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. Male Impregnating index * No. of males Impregnating at least one female/No. of males caged with females. Pregnancy rate No. of pregnant females/No. of females exposed to males. DO 067509 OONF TDFNT IAI YZ0083072 -18Table 3____________ A Oomlnant-Lethai innaiati&n Study With Benzene In Rats 79-7342 _ n,% nimioi inn. _CflrporaJ,utea, Implantation and Implantation Efficiency Data *' ' - -w Post-Treatment Mating Period Group ppm Mean No.,/Pregnant Female Corpora Implantation Lutea Sites Mean Implantation Efficiency Values* % Week 1 A4F K+ I 14.6 13.8 <>, II 15.3 14.3 (1) III 13.5 12.0 (10) V92.7 92.7 85.7 IV 14.6 13.1 (30) 89.4 V (300) 14.2 13.1 WSek 2 91.1 A- K- I 14.7 13.4 (0) V91.4 II (1) 'III (10) 14.7 14.2 12.7 12.8 85.1 89.0 I 14.7 14.2 (30) 95.2 V (300) 14.9 14.0 93.3 tar exolanatl on of stalilstteal tvmhnls A+. mrrr--orr see Appendix 1-5. No statistically significant differences from control, individual female values presented In Appendix C* DO 067510 CONFTOFNTTA! YZ0083073 -19- /y-/342 Table 4 A Dominant-lethal Inhalatlot. Study m</, -----------------wun Mnmm |,, Rati----------------------------------- Inr Uterine Implantation Data Amplified and ^ Mean Mutagenic Ratio Values Group ppm I Mean No ./Pregnant Females Implantation Dead Viable Sites Imp! ants Fetuses Week 1 K+ 13. B K- K+J 1.1 12.7 Mean Mutagenic Ratio* % V7.8 11 14.3 0) III 12.0 (10) 0.7 13.5 0.7 11.3 6.0 7.1 IV 13.1 (30) 1.3 11.8 11.6 V (300) 13.1 1.6 Week 2 11.5 14.9 KI 13.4 (0) K- K0.4 13.0 V4.4 II 12.7 (1) III 12.8 (10) 1.0 11.7 0.8 12.1 10.3 7.3 IV 14.2 (30) 1.0 13.2 8.2 V (300) 14.0 1.5 12.6 11.7 For explanation of statistical symbols (1*e. d, K+, K-, V-) see Appendix 1-5. aMutagen1c 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. DO 067511 CONFIDENTIAL 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. Imp]. VFS Preface Corpora Lutea Implantations Viable Fetuses DO 06751? CONFIDENT IA( YZ0083075 \ Appendix A (cant.) A Danlnent-lethal tnlialatlon Study With Benzene in Rats Pregnancy* Corpora Lute* and Inplantatfm Bata Pre-Treatnent Mating Period* - Group I - Chanter*exposed Control Male Wo. too 101 102 103 1M fOS toe 107 MS 109 110 Penal* Wo. 1308 1310 1331 1332 1333 1334 1335 1336 1389 1390 1408 HM H19 1420 H23 H24 HS3 1454 wee H69 1480 1481 Gorp. lot. 10 W Inpl* 6 H Wot pregnant. 1Z 12 13 13 16 13 17 M 13 10 2 13 12 13 12 18 17 19 19 IS IS wH 19 W Wot pregnant. 10 12 21 17 IS IS 13 15 13 Data not analyzed statistically. Nunter of Dead tool ants 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 1 0 0 0 0 IS 6 14 12 13 13 16 13 2 12 11 IS 19 ts 14 16 11 17 IS 13 13 Main Wo. Ill 112 113 114 115 116 117 m 119 Fenalo wo. W92 1493 1494 1495 1496 1497 1509 1510 I5TS 1516 1539 1540 1551 1552 1560 1561 1564 1565 Total: oin. $.0.: W: Corp. lot. tnol. 15 IS 14 v 14 Wot pregnant. 15 14 Wot pregnant. H 14 Wot oreonant. 17 17 Not pregnant. WOt pregnant. 13 13 IS 14 Wot pregnant. 19 18 Wot pregnant. Wot pregnant. 13 13 458 15.3 2.7 30 411 13.7 3.3 30 79-7342 NMber Of Bead talents . 1FS 01 ' li IS 13 'ft 0 /H f 0 14 0 15 4 13 1 12 0 14 1 17 0 11 0.4 0.9 30 13 400 13.3 3.2 30 SO 1 ** DO 0 6 7 5 1 3 CO NFIDFNTIAI 00 u> I 1 Hate 300 301 302 303 3M 305 306 307 300 309 310 Female Wo. 1326 1327 1341 1342 1343 1344 1347 1348 1377 1378 1385 1386 1391 1395 1402 1403 1404 1405 1406 1407 1415 1416 Corp. let. fnpt. Not pregnant. - 17 16 16 16 16 14 14 M 15 15 18 18 14 13 13 13 17 17 12 12 Not pregnant. 17 7 16 6 19 W 22 15 13 13 K 13 14 14 Not pregnant. Not pregnant. 17 14 ro> o zn o Oat# not analyzed statistically. < r-1 o o g rn w -4-Uy(- Apt ,p~-4 i* IS P / Appendix A (cont.) A Donlnant-Lethal Inhalation stu$y With Benzene In Rats Pregnancy, Corpora Lute* and Inplantation Data Pre-Treatment Hating Period* - Group 11 - (' PP*) Nunber of Dead tael ants 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 1 0 mi 16 16 14 14 15 18 13 13 14 12 7 6 16 15 13 12 M 1 13 Hale Ho. 311 312 313 314 315 316 317 318 319 Female No. 1433 1434 1447 1448 1457 1458 1490 1491 1517 15 1519 1520 1525 1526 1541 1542 1547 1548 Total: Kean: S.D.: N: Corp. Lut'. IH&l Not pregnant. 15 15 14 14 14 14 13 13 Not pregnant. IS 14 13 10 15 15 M 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 79-7547 ! a ffMhrr (If rDead tap! ants its 0 /* 1 2 12 0 14 76 1 13 0 K> Q IS 2 12 1 16 1 11 0 13 1 11 0 12 0M 20 0.6 1.4 32 415 13.0 2.0 32 1! dale do. $00 SOI Stt SOS SOI SOS 5QS SOT SOS 509 sw Feaiale Wo. 1320 1322 13S5 1356 mi 1362 ms 1364 1383 1384 1387 1380 1398 1399 1400 1401 1413 1414 1417 1418 1449 1450 Corp. Let. tail. 14 14 IS 14 13 13 17 14 18 14 16 14 11 4 n7 44 13 13 dot pregnant. IS IS 14 13 20 17 Rot pregnant. 18 IS IS IS 16 16 16 14 15 15 17 17 17 17 Data not analyzed statistically. Appendix A (cont.) A Ooninant-LethaT Inhalation Study With Sentene In (Eats Pregnancy, Corpora Lute* and lagrlantatlon Oats Pre-Treatnent Mating Period* - Group HI - (10 pp) Neater of Dead tailants 0 0 1 0 1 0 0 2 0 0 0 0 0 0 1 1 0 0 1 0 Si 14 14 12 14 13 14 4 $ 4 13 15 13 17 IS 14 IS M 16 16 17 dale do. 511 512 513 5H 515 516 $17 518 519 Fenale do. 1461 1463 1498 1499 1500 1501 1511 1512 1521 1522 1531 1532 1537 1538 1549 1550 1555 1556 Total: dean: S.D.: N: Corp. tot. 'Inel. 92 dot pregnant. W 16 dot pregnant. K dot prei 13 13 13 10 4 14 14 Not pregnant. 11 3 13 13 15 15 not pmplOT* 16 16 14 14 17 IB . 17 8 476 14.4 3.2 33 413 12.5 4.5 33 79-7342 Readier of Dead tail ants 0 0 .t .i 2 J ' 16 0 13 1 12 04 0 14 03 1 12 0 15 0 16 2 ' 12 1K s3 17 0.5 1.0 33 396 12.0 4.7 33 DO 0 6 7 5 1 5 CO NFIDENTIAL I t 4 - oOZ-n OO O *< N u0>0 omZ ^''JJ' ->r~t a 'J Male No. 700 701 702 703 TOO 706 706 707 706 709 710 F nale No. 1303 1301 1317 1319 1353 1354 1357 1358 1365 1366 1369 1370 1375' 1376 1379 1300 1361 1362 1021 1022 1438 1439 Corp. Lot. Inpl. 16 16 12 12 Not pregnant. 19 14 14 14 10 14 15 10 IS 16 16 16 15 10 Not pregnant. 15 M 13 13 12 12 21 15 17 14 61 Not pregnant. 16 16 Not pregnant. 15 14 18 13 *Oata not analyzed statistically. I Appendix A (cent.) A Dominant-Lethal Inhalation Study With Benzene tn Rats Pregnancy, Corpora Lutes and tap) antatIon Data Pre-Treatment Mating Period* - Group IV - (30 pps) Amber of Dead Inolants 0 1 0 0 0 1 0 2 0 4 1 0 2 1 0 0 r 0 l El 16 11 14 14 14 13 15 14 10 10 12 12 13 13 1 16 14 12 Male No. 711 712 713 714 715 716 717 718 719 Female No. 1455 1456 1474 1475 1486 1487 1504 1505 1523 1524 1527 1528 1543 1544 1545 1546 1562 1563 Total: Mean: 5.0.: N: Corp. Lot. '*Inol. 15 15 Not pregnant. 18 15 17 17 61 18 17 13 13 14 13 15 15 17 11 Not pregnant. 14 14 12 12 Not pregnant. 14 14 14 > 14 16 16 15 6 487 14.8 3.0 33 430 13.0 3.8 33 79-7342 Nunber of Dead Inplants 3 0 0 0 0 0 0 0 4 it yVFS 12 / 1 15 17 1 17 . 13 13 15 7 1 13 1 11 0 > 14 1 13 0 16 06 23 0.7 1.1 33 407 12.3 3.0 33 A-S 1 n oa ztl o mo N OmZ Ostni' 00 u> H>-t si I> 00 Male Wo. MO Ml 902 M3 904 MS MW Ml MO 909 910 Female MO. 1313 1314 1337 1338 13(5 1346 1349 1350 1351 1352 1359 1360 1373 1374 1411 1412 1427 1428 1431 1432 1436 1437 Corp. tut._ JRh. 1$ 10 Wot pregnant. Wot pregIMOlm 10 6 12 10 14 14 17 16 16 16 16 14 Wot pregnant. 11 11 IS IS 15 11 13 11 17 15 15 15 Mat preg|MKe 10 M 12 4 12 3 17 15 26 14 Data not analyzed statistically. I Appendix A (cant.) A tafuiit-lttkil Inhalation Study With Benzene In Rati Pregnancy, Corpora Lute* and Implantation Data Pre-Treatment Mating Period* - Group V - (300 ppm) Humber of Dead Inplants 0 1 0 0 1 0 0 1 0 1 0 0 0 2 0 0 0 1 YFS 10 7 to 14 15 16 14 15 10 11 15 15 8 4 3 IS 13 Male Ho. 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.: W: Corp. Lot. Impl. 16 16 Wot pregnant. Not pre 5 2 15 15 Not pregnant. 16 15 14 14 16 16 14 14 18 15 . 16 Nat pregnant. 14 13 15 14 Net pre90n(e 16 16 15 15 455 14.7 3.4 31 394 12.7 3.9 31 79-7342. Ij Member of Dead Implants 1 l r is 0 0 0 0 0 1 1 0 0 0 2 0 12 0.4 0.6 31 !2 ` 15 15 . 14 16 t3 15 16 13 14 14 15 382 12.3 3.9 31 -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: Director, Inhalation Technology Report Preparation: Cecelia A. Bozak, B.A. Study Monitor, Inhalation DO 06751R confidential YZ0083081 t ", w..-* * 'w w' 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 hours/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' HUT females ipjiigmm (target concentration - ppm) t're- treatment Post-trei Mating Period Mating Pe Control - Chamber exposed 20 40 80 Benzene - 1 ppm 20 40 76 Benzene - 10 ppm 20 40 80 Benzene - 30 ppm 20 40 80 Benzene - 300 ppm 20 40 80 ^Treatment schedule - 6 hours/dayj 5 days/week for 10 consecutive weeks. DO CONf IOFnTT*L YZ0083082 I -3 79-734?1 ^ynmlo Im. 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 50*C In Groups II, III, and IV and 80C In Group V. These temperatures were selected as the minimum required to achieve complete volltalIzatlon 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 Instrunents 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 C ONFTDENTIAt. YZ0083083 -4- 79-7342 ____________ Appendix B (cent.) 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 volune of one cubic meter with an effective volune of 760 liters. They were operated dynamically at an air flow between 1S5 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 Instrunent Company, Mlran Long Pathlength Infrared Analyzer Model IA (In-house Identification nunbers "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 Instrunental settings. The calibration curves for this test material are presented on pages 9-17 of this appendix. zM1ran L was used from Day 1 (1/14/80) through Day 59 (3/12/80) of the study. Ml ran 4 replaced Ml ran 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 Ml ran L (1/14/80 - 3/12/80) and the second set corresponding to Ml ran 4 (3/13/80 - 3/21/80). YZ0083084 -------------------------------------- -5Appendix 0 (conty) A Dominant-Lethal Inhalation Study In Rats with Benzene 79-734Juo/ /<Jynmio me . w*v ^ -- - . ^ 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 0>752? CONFIOFNTTAl YZ0083085 -6- 79-7342 Appendix B (cont.) A Domlnant-Letnal Inhalation study in Rats with Benzene nio mioB Inc. Inhalation Report Calculation of Chamber Concentrations (cont.) At the conclusion of the study, samples collected at each sanpllng Interval (1e. 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 perforated for each phase of the study. When It was found that a particular Interval possessed a unique cor- j 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. 3p-test and Student's t-test (modified using Cochran's approximation [tl when variances differ). Snedecor 6.S., and W.6. Cochran, Statistical Methods 6th ed., Iowa State University Press, Ames, 1967, pp 104-106, H4-119. DO 06752-T CONFrOFNTTAL YZ0083086 1 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. 172`hr Mean Background Absorbances (Group 1} Sample Intervals: 1-1/2 hr 2-1/2 hr 3-1/2 hr 4-1/2 hr ~?Tl/2 hr ) t *l r ( 1/14/80 - 3/12/80 1-43 3/13/SO - 3/21/80 44-50 Mean S.D. N O.Q128+ 0.0030 43 $. .Mean 0.0115a 0 0.0042 N7 0.0081* 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 +S1 gnlflcantly different from succeeding values (p<0.05). aDifferent from succeeding values (p>0.05). " -7- DO 0 6 7 5 7 4 C O N F ID E N T IA L 00 u> 00 I 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 v Exposure Nos. Mi hr 1-1/2 hr Correction Factors Sample Intervals: 2-1/2 hr 3-1/2 hr 4-1/2 hr 79-7342 * r l 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 t DO 0 6 7 5 2 5 OONFTDFNTTAl I -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: Densityr Benzene 78.11 amu 0.878 g/ml MIRAN SETTINGS: Wavelength: Pathlength: Range: SI It Width: Meter Response Time: Coarse Zero: 3.3 u - optimized to 3.28 u 21.75 m (14.0 dial reading) 0.1 % (Groups I, II, III) 1.0 R (Groups IV, V) 1 mm 1 second x1 STANDARD: Measured volutes of benzene were Injected Into a closed loop circulation system In order to generate atmospheres Inside the Mlran of known concentration levels. The closed loop system. Including the Ml ran. measures 5.64 liters In volute. DO 0675?f> CONFTDFNT T Al. YZ0083089 10- 79-7342 Appendix B (cont.) A Dominant Lethal Inhalation Study In Rats - with Benzene Inhalation Report Miran Ambient Air Analyzer Closed Loop Calibrations Ml ran L (1/14/80 - 3/12/00) Injection Ranoe Volune Concentration (ul) (ppm) T\ Absorbance *2 ~ 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 % 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 IT 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 067577 CONFTDFNTTAI YZ0083090 t S su> *< N oo 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) icr Absorbance 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 0.002 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 1.0 X 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 JO. 26 0.37 0.46 0.55 0.62 0.63 0.64 0.65 0.68 0.71 0.75 A4 0.000 0.001 0.002 0.003 0.009 0.015 0.020 0.022 0.025 0.033 0.062 0.074 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 06753A CONFTOFNTIAI YZ0083094 Appendix B (Cont.) A Dominant Lethal Inhalation Study In Rats 79-7342 Appendix B (Cont.) 79-7342 I * N Appendix o (Cont.) 79-7342 I 1. I I o00 -18- 79-7342 Appendix B (cont.) Table 3 A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Cixnulatlve Exposure Record Group II - 1 ppm Date Mean Dally Exposure Test Exposure ConcenDay Number tratlon Cumulative Inte- Dally Total grated Dally Goncen- Total Concen- Mean Exposure tratlon Exposure tratlon Concen- SI) Hours Hours Hours Hours tratlon 1/14/80 .1 1/15/80 2 1/16/80 3 1/17/80 4 1/18/80 5 1/21/80 8 1/22/80 9 1/23/80 10 1/24/80 11 1/25/80 12 1/28/80 15 1/29/80 16 1/30/80 17 1/31/80 18 2/1/80 19 2/4/80 22 2/5/80 23 2/6/80 24 2/7/80 25 2/8/80 26 2/11/80 29 2/12/80 30 2/13/80 31 2/14/80 32 2/15/80 33 2/18/80 36 2/19/80 37 2/20/80 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 4.2 106 106.2 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 9.0 156 151.8 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 CONFIDENTIAL 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.) Date Mean Dally Exposure Test Exposure Concen- Day Number tratlon SD Cumulative Inte- Dally Total grated Dally Concen- Total Concen- Mean Exposure tratlon Exposure tratlon Concen- Hours Hours Hours Hours tratlon 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. oo 067536 CONF Mi YZ0083G99 -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 Cumulative Mean inte- Dally Dally Total grated Exposure Dally Concen- Total Concen- Mean Test Exposure Concen- Exposure tratlon Exposure tr'atlon Concen- Date Day Number tratlon SO 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 1.1 6 9.8 2.1 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 51.6 12 103.8 18 159.0 24 203.4 30 255.0 36 312.6 42 366.0 48 417.0 54 475.2 60 544.2 66 618.6 72 681.0 78 741.6 84 800.4 90 856.2 96 917.4 102 993.6 108 1045.2 114 - 1102.2 120 1162.2 126 1218.6 132 1269.6 138 1337.4 144 1395.0 150 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 YZ0083100 -21- 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 (cont.) Date ... Cumulative Mean Inte- Dally Dally Total grated Exposure Dally Concen- Total Concen- Mean Test Exposure Concen- Exposure tration Exposure tratlon Concen- Number tratlon SD Hours Hours Hours Hours tration 2/18/80 36 2/19/80 37 2/20/80 38 2/21/80 39 2/22/80 40 2/25/80 43 2/26/80 44 2/27/80 45 2/28/80 46 2/29/80 47 3/3/80 50 3/4/80 51 3/5/80 52 3/6/80 53 3/7/80 54 3/10/80 57 3/11/80 58 3/12/80 59 3/13/80 60 3/14/80 61 3/17/80 64 3/18/80 65 3/19/80 66 3/20/80 67 3/21/80 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 6.7 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 1574.4 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 55.8 294 2893.8 9.8 53.4 300 2947.2 9.8 Standard deviation of Cumulative Integrated Mean concentration for Group III Is 0.4 for entire study. DO 067538 CONFIDENTIAL YZ0083101 -22- 79-7342 Appendix B (cont.) Table 3 (cont.) ^ A Dominant.Lethal Inhalation 5tudy In Rats with Benzene Inhalation Report Cumulative Exposure Record Group IV - 30 ppm Date Mean Dally Exposure Test Exposure ConcenDay Number tratlon ?D Cumulative Inte- Dally Total grated Dally Concen- Total Concen- Mean Exposure tratlon Exposure tratlon Concen- Hours Hours Hours Hours tratlon 1/14/80 1 1/15/80 2 1/16/80 3 1/17/80 4 1/18/80 5 1/21/80 8 1/22/80 9 1/23/80 10 1/24/80 11 1/25/80 12 1/28/80 15 1/29/80 16 1/30/80 17 1/31/80 18 2/1/80 19 2/4/80 22 2/5/80 23 2/6/80 24 2/7/80 25 2/8/80 26 2/11/80 29 2/12/80 30 2/13/80 31 2/14/80 32 2/15/80 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 44)4.8 150 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 CONFIDENTIAL YZ0083102 23 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study In Rats "" with Benzene 1' Inhalation Report Cumulative Exposure Record Group IV - 30 ppm (cont.) dumulatlve Kean Oally Dally Total Integrated Exposure Dally Concen- Total Concen- Mean Test Exposure Concen- Exposure tratlon Exposure tratlon Concen- Date Day Number J tratlon SD Hours Hours Hours Hours tratlon 2/18/80 36 2/19/80 37 2/20/80 38 2/21/80 39 2/22/80 40 2/25/80 43 2/26/80 44 2/27/80 45 2/28/80 46 2/29/80 47 3/3/80 50 3/4/80 51 3/5/80 52 3/6/80 53 3/7/80 54 3/10/80 57 3/11/80 58 3/12/80 59 3/13/80 60 3/14/80 61 3/17/80 64 3/18/80 65 3/19/80 66 3/20/80 67 3/21/80 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 4766.8 162 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 8721.6 294 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 CONF 0675*0 -cdfntial YZ0083103 -24- 79-7342 Appendix B (cont.) Table 3 (cont.) A Dominant Lethal Inhalation Study In Rats *" with Benzene *" Inhalation Report emulative Exposure Record Group V - 300 ppm Date Kean Dally Exposure Test Exposure ConcenDay Number tratlon SD Cumulative Inte- Dally Total grated Dally Concen- Total Concert- Mean Exposure tratlon Exposure tratlon Concen- Hours Hours Hours Hours tratlon 1/14/80 1 1/15/80 2 1/16/80 3 1/17/80 4 1/18/80 5 1/21/80 8 1/22/80 9 1/23/80 10 1/24/80 11 1/25/80 12 1/28/80 15 1/29/80 16 1/30/80 17 1/31/80 18 2/1/BO 19 2/4/80 22 2/5/80 23 2/6/80 24 2/7/80 25 2/8/80 26 2/11/80 29 2/12/80 30 2/13/80 31 2/14/80 32 2/15/80 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 00 067541 CONFIDENTIAL YZ0083104 25- 79-7342 ,, CV- Appendix B (cont.) Table 3 (cont.) '"`A Dominant Lethal Inhalation Study In Rats with Benzene Inhalation Report Ctmul atlve Exposure Record Group V - 300 ppm (cont.) Date Mean Dally Exposure Test Exposure ConcenDay > Number tratlon Cumulative Inte- Dally Total grated Dally Concen- Total Concen- Mean Exposure tratlon Exposure tratlon Concen- SD Hours Hours Hours Hours tratlon 2/18/80 36 2/19/80 37 2/20/80 38 2/21/80 39 2/22/80 40 2/25/80 43 2/26/80 44 2/27/80 45 2/28/80 46 2/2 9/80 47 3/3/80 50 3/4/80 51 3/5/80 52 3/6/80 53 3/7/80 54 3/10/80 57 3/11/80 58 3/12/80 59 3/13/80 60 3/14/80 61 3/17/80 64 3/18/80 65 3/19/80 66 3/20/80 67 3/21/80 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 8 298 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 32 293 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? CONFIDENT TA|. YZ0083105 26 79-7342 Date Ml ran 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 1/29/80 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 Appendix B (cort.) Table 4 Dominant Lethal Inhalation study In Rats with Benzene Inhalation Report Oally Nominal Concentrations Test Exposure Day Number Nominal Concentration i Group Group Group II III IV .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 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 Group V 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 067543 CONFIDENTIAL YZ0083106 27 79-7342 Appendix B (cont.) Table 4 (cont.) A Dominant Lethal Inhalation Study In Rats _- with Benzene Inhalation Report Dally Nominal Concentrations (cont.) Date Test Exposure Day Number Nomina) Concentration (ppm) Group Group Group IGroup 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 l.l 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 Ml ran 04 3/13/80 3/14/80 3/17/80 3/18/80 3/19/80 3/20/80 3/21/80 Mean S.D. N 60 61 64 65 66 67 68 44 45 46 47 48 49 50 1.2 1.5 1.9 0.8 0.9 1.0 1.0 1.3 0.3 48 11.9 11.0 6.9 9.9 10.3 11.3 10.4 9.8 1.5 49 35.9 32.9 32.0 34.5 34.7 32.4 34.4 30.4 2.2 50 352 362 321 375 355 343 363 323 19 50 Nominal concentration suspect due to possible weighing error* not Included In mean calculation. DO 067544 CONFIDENT!^ YZ0083107 -28- 79-7342 - .TV Appendlx B (cont.) Table S A Dominant Lethal Inhalation Study In Rats ' with Benzene *'* Inhalation Report Mean Dally Nominal Concentration VS. Mean Dally Actual (IRJ Concentrations^ Group II in IV V Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. H Nominal Cone, (ppm) 1.3 0.3 48 9.8 1.5 49 30.4 2.2 50 323 19 50 Actual Cone (Ppm) 1.0** 0.5 50 9.8 1.0 50 30.3 1.2 50 299** 7 50 Significantly different from nominal concentration: **p<0.01. 'Mean dally actual (1R) concentrations were conpared to 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, Ames, 1967, pp. 104-10 i>, 114-119, DO 067545 CONFTDFNTIAI YZ0083108 YZ0083109 Appendix C (coat.) A DalMot-itthi) Inhalation Study Kith Benzene In Rats Individual Nile Body Weights (grjm) Group It <1 pf) 75-7342 ' An. No. 3001 301 302 303 304 30$ 30$ 307 308 309 310 311 312 313 314 315 31$ 317 310 319 Initial 455 419 393 455 417 411 432 415 415 401 410 351 39$ 371 305 379 394 391 44$ 398 Meet i 452 41$ 396 4$3 405 399 425 415 398 394 407 345 397 370 395 396 390 402 451 400 met z 471 42$ 401 488 402 401 428 438 424 385 408 342 40$ 370 402 407 404 403 483 393 Meet 3 493 447 414 491 412 419 445 457 435 388 428 357 418 370 408 410 421 418 473 404 Treatment Period Week 4 Week 5 Meek $ UL^L 50$ 532 539 468 451 479 413 485 430 442 452 459 510 533 535 541 434 452 445 457 439 455 454 456 449 472 482 502 47$ 496 503 515 449 45$ 454 473 393 409 . 409 421 44$ 474 481 481 373 385 400 404 434 459 46$ 472 380 394 391 398 421 44$ 461 465 434 444 453 467 435 45$ 459 474 431 454 455 47$ 498 51$ 521 533 423 444 447 458 Kean 5.0. 8 407 27 20 40$ 27 20 412 31 20 425 35 20 441 37 20 450 39 20 465 471 39 36 20 20 No statistically significant differences free control. Anlnil found dead during Week S of the treataent period. Week g pieefc 5 Tfcelt 10 Change 1A i 419 454 465 485 548 567 489 499 481 495 SM 527 530 533 481 483 437 443 454 498 418 425 482 499 407 419 479 488 473 488 492 501 481 494 533 555 445 47$ 477 491 39 39 19 19 491 474 569 487 495 538 54$ 481 451 461 433 504 410 493 493 45$ 500 546 471 491 40 19 76 79 10$ 82 96 113 131 83 60 74 88 107 40 98 97 66 98 97 71 1- 88 21 19 C-2 Appendix C (eont.) A Daalnant-tethal Inhalation Stndy With Benzene In Rats 79-7342 / Individual Nile Body Weights {grans} Group III (10 ppn) An* Mo* $00 $01 $02 $03 $04 $05 506 507 $08 $09 $10 $11 512 513 514 515 516 $17 $18 $19 initial 406 409 440 416 349 415 45$ 381 414 415 379 404 J78 404 379 404 52$ 354 41$ 39! 396 419 451 428 340 408 466 386 414 420 375 403 392 403 384 400 $34 3S4 418 381 week 2 400 420 44$ 430 3S1 412 480 397 400 431 368 410 396 412 397 406 $40 357 426 391 week 3 411 436 462 451 36$ 434 501 414 430 449 377 417 407 423 416 419 $61 369 443 405 Week 4 423 4$1 479 471 380 446 $22 427 449 465 388 431 417 428 42$ 437 $82 379 461 414 Mean 407 409 413 430 444 SeCl* 38 41 42 44 47 N 20 20 20 20 20 Ha statistically significant differences frm control. Week 5 462 467 $00 482 386 470 $4$ 445 476 48$ 403 457 437 440 444 469 609 392 481 418 464 51 20 Week 6 "Week 7' "Week A 463 478 478 482 $17 527 488 505 393 396 471 480 5S0 568 456 466 49e 498 496 504 413 429 457 471 452 463 452 466 451 467 476 486 616 597 387 400 488 $03 429 443 492 487 536 522 410 491 578 471 511 509 441 478 476 474 480 491 646 408 510 449 Week 9 "Week 1ft $05 507 548 526 419 503 S87 . 481 $33 $24 444 486 484 486 488 $13 664 416 $2$ 455 504 498 556 527 421 513 $87 487 $33 527 438 471 482 490 499 S10 647 419 $22 463 471 481 499 SOS 52 48 54 56 20 20 20 20 $0$ $4 20 Chgfe la Body Itt. f 79 10$/ 99? gl 10$ t 127 102 119 107 63' 68 90 87 ITS 110 113 65 104 79 96 ' 18 20 C-3 YZ0083111 YZ0083112 o 0Z-n so o on O' M z-i-I* uhv>O> p An. Wo. 700 701 702 703 701 70$ 70$ 707 708 709 710 711 712 713 714 71$ 716 717 718 719 M$.0m. 8 IRHK1 JSI 428 419 343 362 419 36$ 38$ 369 421 430 486 379 400 44$ 36$ 437 401 4S4 430 59LL 388 428 . 420 392 363 414 363 391 372 420 419 482 372 412 4S3 377 442 410 456 440 met Z 388 427 406 362 367 408 366 397 381 422 413 497 377 419 456 382 444 414 477 428 406 409 412 37 36 37 20 20 20 Appendix C (cent.) 75.1 A Donlnant-Lethal Inhalation Study Kith Bentene In Rats ___________________ _________ ______________ ____________ j Individual MU Rody Weights (grans) Group I (JO ppm) * neck 3 393 431 421 371 381 423 373 404 388 443 43$ $08 401 446 474 397 457 422 499 462 Mack 4 39) 43$ 433 386 398 449 392 418 414 449 438 $26 41$ 45$ 496 409 472 437 511 471 Week 5 408 4$7 450 405 414 474 401 448 418 474 460 $4$ 431 477 520 418 488 459 536 489 Week 5 "Beefc'T '-VeSta ' Peek 9 "TEsmr 412 416 421 431 457 473 483 497 464 480 496 $14 417 431 437 447 417 431 442 454 479 494 504 $19 407 420 436 442 452 457 457 463 424 442 45$ 4$9 483 494 510 521 461 470 480 491 $52 563 566 572 444 461 482 487 480 497 475 487 S24 540 525 $62 426 433 444 461 494 503 $13 522 461 468 476 479 536 552 57) 567 492 502 501 $16 432 503 $15 454 446 524 440 461 479 518 488 $65 $03 502 $68 461 $26 490 $7$ 514 t Change In 9od** 44 79 4 102 83 110 17 70 107 98 69 83 131 90 115 84 81 83 119 74 426 40 20 440 40 20 459 43 20 464 476 42 43 20 20 484 41 20 495 42 20 498 42 20 96 20 20 Ho statistical)y significant difference from control r C-4 I 1 Appendix C (cont.) A Oonlnant-Lethal Inhalation Study Kith Oenaane In Rats Individual hale tody heights (grans) Group V (300 ppm) 79-7342 An. to. 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 initial <9* 436 431 425 374 376 414 413 391 445 361 353 444 409 389 360 391 401 481 in " Ueek 1 477 430 434 420 377 388 412 410 407 438 380 363 451 407 400 360 409 398 482 375 Meet 2 452 438 436 432 386 389 417 405 416 450 386 366 448 411 398 340 412 391 485 378 TgTT 469 453 461 441 396 399 434 416 427 471 408 375 448 416 415 352 426 401 491 387 Treatment Period _______ TOTT hatfT--ifcek 5 feet T 489 524 531 548 472 492 491 506 475 483 496 511 462 475 493 513 401 437 437 438 424 441 453 467 439 462 468 480 427 446 454 472 440 459 465 478 489 509 521 528 416 439 444 465 387 403 411 424 470 473 499 513 433 442 455 461 420 436 450 463 357 367 373 387 440 454 462 477 420 448 458 466 516 542 556 569 406 427 435 446 Mean 5.0. R 409 38 20 411 34 20 412 34 20 424 3$ 20 439 39 20 458 40 20 468 481 42 43 20 20 ho statistically significant differences frtM control Meet 8 $61 516 514 522 453 481 491 482 479 538 474 432 541 478 478 391 481 483 587 454 492 45 20 Week 568 518 525 539 466 491 496 499 495 557 477 442 544 486 487 401 M2 491 597 459 vSeL'16 569 512 533 549 462 495 500 511 493 561 478 450 559 491 481 403 513 499 590 461 502 505 45 46 20 20 Change In Body Wt. 1-10 9e 82 99 129 to 107 88 101 to 123 98 87 108 84 81 43 104 100 108 86 95 18 20 C-5 tN 0U0> I oD O zn o -oi o^ T1 St z ymi *! N o 0<*0> i Appendix C (cent.) A Oontnant-Lethal Inhalation Study With Benzene In Hats 79-7342 Individual Wale Body Weights (graam) Group I (ChaaOer-Exposed Control - 0 ppai) Animal Wo. 100 101 102 103 104 10$ 106 107 108 109 no 111 112 113 114 11$ 116 117 118 1)9 Mean SJ>. N Mating Period Week 1 Week i A- A512 $36 447 461 $16 $41 $42 551 476 486 456 468 600 614 489 509 $56 $74 474 48$ $19 $26 $45 $74 522 537 460 473 497 523 459 480 $21 $35 539 $47 46$ 472 496 522 505 521 40 41 20 20 teeTT A* $72 477 $62 $74 503 48$ 641 527 $92 498 $38 $9$ 564 486 539 490 544 $61 481 $36 538 45 20 Week 2 A* $90 482 579 595 516 500 656 $34 603 $12 $42 607 575 498 $54 503 SS2 574 486 546 $50 47 20 Week 3 A597 4as 58! $88 $21 502 663 530 619 520 538 619 586 500 547 496 564 577 490 553 5S4 49 20 Post-Hating Period ifeelc 4 Week 5 Week 's A- A A* 604 612 618 497 511 529 599 608 614 615 622 643 537 537 553 $16 528 547 692 700 722 $46 $5$ 566 630 640 654 S43 552 568 562 574 588 629 632 644 $97 599 607 $11 515 538 547 573 $83 $12 511 527 S73 575 506 $86 598 619 502 507 522 $63 $57 $68 $68 575 590 51 51 $1 20 20 20 iteek 7 A634 $38 637 656 557 $51 723 581 671 $74 $93 652 616 539 601 537 594 624 532 575 599 $2 20 Week fl A* 637 $33 628 668 $57 $53 739 $8$ 674 583 597 641 614 $43 G02 $41 599 626 534 580 602 54 20 Week l6 A* 653 $49 641 690 569 557 773 591 699 599 612 666 626 557 619 557 599 634 $40 597 616 59 20 For explanation of statistical sywbol: A-; see Appendix t C-6 n o zTl oo mom o Om' z-i ftn T> Appendix C (cont.) A Doninant-lethal Inhalation Study With Benzene In Rats Individual Nate Body Weights (grans) Group II (T ppa) AnInal hatine Period .Wo. Week 1 Week t week l~ week z week 300* 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316k 523 475 537 491 511 525 554 504 455 496 438 518 415 504 504 490 551 496 581 509 525 540 566 529 476 525 460 541 431 516 512 524 576 511 600 529 544 569 593 546 482 546 466 555 442 536 529 511 592 527 616 555 557 592 606 571 497 556 476 5M 455 552 535 588 533 621 557 557 601 615 579 508 570 481 588 438 548 542 608 550 641 568 591 615 636 593 526 579 492 606 455 565 563 615 555 652 582 591 633 649 601 539 593 502 617 469 579 566 317 318 319 503 531 484 509 553 496 512 577 516 530 591 529 538 605 530 547 606 537 555 620 538 Naan 498 S.O. 34 N 19 518 36 19 534 42 19 551 45 18 556 48 18 571 48 18 58! 49 18 No statistically significant differences from control, hhliil found dead during Weak 8 of the treatment period, bftntaal found daad Week 2 of the post-awtfng period. 637 576 665 591 $09 642 663 621 556 611 513 629 490 588 576 572 620 556 595 48 18 79-m? nar>--ran--hm m 651 654 585 583 679 676 607 597 624 637 659 667 679 60S 630 630 564 565 612 617 521 534 642 651 504 509 603 609 587 602 589 591 639 640 563 561 608 612 49 49 18 18 C-7 ti ssrsssssssmmis YZ0083115 * Appendix C (cont.) A Donlnant-Lethal Inhalation Study With Benzene In Rats 79-7342 Individual Nate Body Weights (grans) Group lit (W ppi) MIlMl Nattnq Period 509 513 523 501 502 513 502 554 575 503 537 552 504 431 439 505 520 527 506 so; 599 496 606 517 500 540 55! 509 528 542 510 446 461 511 476 491 512 486 489 513 496 519 514 514 536 S15 509 522 SK 657 692 517 429 451 SIS 529 551 519 456 471 Mean 511 S.D. 54 N 20 526 57 20 541 542 602 570 454 538 627 538 566 553 473 507 497 537 547 534 736 466 575 484 544 63 20 Week 2 566 563 619 580 468 557 639 544 584 575 480 509 509 542 555 544 758 474 593 492 558 67 20 week 3 570 576 620 589 475 560 642 538 591 576 485 519 518 545 569 555 764 479 591 492 563 66 20 Post-Mating Period week 4 Week 5 Week 5 585 602 591 589 605 617 639 647 652 602 613 634 493 497 510 576 583 596 6S5 665 664 556 567 582 613 626 626 591 589 592 498 498 520 534 550 556 523 519 540 556 550 570 583 594 603 574 578 591 802 806 830 490 498 506 611 619 621 SOS 517 519 579 586 596 71 72 72 20 20 20 BeeT7~ 619 626 659 650 514 616 697 593 649 611 516 567 554 583 617 597 811 512 643 528 608 71 20 Week 8 "Seek VS 623 647 634 661 669 712 652 671 524 526 616 638 700 726 590 612 638 677 603 621 528 539 560 583 556 579 521 597 624 654 590 607 826 823 515 525 632 656 527 543 606 630 75 74 20 20 Wo statistically significant differences Iron control. c-8 00 067553 C0NFT0FNTTAI OoUe> DO 0 6 7 5 5 4 CO NFIDENTIA! no o zo -n -or<n oON'l Z--4 yi *< --A S owe J Appendix C (cont.) A Oaninant-Lethal Inhalation Study With Benzene In Rets 79-7342 Individual Hale Bo^r Weights (grwsl Group V (300 ppn) Anlwl Wo. 900 901 902 903 904 90S 906 907 908 909 910 91! 912 913 914 91S h 917 918 919 Mean 5.0. N Katina Period Ween 1 Week 2 575 605 508 532 532 547 549 560 474 489 497 515 512 502 S13 522 504 524 568 589 472 482 415 449 555 575 502 517 489 517 419 436 516 526 502 516 606 608 463 469 509 524 48 47 20 20 Week 1 616 548 573 580 509 541 SIS 549 536 603 509 475 606 539 524 455 551 536 622 483 544 46 20 Week i 637 560 591 602 520 S5B 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 62S 531 491 617 559 538 476 575 551 646 496 564 49 20 Post-Matlna Period Week 4 Week 5 Week 6 649 667 683 581 586 607 620 625 634 621 637 641 531 531 540 588 553 595 613 557 573 587 604 615 576 579 597 644 645 671 549 562 577 SOB 512 515 643 651 683 570 563 590 543 549 568 498 507 525 583 593 623 578 588 600 651 662 668 511 515 531 579 587 603 48 50 52 20 20 20 Wo statistically significant differences from control t Week 7 701 614 643 664 544 621 591 626 600 676 585 535 664 609 574 533 613 604 681 539 611 SO 20 Week 8 681 614 650 673 561 611 592 631 601 680 565 543 658 610 . 573 539 608 608 681 539 610 49 20 Week 10 717 633 657 686 571 649 610 634 624 697 590 561 709 619 589 544 624 618 698 555 629 53 20 C-IO D-l Appendix 0 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/orxontrol^j^nlmals and are not considered to be compound-related. DO 067556 CONF IDFNTTAl YZ0083119 Group ppm Animal Number 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 0-2 79-7342 Appendix 0 (cont.) A Dominant-Lethal Inhalation Study ' With Benzene In Rats Gross Postmortem Observations - Males Type of Death8 Observations)b T N.O.A. T Lungs: few scattered red foci on all surfaces of all lobes. T N.O.A. T Lungs: few scattered tan foci on all lobes T Lungs: dark red areas (0.3 cm. In dlam.) on left lobe. Liver: accentuated lobular pattern. T N.O.A. T N.O.A. T N.O.A. T N.O.A. T N.O.A. T N.O.A. T N.O.A. T Lungs: scattered red foci on all lobes and 'surfaces. Ear (Rlaht): scab at eartao (0.2 cm.). T Ear (Right): scab. T N.O.A. T N.O.A. T N.O.A. *T Terminal. "N.O.A. No observable abnormalities. DO 067557 CONFTDFNTIAl YZ0083120 Group ppm Animal Number I 0 (cont.) 117 ' 118 119 II 300 1 301 302 303 304 305 306 307 D-3 79-7342 Appendix 0 (cent.) - A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmortem Observations - Hales Type of Death* Observation?s)b T Lungs: scattered tan foci on all lobes and surfaces. Ear (Right): scab (0.2 cm. In dlam.) T N.O.A. T N.O.A.' 5.D. Liver: accentuated lobular pattern of all lob'es and surfaces; si Ightly 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: cerebrim 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 "tboes. 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 lobes and surfaces. T Kidneys (Both): pale brown. Liver: ail lobes pale brown with extremely accentuated lobular pattern. Lungs: multiple scattered red and gray foci on all lobes. *T Terminal; S*D. Spontaneous Death. bN.0.A. No observable abnormalities. DO 067558 CONFTOFNTIAL YZ008312I 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 Death8 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 eartag. Kidneys Both! scattered tan foci, liver a f lobes pale tan. lungs: a 1 lobes dark red with scattered gray, tan and red foci. T lungs: few scattered gray foci on all lobes. T Kidneys!Both): mottled red. lungs: tew scattered red foci on all lobes. T N.O.A. T N.O.A. T lungs scattered red foci on all lobes and surfaces. S.D. Seminal vesicles: 2-3 x larger than normal with surrounding edema (clear). Urinary bladder large (3.2 x 1.8 on.); 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. JT Terminali S.D. Spontaneous Death. DN.0.A. No observable abnormalities. C0O0NF10FNTTAI 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 Death8 Observation s)b II 316 S.O. 1 * (cont.) (cont.) lunos: 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; an lobes pale tan; accentuated 1obular 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 eartag. Liver: all lobes pale tan; moderately accen tuated 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.O.A. No observable abnormalItles. DO 067560 CONFTDENTTAI YZ0083123 Group ppn Animal Number III 10 (cont.) 509 510 511 512 513 514 515 516 517 518 519 IV 700 30 701 702 0-6 79-7342 Appendix 0 (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats Gross Postmorten Observations - Males type "oT........"""ni Death* ______________ Observation^)" T Lungs: scattered red foci on all lobes and surfaces. T Liver; accentuated lobular pattern of all lobes. Lungs; all lobes slightly red: white dlscoioration (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: alllobes andsurfaces 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: few scattered tan foci on all lobes. T TestesfBoth):slightly reddened. Moneys (doth): mottled red. Lungs: red focus on right median lobe. T N.O.A. T N.O.A. T Liver: alllobes andsurfaces slightly pale tan; accentuated lobular pattern. *T - Terminal. "N.O.A. - No observable abnormalities. oo CONF TOFNTTAl YZ0083124 * <* Group PP Animal Number IV 703 30 (cont.) 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* 0bservat1on(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 rethrown; extremely accentuated lobular pattern. T N.O.A. T Liver: slightly accentuated lobular pattern oTall lobes Forellmbs (Both): moderate hair loss. T N.O.A. T N.O.A. T Liver: accentuated lobular pattern of all lobes and surfaces. Unas: few scattered dark red foci on all lobes and surfaces. T Liver: all lobes pale tan; extremely accen tuated lobular pattern. f.ungs: 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. .ungs: scattered red foci on all lobes and surfaces. Kidneys (Both); few depressions (<0.2 cm.). *T Terminal. "N.O.A. No observable abnormalities. AO 06756? CONFIDENT TA! YZ0083125 Group ppm Animal Number IV 715 30 (cont.) 716 / 777 718 779 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 "r " 1~ 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. Lungs: scattered tan and red foci on all tobies and surfaces. T Kidney (Right); embedded cyst (0.4 cm. In dlam.) near hflus; containing clear, thin fluid. Kidneys (Both): pale brown. T N.O.A. T N.O.A. T Testis (Left); slightly reddened. lungs: red focus on ventral surface of left lobe. liver: slightly accentuated lobular pattern UTall lobes. T - Terminal* "N.O.A. No observable abnormalities. DO 067563 rONFTDFNTTAI YZ0083126 Group ppm Animal Number V 300 (cont.) 907 9/ 08 909 910 911 912 913 914 915 916 917 918 0-9 79-7342 Appendix 0 (cont.) A Dominant-lethal Inhalation Study with Benzene In Rats Gross Postmortem Observations - Males Type of Death8 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 ofall lobes. 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) - (0.6 x 1.0 cm.). liver: slightly accentuated lobular pattern TTa)1 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 Of>75&4 CONFIDENTIAL YZ0083127 Group ppm V 300 0-10 79-7342 Appendix 0 (cont.) A Dominant-Lethal Inhalation Study --w ***- - .With Benzene In Bats Gross Postmortem Observations - Males Animal Type of Number Oeatha Observations)1* 919 T f Kidney (Right); renal calculi. Lungs: scattered gray foci on all lobes. Eye (Left): 20* opacity. *T Terminal. bN.0.A* No observable abnormalItfes* DO 067565 CONFTDENTTAI YZ0083128 E-1 79-7342 Appendix E ,, '"--A Dominant-Lethal Inhalation-Study With Benzene In Rats Pregnancy and Implantation Data - Post-Treatment Mating Period Corp. Lut. Impl. / VFS M.R. I.E. Preface Corpora Lutea Implantatlons Viable Fetuses Mutagenic Ratio (for definition see Methodology and Reference; Appendix I) Implantation Efficiency (for definition see Methodology and Reference; Appendix I) co^ YZ0083129 t Appendix E (cont.) A Dominant-Lethal Inhalation Study With Benzene In Ret* Pregnancy, Corpora Lutea and Implantation Data .! 79-7342 Male ho. 100 Female Jtes_ 200 201 Corp. tut. A*F 14 20 111. X+ 13 19 Ml 202 203 16 IS 15 15 WZ 204 205 18 17 Not pregnant. 103 206 207 13 20 13 15 104 208 209 Not pregnant. 15 14 105 210 211 Not pregnant. 15 M Wt 212 213* 19 13 17 14 101 214 21S 15 12 15 12 WO 216 217 15 15 14 15 109 218 219 12 16 11 K no 220 2214 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 98.4 98.2 * Post-Treatment Hating Period (Meek 1) - Group t - Chafer-Exposed Control Number of Dead Implants It0 2 H.R. VFS X 10-2 K*J V13 1.9 17 10.5 Hate Female Corp. l.E. HO. JSts_> tut. lw>1. X 10-2 Ill 222 16 15 ^3.8 223 12 12 97.9 1 14 6.7 112 224 13 13 98.1 0 15 1.7 225 13 13 98.1 0 17 1.5 113 226 9 8 88.9 227 13 g 69.2 0 13 1.9 114 228 13 13 98.1 1 14 6.7 229 15 14 93.3 115 230 Not pregnant. 2 12 14.3 231 14 14 98.2 116 232 12 4 33.3 1 13 7.1 233 14 W 98.2 3 14 17.6 117 234 13 13 98.1 1 13 7.1 235 14 14 98.2 1 14 6.7 118 236 18 18 98.6 0 12 2.1 237 16 15 93.8 0 14 1.8 119 238 13 13 98.1 1 14 6.7 239 12 12 97.9 0 11 2.3 3 13 18.8 o r 14 1.8 10 9 52.6 total: T3--T58--TI5T7S Hean: 14.6 13.8 92.7 S.O.: 2.4 2.8 12.3 N: 36 36 34 number of Dead Implants ; M.R. X 10-2 1 'it 6.7 1 *i 8.3 2 ii 15.4 1 f 7.7 1 7 12.S 0 <9 2.8 a 13 1.9 i 13 7.1 * 2 12 14.3 0 4 6.3 0 14 1.8 1 12 7.7 1 13 7.1 1 17 5.6 0 15 1.7 0 13 1.9 0 12 2.1 ------------ 38------------ ' 451 mr i.i 12.7 7.8 1.8 2.6 9.1 36 36 36 For explanation of statistical symbols: AH., !, K-, K+J, V-; see Appendix I. Tout nuaber of Implants greater than total maaber of corpora lutea; data excluded from calculations of Implantation efficiency. ro> o zn o Mo OO' rr\ s) Z in -IH>H ONl' E-2 't , 1I Appendix E (coot.) A Doalnant-lethal Inhalation Study With Benzene In Rats Pregnancy, Corpora Lutea and Inplantatlon Data 79-7342 Male No. 300* 301 302 303 sot 305 308 307 308 309 310 Fonale No. 400 401 402 403 404 405 408 407 408 409 410 411 412 413 414 415 418 417 418 419 420 421 Corp. tut. - JSL m - 18 17 18 14 13 19 14 18 18 12 12 15 15 15 15 17 15 13 12 Not pn 15 15 14 14 18 16 15 13 Not pregnant. 18 16 18 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 88.7 98.4 93.8 78.9 98.1 Post-Treatiaent Hating Period (Week 1) - Group II - (I ppn) NunOer of Dead Ia1 ants N.R. ws X 10-2 -- Hunber of Male Fenale Corp. I.E. Dead Indents He. No. tut. Inol. X 10-2 . . 311 422 Not pregnant. -- 423 16 12 75.0 1 1 15 6.3 312 424 15 15 98.3 O 16 1.6 425 17 17 98.5 2 0 1 12 7.7 313 426 13 13 98.1 1 13 7.1 427 M 14 98.2 4 2 1 17 5.6 314 428 Not pregnant. 0 12 2.1 429 . 17 15 88.2 0 0 15 1.7 315 430 18 17 94.4 2 13 13.3 431 Not pregnant. 3 0 15 1.7 316 432 Not pirCfRMha 0 12 2.1 433 18 13 72.2 1 317 434 Not pregnant. 0 15 1.7 435 15 15 98.3 1 0 14 1.8 318 438 13 12 92.3 1 15 6.3 437 14 14 98.2 0 0 0 13 1.9 319 438 15 13 86.7 439 Not pregnant. 0 1 15 6.3 TStaTTT53T- 451^ "5770-- ii 0 IS 1.7 Mean; 15.3 14.3 92.7 0.7 S.D.: 1.8 1.8 8.2 1.0 0 10 2.5 N: 30 30 30 30 0 13 1.9 H.ft. ` 9FS1 t X 10-2 11 / 8.3 13 f 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 --sr-191.0 13.5 6.0 1.9 6.5 30 30 *<a1e No. 300 found dead during Week 8 of treatnent period. ao UcTsorjl' "< E-3 C O N F ID E N T IA L Appendix (coot.) A Dotleant-lethal Inhalation Study With Benzene in Rats Pregnancy, Corpora Lutea and Inplantation Data 'i 79-7342 Male No. 500 501 502 503 504 505 505 507 508 509 510 Penal e Gorp. No. Lut. leol. 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* 14 507 10 15 _ 6 60.0 608 K 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 98.6 516 12 617 16 12 97.9 16 98.4 618 12 619 M 12 97.9 12 85.7 620 12 1 $21 Not pregnant. 8.3 Post-Treatment Hating Period (Week 1) - Group III - (10 ppn) Hunter of Oead Inplants 1 2 1 N.R. VF xlO-2 12 7.7 10 16.7 11 8.3 0 9 2.8 Hale Fenale NO. No. 511 622 623 512 624 625 513 626 627 Corp. tut. 13 11 I.E. isL x10-e 3 23.1 54.5 11 11 97.7 12 12 97.9 16 M Not pregnant. 87.5 Hunter of Oead Inolants 4- 0 . f3 0 T 1 10 0 }* 0 (4 N.R. x!0- 8.3 4.2 9.1 2.1 1.8 1 14 6.7 514 628 15 14 93.3 0 H 1JB 2 4 33.3 629 13 11 84.6 1 10 9.1 0 14 1.8 515 630 15 13 86.7 2 11 15.4 1 13 7.1 631 Not pregnant. ' I 16 5.9 516 632 13 13 98.1 0 13 1.9 633 17 17 98.5 0 17 1.5 517 634 635 Not pregnant. 17 13 76.5 0 13 1.9 518 636 11 10 90.9 0 10 2.5 2 16 11.1 637 15 IS 98.3 1 14 6.7 0 12 2.1 519 638 12 12 97.9 2 ,10 16.7 1 15 6.3 639 13 13 98.1 0 rl3 1.9 0 12 2.1 Total: ----------3HT- ---------- 20--------- 340 tustt 1 11 8.3 Nean: 13.5 12.0 85.7 0.7 11.3 7.1 S.D.: 2.1 3.9 22.8 0.8 3.8 6.8 0 1 (K.0) N: 30 30 28 30 30 29 YZ0083132 *10401 nwber of Hgrianti greater than total nwber of corpora lutea; data excluded fron calculation of inplantation efficiency. n oo zo -a o O O' !TS z tn H--C O'O' r E-4 Hale Ho. TOO Female Ho. 800 SOI Corp. Let. 14 12 Hart. 14 10 701 802 803 702 804 805 703 806 807 704 808 809 70S 810 811 70S 812 813 707 814 815 70S 816 617 14 13 Hot pregWnV Hot pregnant. M 10 13 12 Hot preg|nWK 12 w 12 12 14 5 16 1$ 13 13 14 12 Hot pregnant. 15 13 17 16 17 10 709 818 819 710 820 621 W IS 15 15 16 13 K 14 oO o 2n O Mo O^ 2 (NnJ H- O\J x1r-0E-z6 98.2 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 Appendix E (cent.) A. Donlnant-Lethal Inhalation Study Mlth Benzene In Hats Pregnancy, Corpora Lutea and Inplantation Bata 79-7342 Post-Treatnent Hating Period (Week 1) - Group IV - (30 ppe) llunber (rf Dead laolants VFS 1 13 37 x10-2 7.1 30.0 Hale Ho. 711 Female Ho. 822 823 Corp. iix_ I.E. Inpl, xlO-* Not pregnant. ^ Hot pregnant. 1 12 7.7 712 824 16 825 11 15 93.8 11 97.7 713 826 14 W 98.2 3 7 30.0 827 Not pregnant. 0 12 2.1 714 828 14 14 98.2 829 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 pre|nWv 718 836 Not prePlIRha 1 12 7.7 837 18 16 88.9 8 8 50.0 719 838 17 17 98.5 5 5 50.0 839 Not pregnant. i 15 6.3 l 12 7.7 0 15 1.7 l 13 7.1 ""Total: "T71------- ----- OT" 2SM.B-- Mean: 14.6 13.1 89.4 S.O.: 1.7 2.5 13.8 H: 29 29 29 Ntnfter oT Dead Implants * H.lt. W*1 1l r 1 14 , 6.7 2 * (f 18.2 0 14 1.8 1 0 14 1.8 0 M ' 1.8 0 13 1.9 2 14 12.5 1 13 7.1 0 14 1.8 2 W 12.5 0 17 ; 1.5 39 HZ 336.B 1.3 11.8 11.6 1.7 3.1 13.1 29 29 29 E-5 YZ0083133 l Hale Fenale Corp. Wo. ja^ Ut. teal. 900 WOO W01 901 woe* 1003 902 wot 1005 15 n 18 13 12 M 10 20 17 12 12 903 1006 1007 17 16 1I7t 904 WOO 1009 909 10W ion W 12 11 13 16 12 0 13 906 W12 W13 907 ww 1015 12 12 Netprei|MKe 13 13 M 13 900 W16 1017 H 13 . 14 13 909 W W19 13 W 11 2 910 1020 1021 11 It 11 14 t.E. io-< 93.3 90.9 94.4 92.3 97.9 98.5 87.5 98.4 97.9 72.7 98.1 97.9 98.1 92.9 98.2 98.1 84.6 20.0 97.7 98.2 Appendix E (coat.) A Oonlnant-lethal Inhalation Study With hamane In Rats Pregnancy, Corpora lutea and lnplantatton Dote Post-Treatment Kiting Period (Meek 1) - Group V - (300 ppn) 79-7342 + Hunker of Dead Inalants H.R. ffs a10** Kale Fenale Gorp. Ho. He. lot. l.E, Inol. aW-2 Umber of1 Dead Inol ants 1 N.X. VFS' alO-z 3 11 21.4 911 1022 12 11 91.7 0 11' l 2.3 2 8 20.0 1023 13 13 98.1 1 i2 r 7.7 1 19 5.0 912 1024 15 15 98.3 9 6 . 60.0 0 17 1.5 W2S 12 12 97.9 0 W { 2.1 0 12 2.1 913 W26 15 14 93.3 9 5 64.3 0 12 2.1 W27 11 10 90.9 7 3 ' 70.0 0 17 1.5 914 1028 Not pregnant. 0 14 1.8 1029 16 K 98.4 1 15 6.3 2 14 12.5 915 W30 H 13 92.9 2 11 ` 15.4 4 8 33.3 1031 Not pregnant. 1 7 12.5 9K W32 16 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 W35 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 to 28.6 0 11 2.3 Total: 5ZI------ ~<8T"ETOT!-- M ~42S~'35TT 2 0 87.5 Keen: 14.2 13.1 91.1 1.6 11.5 M.9 S.D.: 2.7 3.0 15.5 2.4 3.9 21.6 0 11 2.3 W: 37 37 36 37 37 37 1 13 7.1 CONFIDENT TAl Total Maker of taplanti greater than total amber of copora lutea; data excluded fna calculation of lnplantatton efficiency. oo D> yNil I W t i t appendix E (cont.) A DcnfMnt-Lethal Inhalation Study Kith benzene In Jtats Pregnancy, Corpora lutea and Inplantation Data 79-7342 > Kale Female Corp. Wo* Wo. Mt. ingU 100 240 241 A13 17 K13 16 101 242 243 14 20 14 14 102 244 245 12 12 Wot pregnant. 103 246 247 11 15 It 13 104 248 249 15 13 14 11 105 250 251 14 14 14 M 106 252 253 15 15 IS 15 107 254 255 15 13 Wot pregnant. TOO 256 257 Wot pregnant. 18 14 10$ 258 259 13 14 13 14 no 260 261 14 15 14 15 i-f* *10*2 t98.1 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 (Week 2) - Group I - Chamber-Exposed Control Hunter of Oead Instants K0 1 K.g. VPS 10-? K- 913 1.9 15 6.3 Hale Female Corp. WO. Wo. Ut. Ill 262 IS 263 11 M.E, isL. xlO-* 12 80.0 11 97.7 dumber of Oead Inplants ' i H.6. 9FS f *10-2 0 12 / 2.1 0 11 1 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 1.8 0 14 1.8 IIS 270 Hot pregnant. 0 11 2.3 271 22 15 68.2 0 IS 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 13 1.9 118 276 21 14 66.7 277 16 15 93.8 0 14 1.8 2 10 , 16.7 1 13 7.1 I 14 6.7 119 278 13 13 98.1 1 13 7.1 279 12 11 91.7 0 13 1.9 0 11 2.3 0 13 1.9 1 13 7.1 o 14 1.8 1 14 6.7 ' Total: T35------ --251--~3ni.r ------------15---------- <5; "50----------- Hean: 14.7 13.4 91.4 0.4 13.0 4.4 S.D.: 2.8 1.8 9.4 0.7 1.7 3.8 N: 36 36 36 36 36 36 DO 0 6 7 5 7 ? CONFTDFNT TAl For explanation of statistical symbols: A-. K-, V-; see Appendix I. *< N E-7 M Hale Fenale Carpi. No. No. Jk_ 300** 440 441 - Inol. - - 301 442 443 12 11 Not progfllille 302 444 44$ 15 15 Nat progsvn$a 303 44$ 447 Nat pregnant. 1$ 15 304 440 449 14 13 12 12 305 450 451 12 16 2 306 452 453 307 454 o oo zo ~n i-t o o O'. 1SS 300 45$ 457 309 450 Z-i Nl nW 7> 459 310 4C0 461 M 13 1$ 1$ 24 15 14 13 IS 14 15 15 Nat pregnant. 14 14 15 7 10 1$ I.E. xlQ-? . 91.7 90.3 93.0 85.7 92.3 00.9 15.7 92.9 90.4 62.5 92.9 87.5 90.3 90.2 46.7 80.9 Appendix E (coot.) A Donloant-Lethal Inhalation Study tilth feiiaw In Rats Pregnancy, Corpora lutea and Implantation Data 79-7312 ! 5 Post-Treatment Hating Period (Week 2) - Croup II - (I ppn) Dead holants . 1 0 ffS xlO** 10 9.1 15 1.7 Ha ale Fenale Carp. No. No. Lot. 311 4(2 15 463 13 312 464 11 465 16 313 46$ 19 467 12 Inol. W 13 11 15 15 12 I.fc. xlO-? 93.3 98.1 97.7 93.8 78.9 97.9 T 1 Dead Inolants | i N.R. vrs f'x- 0 14 1.8 1 12 / ?*7 0 11 2.3 1 14 . 5 10 33.3 5 7 41.7 314 468 10 10 97.5 1 14 $.7 469 IS 15 98.3 0 12 2.1 315 470 11 5 45.5 1 11 8.3 471 11 1 9.1 0 16 1.6 316 472 16 15 93.6 2 0 07.5 473 Not pregnant. 0 10 2.5 2 13 13.3 0 5 5.0 1 0 (75.0) 0 15 1.7 0 13 1.9 317 474 16 15 93.8 3 13 IB.8 475 14 14 98.2 1 14 $.7 318 47$ Nat pregnant. 1 12 7.7 477 13 13 * 98.1 0 15 1.7 0 14 1.8 . 0 13 1.9 0 14 1.8 319 478 17 17 98.5 2 13 13.3 479 14 14 96.2 1 16 . 5.9 1 13 7.1 2 m 14.3 0 7 3.6 0 16 1.6 ' Total nso----705 777412" ------------- J1----------- Nean: 14.7 12.7 85.1 1.0 S.O.: 2.8 3.9 23.3 1.3 N: 32 32 32 32 374 11.7 4.0 32 Birr W.3 17.0 31 *Nal No. 300 found dead during Meek 0 of treatment. E-8 8 k-t Male Fault Mo. A. MO 640 641 Cory. Lot. 12 17 J-E. 1SL. x10"* 12 97.9 17 98.5 Ml S42 643 H 13 Not presIMHla 92.9 M2 644 645 IT 16 Not prosPNMC* 94.1 M3 646 647 11 13 9 81.8 5 38.5 $04 640 649 11 11 11 97.7 11 97.7 MS 6M 651 16 K Mot prosPHIICa 98.4 SOS 652 653 Mot presIKIioo 14 14 96.2 507 6S4 655 Not prespnWb* 13 12 92.3 SOS 656 657 17 t2 17 98.5 12 97.9 509 650 659 11 1$ 6 54.5 14 93.3 S10 660 661 13 M 13 98.1 14 96.2 Appendix E (cont.) A BntMiit*litlul Inhalation Study Kith Benzene In hats Pregnancy, Corpora Uitea and Inplantation Data 79-7342 Poat-Treatnent Mating Period (Week 2) - Croup III - (10 ppa) Hatter of Dead Ino! ants 0 1 MVFS 12 2.1 16 5.9 Hale Feuale Ho. -*>t . 51! 662 663 Corp. Lot. 17 W I.|. Inpl. KlO-g 4 v 23.5 5 50.0 PESEer'oT Dead Inplant* 0 0 N.t. SI >10-7 6.3 5.0 1 12 7.7 512 664 19 17 89.5 0 17 1.5 665 IS 15 98.3 0 1.7 li 1 15 6.3 513 666 K IS 93.8 1 6.7 667 Not prcignant. 1 4 5 44.4 514 668 Not pregnant. 1 4 20.0 669 14 14 98.2 0 14 1.8 1 TO 9.1 515 67Q 13 13 98.1 0 13 1.9 1 10 9.1 671 16 16 98.4 0 16 1.6 1 15 6.3 516 672 Not pregnant. 673 13 13 90.1 1 12 7.7 517 674 14 H 98.2 0 14 1.8 0 14 1.8 675 15 M 93.3 1 13 7.1 518 676 15 98.3 1 14 6.7 0 12 2.1 677 20 17 85.0 1 16 5.9 2 15 11.8 SN 678 13 13 98.1 0 13 1.9 0 12 2.1 679 14 14 98.2 1 13 7.1 0 6 4.2 2 12 14.3 1 12 7.7 2 12 14.3 Total: 155------ - m~ 7547:5"" -----------74----------- Mean: 14.2 12.8 89.0 0.8 S.D.: 2.4 3.6 19.1 0.9 N: 32 32 32 32 nr"730" 12.1 7.3 3.6 8.1 32 32 E-9 oo 067574 conftofntt Al 00 w u> i <1 I Appendix (cont.) A Donlnant-Lethal Inhalation Study With flenienc In Rats Presnancy, Corpora Lntea and Inplantation Data ! 79-7342 Male Fenal Mo. Mo. 700 840 841 701 842 843 702 844* 845 703 846 847 701 848 849 705 850 851 705 852 853 707 854 855 708 855 857 709 858 859 710 860 861 Corp. let. lnpl. W 18 13 13 15 14 14 13 15 16 14 12 14 14 Npt 15 15 15 14 11 11 15 15 14 W 14 14 13 9 12 12 IS 14 15 15 18 18 18 18 Not presPMIK* Not pres|Mllb k}IO -34 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-Treatnent Matins Period (Mark 2) . Group IV - (30 ppn) lumber of Dead Implants M.R. IffS w- 1 17 5.6 1 12 7.7 2 12 14.3 0 13 1.9 2 14 12.5 0 12 2.1 Male Fenale Corp. Mo. No. Ut. 711 862 8(3 14 15 712 864 865 13 13 713 866 867 13 15 "" I.t, lnpl. Ht6"* 13 92.9 IS '98.3 13 98.1 13 98.1 11 98.1 14 93.3 Humber of Dead Inolants 0 0 1 1 0 5 N.R. w- 13 , 1.9 15 i 1.7 T 12 7.7 / 7.7 131 1.9 9 35.7 1 13 7.1 714 868 14 14 98.2 867 13 W 98.1 0 15 1.7 715 870 16 14 87.5 0 14 1.8 871 16 1$ 93.6 1 13 7.1 0 13 1.9 1 13 7.1 3 12 ` 20.0 0 It 2.3 716 872 14 14 98.2 0 15 1.7 873 Not pn!9****K* 0 1 13 7.1 717 874 13 13 98.1 2 0 14 1.8 875 15 13 86.7 1 14 1.8 11 15.4 12 7.7 5 4 55.6 718 876 Not preenant. 1 11 6.3 877 19 19 98.7 1 18 5.3 0 14 1.8 719 878 17 16 94.1 0 IS 1.7 879 15 15 96.3 1 15, 6.3 1 14: 6.7 1 17 5.6 2 16 11.1 `Total:**TTC------ ~W 3237.7` ----------- 35----------- Mean: 14.7 14.2 95.2 1.0 S.O.: 1.8 2.0 5.9 1.3 N: 35 35 34 35 451--TSTT 13.2 8.2 2.5 W.6 35 35 Tout of Inplantt fleeter than total of corpora lutea; data excluded frp calculation of Inplantat Ion efficiency, E-K> DO 0 6 7 5 7 5 C O N F ID F N T TAI *< g 0u> u> ]\ Appendix E (cont.) A Ocnlnent-Lethal Inhalation Study With Benzene in Rets Pregnancy. Corpora lutea and Inplantation Data 19-7342 ! r> Oz CoJ HO O O' TI Sj --Zt ONl' r~i (JV Hale Fonale Wo. Wo. Poo 1040 1041 Ml 1042 1043 M2 1044 1049 903 1040 1047 904 1040 1049 905 1050 1051 901 1052 1053 907 1054 1055 9oe 1050 1057 909 1050 1059 910 1000 1061 Corp. Inpl. 14 16 K8 Nat pregnant. Wat pregMHK 15 13 16 16 13 13 13 13 H 14 20 16 W 16 15 14 14 14 IS 15 13 13 IS 15 16 W Nat png 14 14 14 14 15 15 17 17 10~* 90.4 50.0 86.7 96.4 98.1 98.1 98.2 80.0 98.4 93.3 98.2 98.3 98.1 96.3 98.4 98.2 98.2 96.3 98.5 Post-Treataent Hating Period (Week 2) - Group V - (300 ppn) hnl*r of Dead Indents W.|. ' PFS xlO-7 1 15 6.3 0 8 3.1 1 12 7.7 3 13 18.8 0 13 1.9 0 13 1.9 0 14 1.6 0 16 1.6 0 16 1.6 3 11 21.4 0 14 1.8 1 14 6.7 0 13 1.9 2 13 13.3 1 15 6.3 2 12 14.3 0 14 1.8 1 14 6.7 2 15 11.8 Kale Fenate Wo. No. 911 1062 1063 912 1064 1065 913 1066 1067 Corp. tut. w. 17 17 13 12 15 15 Not pregnant. 16 16 13 12 J.E. xIO-* 98.5 92.3 98.3 98.4 92.3 Hunker of Dead Indents t L N.R. "2*r xIO- 4 13 23.5 1 " i 8.3 1 14 * 6.7 1 8 8 50.0 8 4 66.7 914 1068 15 1069 12 14 93.3 12 97.9 0 14 . 1.8 2 10 K.7 915 1070 15 1071 20 15 98.3 8 40.0 2 13 13.3 5 3 62.5 916 1072 U 1073 17 16 98.4 M 94.1 0 16 t.6 1 15 6.3 917 1074 1075 13 15 98.3 13 98.1 1 14 6.7 1 12 7.7 918 1076 12 1077 11 13 11 97.7 1 12 - 7.7 0 11 2.3 919 1078 16 1079 14 16 98.4 12 85.7 0 16 1.6 1 11 8.3 Total: *07--' 505 TZCTTI------------53---------T5Z--mmx Dean: 14.9 14.0 93.3 I.S 12.6 11.7 S.D.: 1.9 2.2 12.9 2.0 3.0 16.0 W: 36 36 35 36 36 36 ToUl nwber of f^lant* greater than total noaber of corpora luteoi data excluded from calculation of inplantation efficiency. N E-11 S wt F-l Appendix F A Dominant-Lethal Inhalation Study With Benzene In Rats 79-7342 Certified Feed Information* Week of Study Interval 1 Acclimation 2 Acclimation 3 Pre-Treatment Mating Period 4 Pre-Treatment Mating Period 5 Female Sacrifices (Pre-Treatment) 6 Treatment Period 7 Treatment Period 8 Treatment Period 9 Treatment Period 10 Treatment Period 11 Treatment Period 12 Treatment Period 13 Treatment Period 14 Treatment Period 15 Treatment Period 16 Post-Treatment Mating Period-Week 1 17 Post-Treatment Mating Period-Week 2 18 Female Sacrifice (Week 1) 19 Female Sacrifice (Week 2) 20 Post-Mating Interval 21 Post-Mating Interval 22 Post-Mating Interval 23 Post-Mating Interval 24 Post-Mating Interval 25 Post-Mating Interval 26 Post-Mating Interval 27 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 5/20/80 5/27/80 6/3/80 6/10/80 Lot. No. Oct 03792B Oct 037928 Oct Z2791D Oct 22791D Oct 22791C Oct 22791C Dec 6791A Dec 06791A Dec 06791A Dec 20791C Dec 20791C Dec 06791E Dec 27792C Dec 27792C Jan 04801C Feb 14801E Jan 31801B Feb 14801C Feb 14801C Feb 14801C Feb 28801F Feb 14801A Feb 14801A Mar 06801E Mar 26801E Mar 19802F Mar 19802F aPur1na Laboratory Chow S002 (Certified Feed) mash; ad llbltun. DO 067577 CONFTDFNTTAl. YZ0083140 " & -w- 6-1 Appendix G A Dominant-Lethal Inhalation Study with Benzene In Rats *j Analysis of Benzene 79-7342 Pre-test Study Interval Prior to Week 6 of the Initiation Treatment of treatment Period Completion of Treatment Date Collected: 12/3/1979 Freezing Point (?C) Purity (t) / 5.5064 99.96 Specific Gravity 0.8845 Total Sulfur by Mlcrocoulometry -ppm <1.0 1/14/1980 5.5084 99.97 0.8845 0.0 2/18/1980 5.5188 99.98 0.8843 0.0 3/21/1980 5.5291 100.00 0.8844 0.0 Sats ppm 42 42 28 45 Toluene ppm Acetone ppm 28 2 28 2 5 26 0.3 Analyses were performed at the Exxon Research and Engineering Company Linden, N.J. DO 067578 CONFTOFNTIAI YZ0083141 H-l Appendix H A Dominant-Lethal Inhalation Study With benzene In Rats DETERMINATION OFPOLLUTANTS IN DRINKING WATER GIVEN ' - T(rANlffSLS~ FOR STUDIES NO. 79-2425 AND 79-7342 Report No.: 80023 Sponsor: Chemical Manufacturers' Association and American Petroleum Institute Washington, D.C. Prepared y: James C. Eschbach Date: September 3, 1980 Issued by: Blo/dynamfcs Inc. Department of Metabolism and Analytical Chemistry Environmental Sciences P.0. Box 43 Mettlers Road East Millstone, New Jersey 08873 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'Grodnlck, Ph.D. DO 067579 D1rector CONFIDENTIAL 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. 79-2425 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 CONFTDFNTTAI YZ0083143 H-3 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. YZ0083144 Appendix K (cont.) A Dominant-Lethal Inhalation Study With Benzene In Rats TABLE I. RESULTS OF DRINKING WATER ASSAYS FOR STUDIES NO. >9-2425 And'79-7342--------------------------- METALS Antimony Beryllium Cadmlim Arsenic Lead Mercury Selenium Assayed 3/17/80 PPb < 1.3 <1.0 <1.0 < 0.5* <1.0 < 0.5* < 1.0* Assayed 6/12/80 PPo < 6.7b < 1.0 < 1.0 < 1.0* 1.2 < 0.5 < 8.3* 80023 P 4 of 4 PESTICIDES Lindane Heptachlor DDT Dleldrtn 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. CONFIDENTS 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 ImplantationData -Post-Treatment Mating Period 1. Number of corpora lutea 2. Number of Implantations 3. Number of dead Implants 4. Total number of dead implants 5. viable fetuses 6. Implantation efficiency (pre-Implantation loss) 7. Mutagenic ratio (post-Implantationloss) 8. Pregnancy rate 9. Male Impregnating Index A A A A A C C B B 7-a 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 analysts 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-dlstrlbutlon to assess significance. If significant differences among the means were Indicated, Ounnett'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 KruskalWallls 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 (l.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 if twosided risk levels. aSnedecor, G.w., and Cochran, W.G., Statistical Methods. 6th ed.,Iowa State Unlv. Press (1967), Hollander and Wolfe, Nonparametric Statistical Methods, John Wiley and Sons, New York (1973), Dunnett. C.W.J. Am. Sta. Assn.. Vol. 50 (1955) and Biometrics, Vol . 20 (1964j. 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 SAC SAC D HAW KAW SAC SAC HAW 00 067584 OONFTDFNTTAl. 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 8b: 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, Armltage's test for linear trend In the dosage groups was performed. I bCh1-square: Snedecor, G.W., and Cochran, W.G., Statistical Methods, 6th ed., Iowa State Unlv. Press. Ames, Iowa, 1971, pp. 250-253. Fisher Exact Test: Bradley, J.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 Co., N.Y. 1966, p.15. Armltage's Test: Armltage, P. "Tests for Linear Trends In Proportions and Frequencies". Biometrics. Sept. 1955, pp. 375-386. DO OONFIOFNTTA1 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. Hie 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 nunber 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 5X and IX 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 C0NFTDFNTTA1 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 Slg p<0.05 p<0.01 Absolute Data 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-Uallls, nonparametrlc). K+ . The means differ significantly (Kruskal-Uallls nonparametrlc). 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 CONFTDFNTTAl YZ0083150 s Appendix J A Dominant Lethal Inhalation Study in Rats with Benzene Histopathology Report Bio/Dynamics Project Humber M15, 79-7342 Submitted tot Dr. A. L. Knezevich Director/ Pathol gy Depi Bio/Dynamics Inc. East Millstone/ N. J. DO 067588 CONFTDFNTTAl YZ0083151 J-2 79-7342 A Dominant Lathal Inhalation Study In Rata 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 067589 CONFTDFNTlAl YZ0083152 J-3 A Dominant Lethal Inhalation Study in Rata with Benzene Histopathology Report 79-7342 1. Introduction A atudy 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 Mo. 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 I animals re ceived unmedicated chamber air. Groups II, III, IV and V animals were administered Benzene by inhalation at doses f 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 preserved for 48 hours in Bonin's fixative and transferred into lot neu tral buffered formalin. After fixation, the specimens from Groups 1 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 CONF T DFNT T Al YZ0083153 J-4 79-7342 , Results and Discussion ' Thd* microscopic findings are presented in Tables I and XI and suntnarized in Table XXX. 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 (Grow 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. YZ0083154 79-7342 A Dominant Lathal Inhalation Study in Rata with Benzene Histopathology Report Summary A study was designed and performed to the muta genic potential of Bensene administered by inhalation to Charles River male rats. Microscopic evaluation of the testes end epididymides f control and treated rats (High Dose Group) revealed few lesions in treated rats. However there was no conclusive evidence indicating compound related injury. N. jonal Coni DO 067592 CONFIDENTIAL YZ0083155 3707 100 3708 3709 102 3710 3711 104 3712 3713 106 3714 107 3715 108 3716 109 J-6 79-7342 Table I W' *__ J, *' . ^ a Dominant Lethal Inhalation Study in Rata with Benzene Control Rats Microscopic Findings Animal Number t Sex Pathology Number 80- Group Testes Epididymides SOT EOT H rt M MM M M MM MM M II1IIIIIII NNNNNNNNNN NNN *NNNNNN Do 06 75 <53 C ONF rDpNTTA( YZ0083156 79-7342 0-7 Table I A Dominant Lethal Inhalation Study in Rats with Benzene Control Rats Microscopic Findings Animal Number Sex Pathology Number 80- Group Testes Epididymides 110 111 112 113 114 115 116 117 I 119 M M M MM MM MMM tn m H r n th o HN r- m p HN CN IN pm* r- r m IN p n CM pn- in in IN p* i*> IN P- l*> I I I I I I I I I1 N N NN NN N N NN * N N N NN N N NN B IT DO 067594 CONFIDENTIAL YZ0083157 606 806 006 79-7342 J-8 Table II A Dominant Lethal Inhalation Study In Rate with Benzene Exposed Rata Microscopic Findings Animal Number Sex Pathology Number 80- Group 901 902 903 904 906 907 in o ON MMMMMM MMMM 6* 00 ON e H N m 9* in %D N NN n n n mm f* r- r- r- f- r>- - r* r- r* n mm VV VV VVVVVV Testes - seminiferous tubules, degeneration - focal syncitial cell formation Epididymides - globular/ayncitial cell formation N N N NNNN N 3 2 NNN*NNN*N 2 DO 067595 CONFIDENT T At YZ0083158 79-7342 Table II A Dominant Lethal Inhalation Study in Rate with Benzene Exposed Rata Microscopic Findings Animal Number Sex Pathology Number Group 80- O H M n <w lA VD r- 60 9k H O' H r4 O' O' 4-4 O' < *e4\ ^4 H o> O' *4 O' H O' MM M M MM M M MM oI- CO O' co ** HN V n T uo *o Pn cto- hco M cro- !* 1^ P* CO CO n n V VVV VV V V VV Testes Epididymides u N NN N N N U NN NNNNN * NN *N 0 067596 cnft dfntiai YZ0083159 J-10 Table III A Dominant Lethal Inhalation Study in Rata with Benzene Summary of Microscopic Findings Organ/Microlcopic Finding Group Testes - seminiferous tubules, degeneration - focal syncitial cell formation Epididymides - globular/syncitial call formation I 79-7342 v 1/18 1/18 1/16 DO 067597 CONFIDENTIAL YZ0083160 K-l Appendix K A Dominant-Lethal Inhalation Study with Benzene In Rats W " 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 TECKNICIAN-IN-CHARGE Kathryn E. Sloan VETERINARIAN Fred A. Hines DIRECTOR INHALATION TECHNOLOGY George M. Rusch SUPERVISOR Gary Hoffman TECHNICIAN-IN-CKARGE 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. 00 YZ0083161 K-2 Appendix K (cont.) A Dominant-lethal Inhalation Study with Benzene In Rati " m .wDEPARTNENT/TITLE PERSONNEL NAME DATA LOGISTICS SUPERVISOR B.J. Guth QUALITY ASSURANCE SUPERVISOR * Craig N. Lamb 79-7342 DEGREE B.A. B.A. 067599 OONFTDFNTTAI YZ0083162