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BUSK;' r,U.- RESEARCH
R. D.'4, V.elbr. Rr-id, Export, "cnr.s ,ivaf.!j 1SB32
CENTER
<412)733-5200
REV 8(E)
FINAL REPORT
ETHYLENE OXIDE DOMINANT LETHAL MUTAGENICITY INHALATION
STUDY ON RATS
JUNE 13, 1984 Project Report 47-87
BUSHY RUN RESEARCH CENTER R.D, #4, Mellon Road Export, PA 15632
WILLIAM M. SNELLINGS, Ph.D, Study Director
DO 14?435 r.ONFTDENTTAL
Bushy Run Research Center A Joint Mellon Institute -- Union Carbide Corporation Operation
Table of Concents
Page List of Table Titles......................................................................................................................... 11
List of Figures............................................................................................................................................HI *
Abstract ................................................................................................................................. .....
1
Objective....................................................................................................................................................
^
Materials and Methods........................................................................................................................ Test Material and Positive Control Chemical........................... Liquid Ethylene Oxide Chemical Analysis...................................................................... Ethylene Oxide Vapor Generation........................................................................................ Inhalation Chamber Description and OperationalCharacteristics .... Ethylene Oxide Vapor Concentration Determination .............................................. Target Chamber Concentrations and Triethylenemelamlne Dosage ...................
Animals and Animal Husbandry ............................................................................................. Quality Control............................................................................................................................. Group Assignment and Number of Animals per Group .................................................
Experimental Design.................................................................................................................... Sacrifice and Necropsy................................................................ Experimental Data Evaluated................................................................ Statistical Analyses ............................................................................................................... Records...............................................................................................................................................
2 2 2 2 3 3 3
4 ^ 4
5 ^ 6
Results and Discussion .................................................................................................................... Test Material Purity and Stability ...................,.......................................................... Chamber Concentrations, Temperature and Relative Humidity............................
Observations for Toxicologic Effects .......................................................................... Body Weight...................................................................................................................................... Fertility.............................................. .... ..............................................*..................................... Necropsy ........................................................................................................................................... Preimplantation Loss ............................................................................................................... Resorptions and Live Implants............................................................ Mutagenesis Data Tabulated in Reference to theMale Rat................................. Discussion and Summary ..........................................................................................................
^ 7 2
7 9 9
Reviewed and Approved......................................................................................................................... H
Acknowledgements................................................................................................
H
References............................................................................................................................................... ^
Tables..................................................................... .... ..............................................................................
Figures........................................................................................................................................................ ^
Inhalation Test in Rats......................................................................................................Appendix A
Individual Animal Results of Dominant Lethal Data..........................................Appendix B
List of Tables
Table No.''
*age
1 Physical Properties of Ethylene Oxide ............................................................. 13
2. Gas Chromatographic Conditions Used for the Exposure Chamber Analysis of Ethylene Oxide....................................................... ^.....................
14
3. Chemical Analyses and Specifications for Drum 7JNC45 G581 of Ethylene Oxide............................................................................................................... 15
4. Chloride Content of Liquid Ethylene Oxide ................................................... 16
5. Inhalation Chamber Ethylene Oxide Vapor Concentration Determination ............................................................................................................... 1^
6. Inhalation Chamber Temperature.............................................................................. lg
7. Inhalation Chamber Relative Humidity................................................................. 19 8. Body Weight Changes of Male Fischer 344Rats................................................. 20
9. Fertility Data................................................................................................................... ^1
10. Summary of Dominant Lethal Data..........................................................................
11. Mutagenesis Data for the High Exposure and Control Groups Tabulated According to the Male Fat Used in the Mating...................
22 24
confidents
List of FiEures Figure No.
1 Preimplantation Losses Second Week of Mating................... 2 Preimplantation Losses Third Week of Mating................... ... 3 Early Resorption Sites Second Week of Mating................... A Early Resorption Sites Third Week of Mating ................... 5 Early and Late Resorption Sites Second Week of Mating 6 Early and Late Resorption Sites Third Week of Mating.
p *j r- " r
25 26 27 28 29 30
iii CO C-ONF 'C:Fs~
BUSHY RUN RESEARCH
R. D 4, Me!!cn Road. Export, Pennsylvania 15632
UCC BUSINESS CONFIDENTIAL: Not to be released without the written consent of the Chairman of the Ethylene Oxide Steering Committee.
CENTER
(412) 733-5200
Project Report 47-87 Page 1 of 31 June 13, 1984
Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Study on Rats
Sponsor: Ethylene Oxide Producers
** ***
ABSTRACT
Groups of sexually mature male Fischer 344 rats were exposed to ethylene oxide (E0) vapor for 6 hours per day, 5 days per week for 10 weeks. The target exposure concentrations were 100, 33, and 10 ppm of E0 which are equivalent to 180, 59, and 18 mg/m3, respectively. No effects of treatment were observed from body weight determinations, clinical signs, or gross lesions at necropsy. At the end of the exposure period, the males were mated with sexually mature, unexposed, females. Low fertility rates were recorded for all treatment and air-control groups particularly, though not exclusively, during the first week of mating. Values for preimplantation loss were numerically increased in the 100 ppm exposure group in comparison to either air-control group. For the second and third weeks of mating, the number of resorptions were within the range for the two air-control groups. Although there were considerable differences between the two air-control groups for the number of resorptions, the values were increased in the 100 ppm group above the range for the controls; this was particularly noticeable during the third week. Other observations which could be indicative of a dominant lethal effect were so variable as to cake it inconclusive that the resorption data were a threshold indication of a dominant lethal effect. Within the constraints of the variability within the various groups, 33 and 10 ppm are considered to be concentrations not resulting in a dominant lethal effect.
OBJECTIVE
The purpose of this study was to evaluate the potential of ethylene oxide vapor, inhaled during a period of active spermatogenesis, to produce a mutagenic effect in the germinal cell line of male rats resulting in nonviable zygotes or embryos.
Bushy Run Research Center A Join! Mellon Institute -- Union Carbide Corporation Operation
Report 47-87 Page 2
MATERIALS AND METHODS
This study was conducted according to the protocol [CHF Jpb #39-598 (B), dated December 1, 1976 and amended February 23 and 24, 1978, and March 29, 1978] prepared for the evaluation of possible dominant lethal mutagenicity resulting from the Inhalation of ethylene oxide vapor. Any deviations to the protocol and amendments have been indicated in this report.
Test Material and Positive Control Chemical
Liquid ethylene oxide (CAS No. 75-21-8) was received in a 55-gallon drum from Union Carbide Corporation (UCC) on April 25, 1977 with the UCC identification number 7JNC45,G581. This drum was kept in a sheltered area outdoors for the duration of th study. This material was assigned the Chemical Hygiene Fellowship (CHF) Sample Number 40-163. The production site of the ethylene oxide (EO) was Union Carbide Corporation, Seadrift, TX. The tank car from which this sample was obtained was labelled Number GATX84731. Pertinent physical properties of EO are presented in Table 1. Triethylenemelamine (TEM) (CAS No. 51-18-3) which was used as the positive control chemical was received from Polysciences, Inc., Warrington, PA, on January 13 1978. This chemical was assigned the CHF Sample Number 41-29.
Liquid Ethylene Oxide Chemical Analysis
Prior to and following the termination of the inhalation exposure, EO was chemically analyzed at the Research and Development Department, UCC, South Charleston, WV to determine the purity and stability of the chemical.
Ethylene Oxide Vapor Generation
Liquid aliquots (approximately 5 pounds) from the storage drum were transferred to a stainless steel cylinder. This cylinder which was attached to the generation system was maintained at approximately 35C by means of a constant temperature recirculating bath. The ethylene oxide vapor pressure generated at that teraperaturi was utilized to conduct the gas through stainless steel tubing and a pressure-reducing regulator to maintain approximately three psl internal pressure.Manifolds of stainless steel tubing directed the vapor through control valves and flowmeters to the chamber air-inlet duct. Ethylene oxide vapor was diluted at the inlet duct with exposure room air and drawn into the Inhalation exposure chamber.
^ 1 4P>44q Ct')N,FlOfrNr7A|
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. Report 47-87 Page 3
Inhalation Chamber Description and Operational Characteristics
All five inhalation chambers were located in Room 139. Each chamber was rectangular in shape and had a volume of approximately 4350 liters with internal dimensions of 2.1 meters long, 2.1 meters high, and 1.0 meters wide. The chambers were constructed of stainless steel and contained glass windows for animal observation. Inside each chamber, a delivery duct with holes equidistantly spaced was mounted on one of the walls and extended lengthwise along the top. An identical duct for chamber exhaust was located near the floor of the chamber, directly beneath the inlet duct. Artificial light from the room was the only source of light in the inhalation chamber. Each chamber held one carrier with animal cages.
The airflow through each chamber was approximately 2000 liters per minute, which is approximately 27 air changes per hour. During each 6-hour exposure, chamber temperature, relative humidity, and airflow rate were recorded approximately four times a day.
Ethylene Oxide Vapor Concentration Determination
All chambers, including the control chambers, were monitored for E0 concentration by means of a Varian 2700 gas chromatograph (GC). Pertinent information regarding the operating conditions of the GC is presented in Table 2. An automatic sampling and recording system connected to the GC acquired the sample and recorded the data on a printer and on magnetic tape. A known standard concentration of SO was sampled several times before the start of daily chamber analysis to check the monitoring system. Furthermore, during each cycle of chamber analyses, the same standard was reanalyzed. Approximately eight samples were analyzed from each chamber each day. Conversion of parts per million, ppm (expressed as volume/volume) to mg/m^ is 1 ppm *1.8 mg/m^ (25C and 760 mm Hg).
Target Chamber Concentrations and Trlethylenemelamlne Dosage
Target concentrations of 100, 33, and 10 ppm of EO were selected for the exposure concentrations based on the results of an eight-week inhalation study (report 45-139) on rats. In this study, young rats exposed to 100 ppm were noted to have throughout the study a lower gain in body weight than the controls, whereas only a transient change in body weight was noted for rats exposed to 50 ppm. The target exposure concentrations for the one generation reproduction (report 42-7) and teratology (report 42-13) studies were the same as the present study, since the animals from each of the three studies were exposed at the same time. There, were two separate groups of animals which were exposed to room air (0 ppm of EO) and served as the negative or air-control . groups (air-control I and air-control II). The purpose of having two control groups was to allow for better assesment of the variability in this study between non-chemically treated groups of animals, A sixth group of animals was treated with triethylenemelamine (0.25 mg/kg) in water by a single intraperitoneal injection 48 hours before the first day of mating. [Note: This is a deviation from the protocol which states injection will be given 24 hours before the first day of mating.]
OO 14,2441 GONFTDFNTTA!
Report Page 4
Animals-and Animal Husbandry
Thirteen-week-old virgin male Fischer 344 rats (F-344/Ma: fBR) and three separate groups of four-week, five-week, and six-week-old virgin female Fischer 344 rats were received February 14, 1978 from Microbiological Associates, Inc., Walkersville, MD. They were housed in Room 139. All animals were individually numbered by using a toe-clipping method of identification, t&ch animal of the same sex was given a different number. Listed on each cage card were the Identification numbers for the animals within the cage.
During the non-exposure period, food (Wayne Lab Blox F-6 Laboratory Animal Diet, Allied Mills, Inc., Chicago, IL) and water (Municipal Authority f Westmoreland County, Greensburg, PA) were available ad libitum. The artifical fluorescent lighting was on a 12-hour light and 12-hour dark cycle (approximately 6:00 a.m. to 6:00 p.ra). When animals were in the room, the temperature and humidity controlling devices of the non-recirculated air supply Co the room were set to maintain the environment between 66 to 77F and 30 to 70% relative humidity.
Male rats were housed and exposed two per cage in stainless steel, wire-mesh cages (35 cm x 17 cm x 18 cm high), and females and positive control males were housed separated by sex, three per cage. In stainless steel, wire-mesh cages (23.5 cm x 20 cm x 18 cm high). Stainless steel shelf pans with a layer of Deotized Animal Cage Board (Upjohn Co., Kalamazoo, MI) were placed under each row of cages to prevent urinary and fecal contamination of lower levels. The paperboard along with the food and water were removed during each six-hour exposure for the male rats. During the mating period, one male and one female '--ere housed In stainless steel, wire-mesh cages (23.5 cm x 20 cm x 18 cm high).
Quality Control
Immediately upon arrival and during a two-week quarantine period, the general health status of the rats was assessed. Quality control examinations which included body weight determinations, fecal examination for Intestinal parasites, blood serology, evaluation of the nasopharynx and lung for aerobic bacteriologic flora, and micropathologic examination of selected tissues were performed on a portion of the group of rats upon receipt. The results of the quality control were within normal limits for commercially available, specific pathogen-free rats; therefore, the animals were determined to be of suitable quality for the inhalation study.
Group Assignment and Number of Animals per Group
At the time of random assignment of animals into exposure groups, only the rats whose body weights were within + two standard deviations from the mean weight of all the rats of that sex or age group were selected. Any rat having signs of poor health during the preliminary observation period was rejected. Male rats were randomly assigned to one of six groups of 10 each. The females were randomly assigned into three groups of 60 each.
CO 142442 CONF I DENT T A!
Report 47-87 Page 5
Experimental Design
A modification of the method for dominant lethal testing %s presented by Green, et al. ( 1977) was followed in this study. .The initial day-of the EO exposures was on February 27, 1978. All groups of males were exposed six hours per day, five days per week for ten weeks. The last exposure day was May 5, 1978. The injection of TEM occurred on May 4, 1978. Animals were observed twice daily for possible toxic effects from ethylene oxide exposure or TEM treatment. Body weights of the males were determined prior to the initiation of the exposures, every two weeks during the exposure period, and immediately preceding the first mating period.
The first of three sequential mating weeks for all males began the day (May 6, 1978 which is one day later than that stated in the protocol) after the last exposure. At the beginning of each mating week, a virgin female, approximately 18 weeks old, was randomly assigned to a mating cage with one of the treated male rats. In anticipation of obtaining higher percentages of pregnancy, the females which had not thrown a vaginal plug within four days of mating were switched to another male within the same treatment group. The female remained with this male until a vaginal plug was noticed or until the end of the mating period for that week (i.e. 3 days). The day a vaginal plug was observed was designated as day 0 of gestation and the female was separated from the male.
Sacrifice and Necropsy
Females were sacrificed on the 13th day of gestation, or if no vaginal plug
was observed, 14 days from the midweek of mating. The females were sacrificed
by exsanguination through severing of the brachial vessels following the
induction of anesthesia with methoxyfluorane. All gross'lesions were recorded.
The corpora leuta of each ovary were counted if present. The uterus of each
female was examined for the number of implantation sites, and the number of
early and late resorption sites. The position of implantation sites relative to
the uterine horn was recorded.
>
The uterus, ovaries, and urinary bladder and any gross lesions were removed at necropsy and placed in 107. neutral buffered formalin. The males were sacrificed by CO2 asphyxia. The testes and epididymides were retained in 10% neutral buffered formalin.
DO 142443 CONFIDENTIAL
6
Experimental Data Evaluated
The'appropriate records were maintained in order to determine the following:
a) Female Fertility Index * No. of females with implantations x jqq No. of females mated
b) Male Fertility Index No. of males successfully impregnating a female x jqq No. of females mated
c) Pre-implantation Loss No. of pre-implantation loss per female x jqq No. of corpora lutea per female
d) Proportions of the females with one or more or with two or more resorption sites;
e) Mutagenic Index (1) No. of resorption sites per female v iQONo. of implantation sites per female
Mutagenic Index (2) * No. of early resorptions per female x iqq No. of implantation sites per female
Statistical Analyses
The fiducial limit of 0.05 (two-tailed) was selected as the critical level of significance. All data of each exposure group were compared statistically to the air-control group by using the following tests: Continuous variable data (such as body weight gain) were analyzed by Bartlett's test for homogeneity of variance (Sok; and Rohlf, 1969), analysis of variance (Sokal and Rohlf, 1969), and Duncan's multipl range test (Duncan, 1955, 1957; Harter, 1960). Whenever the F value for analysis of variance was significant and Bartlett's test indicated homogeneous variance, Duncan' multiple range test was used to delineate which groups differed significantly from the control. When Bartlett's test indicated heterogeneous variance, the F-test (Sokal and Rohlf, 1969) was employed to compare each exposure group with each air-control group. The type of t-test then used was selected according to the significance of the F value. The Student's t-test (Sokal and Rohlf, 1969) was used when the F value was not significant. The Cochran t-test (Cochran and Cox, 1957) w used when the F value was significant. Contigency data were analyzed by using Fischer's Exact Test (Sokal and Rohlf, 1969).
Because there were not enough animals pregnant per group for appropriate statistical analyses, only mean or median values were reported for the following: preimplantation loss; proportions of females with one or more or with two or more resorption sites; and the two mutagenic indices.
Records
The final report, specimens, slides, and all raw data are retained in the Archives of the Bushy Run Research Center for possible further reference.
DO 1, 40444 IF T Dot'll T Al.
COh
( V
F tivcr t ?ge 7
RESULTS AND DISCUSSION
Test Material Purity and Stability
The purity of the ethylene oxide test material was stated to be greater than 99.9% ethylene oxide (P. H. Wadia letter dated October 21? 1981). Table 3 lists the results of chemical analyses which were perofrmed prior to and following the inhalation exposure period. Based on these analyses the test material was stable through this study.
Before the initiation of the study, the chloride content of the ethylene oxide was measured by UCC at the Institute Plant Laboratory, South Charleston, WV (Table A). The organic chloride compounds were identified by gas liquid chromatography as ethyl chloride, ethylene chlorohydrin, ethylene dichloride and vinyl chloride. The chloride content of each compound was calculated. The total of these calculated values, 10.1 ppm, agreed with the total chloride content, 10.1 ppm, as determined by a Dohrmann Chloride Analyzer.
Chamber Concentration, Temperature and Relative Humidity
The results for chamber EO concentration determinations are summarized in Table 5. The actual chamber concentrations were close to the target concentrations of 100, 33, and 10 ppm of ethylene oxide with little day-to-day variation. Mean chamber concentration values for the 100, 33, and 10 ppm ethylene oxide exposure groups were 98, 33, and 10 ppm, respectively. The respective coefficients of variation were 3, 3, and 4%.
Less than 5% of the total number of analytical samples from either of the air-control chambers were greater than the 0.05 ppm minimum detection limit. No ethylene oxide leaks from the vapor generation system were detected in the room where both the test and control chambers were located. Consequently, it is believed that these few analyses represent analytical anomalies.
Weekly means of chamber temperature and relative humidity are presented in Tables 6 and 7, respectively. The mean of the weekly means for chamber temperature ranged from 71 to 73*F and chamber relative humidity ranged from 49 to 50%. Dally mean chamber temperature values varied from 70 to 76"F and relative humidity values varied from 45 to 59%.
Observations for Toxicologic Effects
No deaths occurred in any group. No treatment-related adverse effects on apperance or demeanor were observed.
142445 '"-ONFXO ENTTAL
Report 67-67 Page 8
Body Weight
The mean body weight change from Day 0 for each group during the 10 weeks of exposures are given in Table 8. There were no apparent treatment-related effects on body weights. Some statistically significant differences were noted between the E0- and air-exposed groups, but these differences either disappeared by the next weighing interval or were not representative of a dose-related response.
Fertility
Presented in Table 9 are the results of the female and male fertility index determinations. For all three weeks of mating there were no statistically significant differences betwen any treatment group, including the positive control group, and either air-control group.
It was noted that the fertility indices were particularly low for all groups, including the air-control groups, during the first week of mating. The results of the mating for the first week are presented in the tables, but no interpretation of the data were made because of the insufficient number of pregnant animals. Mating during the second and third weeks resulted in fertility rates of only 50 and 60% for some of the ethylene oxide and air-exposed groups. These are less than the 75% fertility rates observed in other studies with this strain of rat. Individual female and male fertility records are presented in Tables 1 and 2, respectively, of Appendix B.
Nec ropsy
There were no gross lesions noted at necropsy for the male rats exposed to ethylene oxide. Only a few gross findings were noted in the female rats. It is not uncommon to have low incidences of certain observations, which included mostly distention of uterine horns, in non~exposed rats. A complete listing of the gross findings for the females is presented in Table 1 of Appendix A.
Preimplantation Loss
The results for the percentages of preimplantation losses are given in Table 10. Median values for the 100 ppm and positive control groups were numerically greater in the second and third weeks In comparison with the air-controls and low and intermediate ethylene oxide groups. These results are graphically presented for Individual animals In Figures 1 and 2. Given in Appendix B, Table 1, are the individual values of each pregnant animal for the number of corpora lutea and the number of implantation sites.
DO 142446 '-ONFTDFNTTAL
Report 47-87 Page 9
ResorptloflS and Live Implants
Results of the total number of resorptions and number of live Implants per pregnant female are presented In Table 10. For the second and third weeks of mating, all values of the 33 and 10 ppm groups were within the range of values for the two air-control groups; however, the values of the 100 ppm group, particularly for the third week of mating, were beyond the ranjfe of values for the air-controls. The number of early and late resorption sites are given for each pregnant animal in Appendix B, Table 1.
The values for the proportion of pregnant females with one or more resorption sites or with two or more resorption sites (Table 10) were either high for both air-control groups or varied considerably between the two air-control groups, consequently making interpretations of the results difficult. Similar problems in variation between air-control groups are present in the interpretation of the results for the number of resorptions per implantation site and number of early resorptions per implantation sites (Table 10). The individual results of these last two observations for all animals are graphically presented in Figures 3 through 6.
Mutagenesis Data Tabulated in Reference to the Male Rat
Presented in Table 11 are the results for preimplantation losses and resorptions per implantations listed with reference to which male contributed to these results. The median values are given for mating weeks 2 and 3. For the males with two values in one week (i.e. because of two impregnations), when either the highest or lowest values are eliminated, the median results indicate the same trend as does just the median value for the group. That is, for preimplantation losses, the values of 100 ppm group were numerically greater than either the values for air-control I or II for mating weeks 2 and 3, and the values for resorptions per implantations of the 100 ppm group were numerically greater than the values for either air-control I or control II for mating week 2 but only greater than air-control I for week 3.
Discussion and Summary
Ethylene oxide exposures at concentrations as high as 100 ppm did not result in any treatment-related effects on body weight or clinical signs in sexually mature male rats exposed for 10 weeks. At necropsy, there were no gross lesions observed in these animals that could be related to treatment.
Fertility indices were low in all groups during the first week of mating. While there was improvement in fertility during the second and third weeks, in many cases the indices were less than those seen historically in animals kept in mating boxes for up to 7 days. It is possible that the shortened mating periods in this study (3 or 4 days) may have been a factor contributing to the lower fertility rates.
OO
com
F.c-pcr:. *4 / Page 10
For some unexplainable reason, many of the values which indicate a dominant lethal event were elevated in air-control group II during the third week of mating, which is demonstrated in Figures 4 and 6. This phenomenon, along with the low numbers of pregnant females for all groups, consequently makes interpretation of the treatment-related effects difficult. What is apparent is that there were also elevated values for the 100 ppm group, but generally, the values for the 33 and 10 ppm groups were similar to those of air-control I.
In summary, values for preimplantation loss were numerically Increased in the 100 ppm exposure group in comparison to either air-control group. However, other supporting evidence of dominant lethal effects, (i.e_. decrease in fertility rate or increase in resorption per implantation) were not sufficiently elevated over both controls to make a firm conclusion of the dominant lethal effects from treatment. Without the results of air-control II, 100 ppm exposure concentration would have been considered an effect level. However, with the results of air-control II considered and with the problem of low fertility rates for all groups, these results should be considered inconclusive in establishing the minimum effect level. Within the constraints of the variability of the result, it appears probable that the exposures to 33 and 10 ppm did not result in a dominant lethal effect in male Fischer 344 rats.
X
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Tr>ETNTT4L
page ::
Reviewed and Approved by:
\fTH >
Wilillilaizm M.Snellings, Ph.D. Study Director, Project Manager
>
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,( ` /<f.__
Fred R. Frank,PhTu. Director
t/y.i/z!/--
Acknowledgement s Analytical Chemistry Animal Care Animal Husbandry Investigator Technical Support
Nee ropsy Report Review
Report Writing
Veterinary Pathology
George W. Klein, M.S.
Denise L. Eisler, AALAS Cert. II
Paul H. Bramson, DVM
Janice P. Zelenak, B.S.
Jeffrey D. Dorko, B.S. William J. Kintigh, B.S.
Norbert J. Smith, M.S.
Carrol S. Weil, M.A. Bryan Ballantyne, M.D., D.Sc., Ph.D.
William M. Snellings, Ph.D. Louan C. Longo, B.S.
Robert R. Maronpot, DVM
00 1,4244'? CONFTDFNTTA!.
REFERENCES
Cochran, W. G., and Cox, G. M. (1957). Experimental Designs. Wiley, New York.
Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics
U_:l-42.
Duncan, D. B. (1957). Multiple range tests for correlated and heteroscendastic means. Biometrics 13:164-176.
Green, S., Moreland, F. M., and Flamm, W. G. (1977). Short communication. A new approach to dominant lethal testing. Toxicol. Appl. Pharmacol,. 39: 549-552.
Harter, H. L. (1960). Critical values for Duncan's new multiple range test. Biometrics 16:671-685.
Sokal, R. R., and Rohlf, F. J. (1969). Biometry. Freeman, San Francisco.
DO 142450 CONFTOFNTIAi.
?.eport - 7-S7 Page 13
Table 1
Physical Properties of Ethylene Oxide* Ethylene Oxide Dominant Lethal Mutagenesis Teat on Rats
Molecular Formula:
CH2CH20
*
Molecular Weight:
44.05
CAS Registry Number
75-21-8
Boiling Point at 760 mm Hg:
10.4C (50.7 F)
Specific Gravity at 20/20C (H2O " 1):
0.8711
Vapor Density (air * 1):
1.49
Percent Volatiles by Volume:
100
Appearance and Odor:
Colorless gaa
Vapor Pressure at 20C:
1095 mm Hg
Solubility in Water:
Complete
Evaporation Rate (butyl acetate * 1):
>1
Flash Point (Tag open cup):
< 0 F; ASTM D 1310
Flash Point (Tag closed cup):
< 0F; ASTM D 56
Concentration Equivalency (3 2 5C and 760 mm Hg:
1 ppm*1.8 rag/m^ 1 mg/liter = 556 ppm
* Information obtained from Material Saf ety Data Sheet, Union Carbide Corporation F-4313QB. 4/78-4M
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Table 2
Ga Chromatographic Conditions Uaed for the Exposure Chamber Analysis of Ethylene Oxide Ethvlene Oxide Dominant Lethal Mutagenesis Test on Rata
Column Column packing Conditions
Carrier gas Flame detector gases
Sample size Lower limit of detection
Stainless steel tubing 1/8" o.d. x 20 ft.
30% TERGITOL - TMN and 3% sodium methylate on Cbromosorb U-NAW 60/80 mesh
Column oven and injector at 80C Injection loop at room temperature Interface and detector 130C
Nitrogen at 35 ml/min
Air 550 ml/min Hydrogen 42 ml/min
lml
0.05 ppm
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Report ^7-ST Page 16
Table 4
Chloride Content of Liquid Ethvlene Oxide Ethylene Oxide Dominant Lethal Mutagenesis Teat on Rats
Ma terial
Organic Chloride Content^(ppm, v/v)2
Calculated Chloride Content
(ppm CL, wtv)1
Organic Compound Ethyl chloride Ethylene chlorohydrin Ethylene dichloride Vinyl chloride
12.2 3 2 1.3
6.7 1.3 1.4 0.73
Determined by gas liquid chromatography (glc) 2 To convert from w/v to v/v: multiply by reciprocal of the density of the
organic chloride at 20C, i.e.,
(w/v value x density at 20 C (v/v value)
DO 142453 OONFTDENTTAI
Report 67-87 Page 15
Table 3
Chemical Analyses and Specifications for Drum 7JNC45, G581 of Ethylene Oxide^ Ethylene Oxide Dominant Lethal Mutagenesis Test on Rats
Chemical Analysis
Analysis Date April 23, 1977J - July 18, 1978
Maximum
Commercial Specifications^
Acidity (X by wt)4
0.0016
0.0014
0.002
Aldehydes (ppm)5
21
19
30
Acetylene (ppm)
Nil
Nil
Nil
Water (X by wt)
0.09
0.005
0.03
Residue (g/100 ml)
0.0012
0.0004
0.005
Color (Pt-Co)^
3
1
10
Odor
Nonresidual
Nonresidual
Nonresidual
Suspended Matter
Substantially Free
Substantially Free
Substantially Free
^ Analyses performed at the Research and Development Department > Union Carbide Corporation, South Charleston, WV.
2 Commercial specifications of Union Carbide Corporation. ^ Maximum of two analyses.
H Measured as acetic acid. ^ Measured as acetaldehyde.
6 Platinum-cobalt color test.
0 0 142454 '"-ONF T !jF.NT T AL
Ke tort - -o. Fa ft 17
Table 5
Inhalation Chamber Ethylene Oxide Vapor Concentration Determinations Ethylene Oxide Dominant Lethal Mutagenesis Test on Rats
_______________________ Ethylene Oxide Target Chamber Concentrations*
Target concentration, ppm
100 ppm
33 ppm
Mean of daily means of measured concentration, ppm^
Coefficient of variation^, %
97.6 3.3
33.0 2.9
30 PP
9.9 3.8
Air Control Chamber Concentrations
Air control chamber Target concentration, ppm
I 0
Medciaonncoefntthraetiodna,ilyppmm1edians of measured Number of individual analyses
e 0.053 373
Number of analyses above minimum limit of detection
13
Mean concentration of analyses above mini;turn limit of detection
0.19
Standard deviation of analyses above minimum limit of detection
Maximum concentration determined, ppm
+ 0.16 0.70
II 0
e 0.053 367 12
0.24
+ 0.20 0.77
Approximately 8 analyses per chamber were made each exposure day of the days that a test atmosphere was generated.
Measurement is based on the standard deviation and the mean. It is a measure of variability of the daily means.
Gas chromatograph minimum detection limit.
DO 14?a35 CONFIDENTIAL
1c
Table 6
Ethvlene Oxide Dominant Lethal Mutagenesis Test in Rats
i Inhalation Chamber Temperature
*
Exposure Week
1 2 3 4 5 6 7 8 9
10
SD
100 pom X SD 2
72 i 72 i 71 4 i 72 i i 70 4 0 70 + 1 72 4 1 70 i- 0 71 4 1 70 0
71 1
iL?SS.
xt SD
72 -k 1 74 3 72 4; 1 72 4 0 72 4 1 73 4 1 74 4 1 74 4 1 73 1 74 0
Target Concentration
10 ppm
0 ppm (CX)
x SD
3c SD
74 4 1 73 4 1 73 4 1 72 4 1 73 4 1 73 4 1 73 4 1 74 4 1 72 4 1 72 4 0
73 4 1 72 4 1 73 4 1 72 4 1 72 4 1 72 4 0 73 4 0 73 4 1
72 4 1 72 4 1
73 4 1
73 4 1
72 4 0
^ Units = Farenheit 2 5 t SD Kean standard deviation
0 ppm (CII) x SD
73 4 1 72 4 1 73 4 1 72 k 1 72 4 1 72 4 1 72 4 1 74 4 0 71 4 1 71 * 1
72 4 1
DO 142456
Table 7
Ethvlene Oxide Dominant Lethal Mutagenicity Inh. Test in Rats
Inhalation Chamber Relative Humidity ^
Exposure Week
1 2
3 4 5
6
7 8 9 10
x SD
100 ppm x SD 2
46 4 1 46 4 0 49 4 3 49 4 3 48 4* 2 54 4 3 52 4 4 52 4 4 52 4 4 50 4 4
50 4 3
33 ppm x i SD
46 4 1 46 4 0 48 4 3 50 4 4 48 4 2 55 4 3 52 j- 4 52 4 4 51 4 4 50 4 4
50 4 3
Target Concentration
10 ppm
0 ppm (Cl)
x t SD
x SD
46 4 1 46 4 1 49 4 3 49 4 2 48 4 2 54 4 2 52 4 3 53 4 4 52 4 4 49 4 4
45 4 1 45 4 0 48 4 3 49 4 4 47 4 2 53 4 2 51 4 5 53 4 3 51 4 4 49 4 4
50 4 3
49 4 3
1 Units * Z Relative humidity ^ x SD = Mean standard deviation
0 ppm (CI1) x SD
46 4 0 47 4 0 49 4 3 50 4 4 48 4 2 54 4 3 54 4 3 53 4 4 51 4 3 49 4 5
50 4 3
DO 142457 CONFIDENT! Al
Report 47-87 Page 20
Table 8
Body Weight Changes of Male Fischer 344 Rats^ Ethylene Oxide Dominant Lethal Mutagenesis Test on Rats
Exposure Week
100 ppm Mean ;+ SD2
Target Exposure Concentrations
Air Control 1
33 ppm
10 ppm
0 ppm
Mean + SD
Mean + SD
Mean + SD
Air Control II 0 ppm
Mean + SD
Mean Body Weight, Grams, On Day 0
266.6
+ 18.4 273.2 + 17.0
271.2
+ 14.3 274.4 + 13.2 274.0 + 16.4
Body Weight Change, Grams From Mean Weight On Day 0
2
13.4
+ 10.5
14.0+ 4.6
9.8b,a + 5.0
16.1 + 6.5 14.3 + 5.4
4
20.0"* 3 + 11.6
25.1+ 6.4
20.5-'3 + 7.1
25.1 + 8.2 30.0 + 9.0
6
31.6
+ 14.1
39.3 + 10.1
30.7 + 8.6 32.6+ 9.5 38.6 + 6.5
8
46.4
+ 13.1
52.4+ 9.5
45.2
+ 8.1
49.4 + 10.2 52.1 + 7.9
10
51.2
+ 13.2
57.5 + 10.4
49.4 + 9.0 55.7+ 8.4 58.1 + 8.0
a = p < 0 .05
b - 0.05 > p > 0.01
1 N = 10 rats per group 2 SD = Standard deviation
l 4^4 to C0NrT0?Nrl
Week
1 2 3
Table 9
Ethylene Oxide Dominant Lei:hnl Mutagenicity Inhalation Test In Rats -F---e--r--t--i-l-i-t-y-----D---a--t-a-1
<
100 ppm
20 (2/10) 70 (7/10) 50 (5/10)
33 ppm
Treatment
10 ppm
0 ppm (Cl)
0 ppm (CII)
TEM (0.25 m g/kg)
Percentage (ratio) Pregnant Females
~No. of females pregnant L No. mated
U (1/7) 70 (7/10) 50 (5/10)
56 (5/9) SO (8/10) 100 (10/10)
12 (1/8) 60 (6/10) 70 (7/10)
33 (3/9) 90 (9/10)
60 (6/10)
56 (5/9) 60 (6/10) 60 (6/10)
i-'-t 'N "O1t0n1 oo
I> 0
Percentage (ratio) Males that Successfully Impregnated One or Two Females
1
20 (2/10)
10 (1/10)
2
60 (6/10)
50 (5/10)
3
50 (5/10)*
50 (5/10)
-t--a--- ;---------- rsuccessfully mated with two females.
50 (5/10) 70 (7/10)* 100 (10/10)
10 (1/10) 60 (6/10) 60 (6/10)
30 (3/10) 70 (7/10) 60 (6/10)*
56 (5/9) 60 (6/10) 50 (5/10)
* Value may be Incorrect since for one pregnant female in this group no vaginal plug was detected, consiMpient1 it is unknown which male impregnanted her.
4
'tan'i *>)
IIM
OI )I
Table 10
Ethylene Oxide Dominant Letlia j_Mutagenicity Inhalation Teat in Rats Summary of Dominant Lethal Data* I
Week
100 ppm
33 ppm
Treatment
10 ppm
0 ppm (Cl)
0 ppm (CII)
* TEM (0.25r.ig/kg)
Median (Quartile Deviation)*Preimplantation Lobs (No. preimplantation loss/No. corpora lutea/Female X1001
1 62 (33) 2 33 (30)
__ irk 9 (34)
40 (34) 0 (8)
--
0 (1)
30 (31) 8 (13)
11 (30) 30 (22)
3 42 (34)
8 (17)
8 (15)
8 (6)
14 (20)
70 (2b)
Total Resorptions / No. Pregnant Females
1 3.0
1.0
0.2
0.0
2.0
5.0
2 1.4
0.1
0.8
1.3
0.6
3.7
3 4.0
1.4
0.9
1.0
2.7
3.5
Total Live Implants / No . Pregnant Females
1 0.5
6.0
5.6
12.0
4.7
2.0
2 5.0
7.7
9.4
9.7
8.8
2.2
3 2.6
8.6
8.4
9.3
4.5
0.3
r> oo Zo n
h -*
o m \> z H O'
O 1>
(Continued)
1u1 -nj>i <<nv 'dJ I inM
t-I
I ,10 1 U J 1 ' ^OUII L i IlllOu)
Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Test in Rats Summary oF Dominant Lethal Data
Week
100 ppm
33 ppm
Treatment
10 ppm
1 ppm (Cl)
0 ppm (CII)
TEM (0.25 inc/Vg)
Proportion of Pregnant Females with One or More Resorption Sites (X100)
1 100
____
20
2 71 14 50
3 100 60 60
--
67 86
67 44 83
Proportion of Pregnant Females with Two or More Resorption Sites (X100)
1 50
0
2 43
0 12
3 60 40 20
--
50 14
33 11 67
Median (Quartile Deviation) No. Reaorptions/No. Implantation Sites/Female (X100)
1 92 (19) 2 20 (30) 3 44 (38)
---
0 (0) 10 (13)
0 (12) 4 (5) 9 (9)
--
13 (11) 9 (3)
9 (42) 0 (6) 42 (38)
Median (Quartile Deviation) No. Early Resorptions/No. Implantation Sites/Female (X10_0)
1 42 (42) 2 8 (22) 3 14 (33)
----
0 (0) 10 (13)
0 (12) 0 (4) 0 (6)
---
4 (10) 9 (3)
0 (42) 0 (6) 34 (38)
* Median Is the 50th percentile. Quartile deviation is (75th - 25th percentile) / 2. **Dashes indicate that values could not be determined because only one animal was pregnant.
100 67
100
too 67 tn
100 (24) 76 (45)
100 (12)
100 (29) 60 (43)
100 (20)
tft-i Tf.V
T<Oll XOf
N.
'M
r*
C O N F ID E N T
I
A
l .
Table U Mutagenesis Oata for the High Exposure and Control Groups Tabulated
According to the. Hale Rat Used In the Hating Ethylene Oxide Homliimit Lethal Mutagenesis Test on Rata
Hale
100 ppm EO Mating Week
12
An I PL/CL* 8/1 ** PL/CL 8/1
1 3
PL/CL 8/1
Male
1
An I PL/CL R/I
Treatment 0 ppm Cl Hat tng Week
2
PL/CL R/l
3 PL/ CL R/l
4786 33
4847 11
90
63 100
90 100 17 20
16 44 63 100
0 100
2741 0 0 M
00
2838
10 0
2854 2896
It
60 0 50 60
08
73 33 42 14
2799 4822
II
0 22 0 18 08
4774
33 0
4803
0 25
2771
median median* median1
OO zO T1 l-t "*
O f* rq i'J
Z f* H O' --I M
>
0 43
33 20 25 14 42 32
42 44 30 38 5B 38
2903 2722
0 13 08 0 18
* Prelmplantatton losa/corpors lutea X100. ** Early and late resorptions/implantation sites X100. nd-not determined
1 median with the highest values of the double mating (e) eliminated. 2 median with the lowest values of the double mating (a) eliminated.
9 10 09 12 14 17 0 8 18
89 08
89 89 8 10
Male
1
An 1 PL/CL R/l
0 ppm CIl Hating Week 2
PL/CL R/l
3 PL/CL R/J
4788 62
4775
0
2819
2829 II
2862 1
30
4838 1
2807 2830
0 9
83
31 0 10 0 80 0 22 14
0 17 80 09 09
00
36 43 55 40 10 U
14 100 00
nd 80
8 0 14 42 *0 0 23 10 0 25 41
tj t-
Fiqure 1 EO DOMINANT LETHAL MUTAGENESIS STUDY
PREIMPLANTATION LOSSES SECOND WEEK OF MATING
- Median and actual data point
i
NO. PR EIM PLAN TATIO N LOSSES/CORPORA LUTEA PER PREGNANT FEMALE X 1000
Figure 2 EO DOMINANT LETHAL MUTAGENESIS STUDY
PREIMPLANTATION LOSSES THIRD WEEK OF MATING
PPM OF ETHYLENE OXIDE ^= Median and actual data point
Figure 3
EO DOMINANT LETliAL MUTAGENESIS STUDY EARLY RESORPTION SITES SECOND WEEK OF MATING
EARLY RESORPTION S IT E S /N O . IM PLAN TATIO N S IT E S PER PREGNANT FEMALE X 1000
o az go
Orrt &Z Z--i J' r-t '/I
x-
*
O z
0
100 33
10 0 0 POSITIVE
CONTROL I CONTROL II
CONTROL
TEM
PPM OF ETHYLENE OXIDE
^ = Median and actual data point ^- Median
)fOt)ji* orOo
rVj)
r
I'
I t--oJ
Figure 4
EO DOMINANT LETHAL MUTAGENESIS STUDY EARLY RESORPTION SITES THIRD WEEK OF MATING
i-
Median and actual data point Median
\) trJ
CU fl
CfPt>4 "OO
tOl' rt II*-1
o
oo
zo
T|
o^ rn \> Z -fi --I O'
Ni
I>
NO. EARLY AND LATE RESORPTION S IT E S /N O . IM PLANTATIO N S IT E S PER PREGNANT FEMALE X 1000
1000 900800 700600500-
400 -
300 200 100
04
Figure 5
EO DOMINANT LETHAL MUTAGENESIS STUDY EARLY AND LATE RESORPTION SITES SECOND WEEK OF MATING
*
t
*
*-jilt* t "W-
**
**
***)((* * ****
0000^ 00`
100 33
10
0
0 POSITIVE
CONTROL I CONTROL II CONTROL
PPM OF ETHYLENE OXIDE
TEM
Median and actual data point Median
R e p o rt
Pace 29
r* -1 i co
orn -*-> Z > H O'
a>
NO. EARLY AND LATE RESORPTION S IT E S /N O . IM PLANTATIO N S IT E S PER PREGNANT FEMALE X 1000
1000900' 800700 600- 500- 600300- * 200 100 0
Figure 6 EO DOMINANT LETHAL MUTAGENESIS STUDY
EARLY AND LATE RESORPTION SITES THIRD WEEK OF MATING
** *
iI***
*
* 1*
*
*
**
*
* *
**
*
ft J* . _ ______ ft ft**
* *
100 33 10 0 0 POSITIVE
CONTROL I CONTROL II
CONTROL
PPM OF ETHYLENE OXIDE
TEM
Median and actual data point
K tp o r
Pare
O t> *
t
`l
cry
i
f. ~ r * * ***"/ Ft 31
BUSHY RUN RESEARCH
R. D. 4, Mellon Road. Export, Pennsylvania 15632
CENTER
Teiepnone (4l2i 733-5200
Quality Assurance Unit Study Inspection Summary
Test Substance: Ethylene Oxide
Study: Dominant Lethal Mutagenicity Inhalation on Rats
Study Director: William M. Snellings, Ph.D.
The Quality Assurance Unit of BRRC conducted the inspections listed below and reported the results to the study director and to management on the cates indicated. It is the practice of this Quality Assurance Unit to report the results of each inspection to both the study director and management.
Inspection
Date
Tvpe
10-22 to 10-30-79
9-1-81
1-17 to 1-31-34
Pathology Data
Event-Final Report Raw Data, Pathology Report and Final Report
To Studv Director To Management
10-30-79
1-17-80
9-1-81 1-31-84
11-3-81 2-28-84
DO 142469 CONFIDENTIAL
Q
Linda J. CalistY Quality Assurance Unit Supervisor
Bushy Run Research Center A Joint Mellon Institute --Union Carbide Corporation Operation
c-Z/s/i /
Date
APPENDIX A Pathology Report Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Test in Rats
D M747 o
CONrTOfNTT
Report 47-; Appendix A. Page 1 of 7
Ethylene Oxide Dominant Lethal Mutagenesis Test in Rats
Pathology Results
Males. There were no gross lesions observed in treated or control rats with the exception of a Control II animal (#78-2883) which had a nodule in the peritesticlar fat grossly compatible with focal fat necrosis secondary to infarction. The pathogenesis of this common lesion is believed to be initiated by torsion of tags of adipose tissue leading to localized ischemia.
Females. A few gross lesions were documented in female rats and these are summarized in Table 1. Several females had bilateral dilation of uterine horns compatible with estrus; none of these rats had implantation sites. The type and frequency of gross findings should not affect the results of this study.
able 1
Report ^ iv A
?2Cr 2 pf 1
Ethylene Oxide Dominant Lethal Mutagenesis Test in Rats
Gross Findings in Females
Dose and Rat Number
First Week of Mating
Gross Findings
100 ppm 0
78-3643 78-3659 78-3669 78-3670 78-3671 78-3657 78-3679 78-3683 78-3685
78-3686
78-3701 78-3724
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. Distention of uterine horns
compatible with estrus. Distention of uterine horns
compatible with estrus. No gross lesions. No gross lesions.
33 ppm 0
78-3655 73-3678 78-3690 78-3699
78-3702 78-3708 78-3712 78-3714
No gross lesions. No gross lesions. No gross lesions. Polycystic right kidney and
pitted surface on left kidney compatible with nephritis. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
10 ppm E0
78-3644 78-3647 78-3660 78-3662 78-3668 78-3672 78-3693 73-3694
73-3709 78-3716 78-3 720
do I*?472 , CONFIDE NTT A
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. Corneal opacity of right eye
compatible with keratitis. No gross lesions. No gross lesions. Distention of uterine horns
compatible with estrus.
(Continued)
-w
---f\J
Co*
Table 1
Report 47Appendix A Page 3 of 7
''Ethylene Oxide Dominant Lethal Mutagenesis Test in Rats
Gross Findings in Females
First Week of Mating
Dose and Rat Number
(Continued)
Gross Findings
Control I
78-3648 78-3656 78-3665 78-3666 78-3681 78-3689 78-3704 78-3707 78-3719
'
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
Control II
78-3642 73-3664 78-3676 78-3688 78-3692 78-3695
78-3697 78-3710 78-3721 78-3722
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. Distention of uterine
compatible with estru:
No gross lesions. No gross lesions. No gross lesions. No gross lesions.
Positive Control (0.25 og/kg TEM)
78-3673 73-3674 73-3675 78-3680 73-3632 78-3634 78-3700 78-3703 73-3706 78-3723
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
(Continued)
t > <w J \ "+ t
' '*
ONFinfr'NTT AL
Table 1
t t-
Repcrt 47--87 Appendix A Face - of 7
Ethylene Oxide Dominant Lethal Mutagenesis Test in Rats
Gross Findings In Females
Second Week of Mating
Dose and Rat Number
(Continued)
Gross Findings
100 ppm EO
78-3565
78-3566
78-3567 78-3570 78-3572 78-3575 78-3607
78-3627 78-3628 78-3561
Distention of uterine horns compatible with estrus*
Distention of uterine horns compatible with estrus.
No gross lesions. No gross lesions. No gross lesions. No gross lesions. Distention of uterine horns
compatible with estrus. No gross lesions. No gross lesions.
No gross lesions.
33 ppm EO
78-3551 73-3554 78-3560 78-3581 78-3589 73-3591 78-3599
73-3605
78-3618 78-3630
10 ppm E0
78-3558 78-3559 78-3569 78-3576 78-3577 78-3578 78-3585 78-3609 78-3612 78-3621
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. Distention of uterine horns
compatible with estrus. Vaginal and vulval hemor
rhage. No gross lesions. No gross lesions.
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
.
_ _ ____ -
(Continued)
DO \42474 CON roe^TT AL
able 1
Report
Append Page 5
Ethylene Oxide Dominant Lethal Mutagenesis Test in Rats
Gross Findinzs in Females
Second Week of Mating
(Continued)
Dose and Rat Number
Gross Findings
Control I
78-3552 78-3553 78-3573 78-3582 78-3584 78-3598 78-3613 78-3614 78-3629 78-3635
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
Control XI
78-3563 73-3574 78-3580 78-3588 78-3592 78-3602 78-3603 78-3606 78-3616 78-3622
Positive Control (0.25 ag/kg TEM)
78-3549 78-3564 73-3586 78-3590 78-3594 78-3596
78-3601 78-3610 78-3611 78-3633
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
S
No gross lesions. Periovarian cyst. No gross lesions. No gross lesions. No gross lesions. Distention of uterine
compatible with ec' No gross lesions Mo gross lesions No gross lesions No gross lesions
(Continued)
C'O (4P>4?f; CONFIDENTIAL
Table 1
-7 * -
--
E'.-T'O rt 4*-67 en;::-: A e O OZ i
Ethvlene Oxide Dominant Lethal Mutagenesis Test In Rats
Gross Findings In Females
Third Week of Mating
(Continued)
Dose and Rat Number
Gross Findings
100 ppm EO
.78-3073 78-3082 78-3111 78-3123 78-3179 78-3246 78-3469 78-3480 78-3484 78-3486
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
33 ppm EO
78-3064 78-3101 78-3201 78-3249 78-3467 78-3475 78-3481
78-3485 78-3522 78-3526
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. Distention of uterine horns
compatible with estrus.
No gross lesions. No gross lesions. No gross lesions.
10 ppm EO
7 8-3096 78-3219 78-3245 78-3464 73-3465 78-3473 78-3477 78-3487 78-3528 78-3529
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
.
(Continued)
DO 14247b CONFIDENT! A1
T&bXfi 1
Ethylene Oxide Dominant Lethal Mutagenesis Test In Rats
,^
Gross Findings in Females
Rf' ort
--
Arr*tnci>i ~
*'3 iZ t1 / c!
Third Week of Mating
Dose and Rat Number
(Continued)
% _________Gross Findings
Control I
78-3059 78-3074 78-3131 78-3207 78-3210 78-3462 78-3466
78-3476 78-3483 73-3524
No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. Distention of uterine ho
compatible with estrus. No gross lesions. No gross lesions. No gross lesions.
Control II
78-3063 78-3105
73-3110 78-3140 78-3146 78-3157 78-3206 78-3470 78-3488 78-3527
No gross lesions. Distention of uterine ho;
compatible with estrus. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
Positive Control (0.25 mg/kg TEM)
78-3070 78-3078
78-3114 78-3222 78-3241 78-3250 78-3460 73-3463 78-3471 78-3482
No gross lesions. Distention of uterine hoi
compatible with estrus. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions. No gross lesions.
EO =* Ethylene Oxide TEM * Triethylenemelamine
DO 1.42 A? 7
confidential
p.epor 47-S7
* APPENDIX B Individual Animal Results of Dominant Lethal Data Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Test in Rats
DO 14-473 LONF 7 rF.\'T 7 A1
IN^CidNO O
3J0da-J
Table 1 Ethylene Oxide Dominant Lettt;11 Mutagenicity Inhnlatlon Test In Rat3
Individual Animal Results of Dominant Lethal Data
Ma ting Week Number
Female An. No.
Mated With Male
An. No.
First, Second
1 1 1 1 1 1 1 1 1 1 1 1
2 2 2 2 2 2 2 2 oo 2 2
*j
f t 'i Is
3643 3701 3724 3659 3669* 3670 3671 3657* 3679 3683 3685 3686
3628 3570 3572 3575 3627 3567 3561 3565 3566 3607
S'
4847 4774, 4786 4786 2710, 2771
2734, 2710 2771, 2734
2842 2854, 2842 2898, 2854 2899, 4774
4847, 4786 2854 2898 2899, 2898 4786, 4774 2842, 2771 2710, 4847 2734, 2710 2771, 2734 4774, 2899
Number Corpora Lutea
Number Implantation Sites
Number Preimplantation Losses
100 ppm Ethylene Oxide
10 1 96
---- ----
---- -- __
-- _-- ---- -- ---- ----
10 1 10 4 10 5
8 12 96 77 12 10 ---- ---- ----
9 3 -- --
-- --
-- -- -- --
9 6 5 0** 3 0 2 -- -- --
(Continued)
Number Early Resorption Sites
0 5 --: --
-- --
-- -- -- --
0 0 3 1 0 3 1 -- -- --
--
Number Late Resorption Sites
Vaginal Plug
Do tor ted
1 0 -- --
-- --
-- -- -- --
1 0 0 0 0 0 1 -- -- --
t
Y Y Y N
N N
Y Y N Y
Y Y V V Y N* Y H N Y ..
) .I
i*:*
'1 r*
r-j
Table 1 (Continued) Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Teat in Rate
Individual Animal Results of Dominant Lethal Data
Mating Week Number
Female An. No.
Mated With Male
Tin. No. First, Second
Number Corpora Lutea
Number Implantation Sites
Number Preimplantation Losses
3 3484 3 3073 3 3179 3 3246 3 3469 3 3486 3 3082 3 3111 3 3123 3 3480
1 3714
1 3655
1 3678
1 3690*
1 3699
1 3702
1 !"1
3708
1 b o 3712
Zo
"0
OT1 ^X) Z ^00
r-t O
1>
4847 2710, 4847 2854 2898, 2854 2899, 2898 4786 2734, 2710 2771, 2734 2842, 2771 4774
4797, 4811 2715, 2726 2779, 2747
2826, 2779 2841, 2826 2364, 2841 2865, 2864
100 ppm Ethylene Oxide (Continued)
12 2 11 12 11 3
--
--
12 7
11 9 ----
----
----
10 0** 8
--
5 2 --
-- --
33 ppm Ethylene Oxide
97 ---- -- ----
---- ---- ---- ----
2 -- ----
-- -- -- --
(Continued)
Number Early Resorption Sites
2 0 0 -- 1 3
--
-- --
1 --
-- -- -- --
Number Late ResorptIon
Sites
V.ipln Pip
0 v 12 i;1
1V -- tl
0V 1Y -- t; -- i; -- ii
Y
0 tl' --N """ II
-- II -- II _-- n ---- H
hh
rIirh-*
* *
i
I
:j m I ) fi* r
O.
Table 1 (Continued) Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Teat In Rats
Individual Animal Results of Dominant Lethal Data
Mating Week Number
Female An. No.
2 3630 2 3618 2 3581 2 3605 2 3551 2 3560 2 3554 2 3589 2 3591 2 3599
3 3526
3 3522
3 3064
3 3101
3 3467
3 3201
3 324 9
3 3475
3o
3481
3
oo zo
3485
n
h -
a
m s\>
z^
h oo
i--t i--*
Mated With Male
An. No. First, Second
Number Corpora Lutea
Number Implantation Sites
Number P reImplant a tion Losses
4811, 4797 4797 2779, 2747 2865 2715 2747, 2726 2726 2826, 2779 2841, 2826 2864, 2841
4811 4797 2715 2726 2865 2747 2779, 2747 2826, 2779 2841, 2826 2864, 2841
33 ppm Ethyl ene Oxide (Continued)
12 2 10 10
93 89 11 10 11 10 11 11 ---- ----
----
10 0 6 0** 1 1 0
__ --
--
12 5 12 10 12 11 10 12 13 12 ---- ---- ----
--
----
7 2 1 0** 1 -- -- -- ----
--
(Continued)
Number Early Resorption Sites
0 0 0 0 0 0 1 ---- __ --
0 1 2 0 4 -- ----
--
Number Late Resorption Sites
V.ipi no. Plug
Del.ec t
0Y 0Y 0 f! ' 0V 0Y 0 ' 0Y
j; __ i * --N
0Y 0Y 0Y 0V 0Y --V --M -- tt tlmm mm '--- II
ci''.t > nT>O
Table 1 (Continued)
Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Teat in Rate Individual Animal Results of Dominant Lethal Data
Mating Week Number
Female An. No.
Mated With Male
An. No.
First, Second
Number Corpora Lutea
Number Implantation Sites
Number Preimplantation Losses
1 3709 1 3693 1 3660 1 3662 1 3716 1 3644 1 3720 1 3672 1 3647* 1 3668 1 3694*
2 3621
2 3558
2 3578
2 3577
2 3612
2 3585
2 3569
2 r> o 3576
2 2
"n o
3559 3609
o m
2
H 3)
-I>h \)
2863, 2795 2795, 2794 2731, 2735 2735, 2740 4810 4842, 4795 4795, 4810 2746
2740, 2746
4842 2731, 4842 2795 2794 4810, 2863 2863 2740 2746, 2740 2735 4795, 4810
10 ppm Ethylene Oxide
12 2
--
--
82
--
--
10 6 10 6
--
--
10 11
----
10
--
6
4 2
--
0**
TM
7 11 11 11 10 . 12 10
-- 13 --
7 12
9 9 9 12
10 -- 13 --
0 0** 2 2 1 0 0 -- 0 --
(Continued)
Number Early Resorption Sites
0 __
1
0 0 -- 0
--
3 0 0 0 0 0 1
0 --
Number Late Resorption
Sites
V-ic.tnal I11 uf; 1 Voiced
0Y __ II
0Y --N
0V 0V --N 0Y
--- N
0Y 1Y 1Y 0Y 0 N1 0Y 0Y --N 0Y --Y
---- >ri >
n-
-.1
Report
Mating Week Number
3 3 3 3 3 3 3 3 3 3
o
X--
a-n ?t--,>' m b>
w v
1 1 1 1 1 1 1 1 1
Table 1 (Continued) Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Test in RatB
Individual Animal Results of Dominant Lethal Data
Female An. No.
Mated With Male
An. No. First, Second
Number Corpora Lutea
Number Implantation Sites
Numbe r P reimplan ta tIon Losses
3465 3477 3487 3528 3096 3473 3529 3245 3464 3219
3656 3648 3665 3666* 3681 3689 3704 3707 3719
4810 2794 2795, 4795 2731 2746 4842 2740 2863 2735
2794
2741 2722, 2741 2768, 2799
2799, 2812 2812, 2838 4803, 2903 4818, 4803 4822, 4818
10 ppm Ethylene Oxide (Continued)
11 5 11 6 10 B 12 9 11 10 11 11 11 11 12 11 11 11 11 11
6 5 2 3 1 0 0 1 0 0
Air Control I (0 22HL EO)
11 12 ---- ----
0** -- --
(Continued)
Number Early Resorption Sites
0 3 0 1 0 2 0 1 0 0
0 -- --
Numbei Late ResorptInn Sites
'-i-ii-.l fl.lt;
l1 i>i
l 0 0
0 0 0 0 0Y 07 1Y
0V --Y --N
N H
tl t; N
C*l' ->1 *T>
rj -.1
r* O
r
fjj \
r*
on '-t
Table 1 (Continued)
Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Test in Rats Individual Animal Results of Dominant Lethal Data
Hating Week Number
Female An. No
Hated With Male
An. No. First, Second
Number Corpora Lutea
Number Implantation Sites
Number Preimplantation Losses
2 3598 2 3613 2 3584 2 3552 2 3635 2 3629 2 3573 2 3553 2 3582 2 3614
3 3476 3 3074 3 3131 3 3207 3 3524 3 3483 3 3462 3 3059 3 3210 3 3466
n oz ao
71
z> P m -VJ z^ ~i 0)
fi 3>
Air Control I (0 ppm EO - Continued)
2838 2903, 2838 2812, 2799 2722, 4822 4822 4818, 4803 2768, 2741 2741, 2722 2799, 2768 4803, 2903
10 --
9 10 13
9 12
--
--
--
9 --
9 11 13 12 12
--
--
--
1
--
0 0** 0 0** 0
--
--
--
2838 2741 2768, 2741 2799 4822 2903 4803, 2903 2722 2812
4818, 4803
,
8 11 11 12 12 12 --
11 -- --
7 10 11 10 11 11 --
12 --
--
1 1 0 2 l 1 --
0** --
--
(Continued)
Number Early Resorption Sites
Number Late Resorption
Sites
v.-ip.Inal n
0
--
2 2 1 0 0
--
--
--
1 1 1 0 2 1 -- 1 -- --
#
0
--
0 0 0 3 0
--
--
--
0 0 0 0 0 0 -- 0 -- --
Y tl Y M1 Y
Y tl M N
Y Y Y Y Y Y N Y N N
C0*TT3) *-T>JJ
fp <t*
\nm3'.
o x <'iJr
JJ
Reror
Table 1 (Continued)
Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Test in Rata Individual Animal Results of Dominant Lethal Data
Hating Week Number
Female An. No.
Mated With Male
An. No. First, Second
Number Corpora Lutea
Number Implantation Sites
Number Preimplantation Losses
1 3642 1 3722 1 3697 1 3710 1 3721 1 3664* 1 3676 1 3688 1 3692 1 3695
2 3580 2 3592 2 3588 2 3616 2 3606 2 3602 2 3603 2 3574 2 3622 2 3563
o oo zo TT >H t-1 O m \>
Z f*
ha n tn j>
4838, 4788 4788, 2819 2862 2883, 4775 4775, 2807
2807, 2711 2819, 2829 2829, 2830 2830
2819 2830, 2829 2829 4788 4775 2862 2883, 2862 2807 4838 2711, 4838
Air Control II (0 ppm EO)
83 __ -- --
10 6 11 11
--
--
5
___
3 0
--
__ __ --
--
--
--
10 2 97
12 12 13 9 10 9 12 11
9 11 11 12 11 11 ----
--
--
8 2 0 4 1 1 0** o**
0
--
(Continued)
Number Early Resorption Sites
0
5 0
--
__
--
--
0 1 2 0 0 0 0 0 1
--
Number' Late Resorption Sites
Vn j* Inal
PI up. Deter' >`d
0Y II
0Y 1Y N----
N__ D
--N --Y
0Y 0 .Y 0Y 0V 0Y 0Y 1V 0Y .0 Y --N
>t; > VJ
O'
itnt
"v!
r:
H
n
l
*-- rn
Tahiti 1 (Cont inued)
Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Test in Rats Individual Animal Results of Dominant Lethal Data
Mating Week Nutnbe r
Female An. No.
Mated With Male
An. No. First, Second
Number Corpora Lutea
Number Implantation Sites
Number Preimplantation Losses
Number Early Resorption
Sites
t Number Late Resorption Sites
Vap.f'i.. I [VI > / i d
3 3470 3 3146 3 3157 3 3527 3 3063 3 3488 3 3110 3 3140 3 3105 3 3206
1 3706
1 3682
1 3684
1 3700
1 3675
1 3673
1 3674
1 3680
1 r\ 3703*
1
oo zo
3723
o m \> zp h co
O' ir>~
Air Control II (0 ppm E0 - Continued)
4775 2830 2862, 2830 4838 2711, 4838 4788 2819 2829, 2819 2807, 2711 2883, 2862
11 nd
--
7 10 11 10
--
--
--
5 5 ---- 6 11 7 9
--
--
6 nd
--
1 0** 4 1
--
--
--
Positive Control (TEM, 0.25 mR/kg)
2857 2793, 2818 2818, 2848 2848, 2857 2775 2745 2774, 2745 2791, 2793
4844, 2775
9 8 9 -- 10 12 -- --
--
2 4 8 -- 10 11 -- --
--
7 4 1 -- 0 1
--
--
--
(Continued)
20 40 ----
60 00 21 10 ---- ----
----
20
40
80
----
41
5*
1
----
----
--"
V V
II
V
II1 V Y H N N
Y Y Y N Y Y N N
N
Trn.i) *r*.jj *:; *< zi:i oM
7> Cl n
O**tl
X
CCf
C^s1Oi
K f
Table 1 (Con 11nued) Ethylene Oxide Dominant Lethal Mutagenicity Inhalation Teat In Rats
Individual Animal Results of Dominant Lethal Data
Mating Week Humber
2 2 2 2 2 2 2 2 2 2
3 3 3 3 3 3 3 3 3 3
Female An. No.
Mated With Male
An. No. First, Second
Number Corpora Lutea
Number Implantation Sites
Number Preimplantation Losses
3633
3549 3586 3564 3611 3590
3601 3610 3594 3596
Positive Control (TEM, 0.25 mg/kg - Continued)
4844 2738, 4844 2774, 2745 2745 2857, 2848 2775 2818 2848, 2818 2791 2793, 2791
10 10 10 --
8 10
10 -- --
--
2 7 4 --
6 7
9 -- -- --
8 3 6 --
2 3 1 --
-- --
3241 3250 3471 3463
3460 3222 3482 3070 3078
3114
2791 2793, 2791 4844, 2857 2857 2848 2775 2818 2738, 4844 2745 2774
10
--
11 6
10 16 11
--
--
1
--
2 5 3 5 7
--
--
9
--
9 1 7 11 4
--
--
Number Early Hesorptlon Sites
0 6 3 -- 0 6
4 -- -- --
1
--
2 5 2 2 7
--
-1
Number Late Resorption
Sites
Vagina Plug
Pol net
0Y 0 N1 1Y --V 0Y 0Y 2Y --N --Y --N
0Y --H
0Y 0Y 0Y 2Y 0Y --N --Y
Y
DO 1 4 7 4 8 7
COMF1 DENTTAl
* Female was Incorrectly mated with two males, each from a different treatment group, consequently, results were not included.
** More implantation sites than corpora lutea 1b defined as no (0) preimplantation losses, nd - not determined; Y - yes; N - no
1 The.,number of early and late resorption sites may be,/<ncorrect since no vaginal plug was detected, and
'
.t 4 s
r
/
> i>
<s'
4<
.i
fci 'D (' fl>
.p r* i1 X
it)
np^tfr.cix s
Tair 10 of i:
Table 2
Ethylene Oxide Dominant Lethal Mutogenicitv Inhalation Test in Rats Individual Fertility Results for Males
Male An. No.
Sueessfully Mated One or Two Females during
Mating Weeks
1
2.
3
4847 4774 4786 2710 2734 2771
2842 2854
2898 2899
100 ppm Ethylene Oxide
YY Y
YY
--"
----
?*
7
Y Y ----
Y
"
Y
7
"
-
Y Y "
4797 2715 2779 2826 2841 2864 2865 2726 2747 4811
33 ppm Ethylene Oxide
7Y -Y -9 ---Y -Y -7 7-
Y Y
-- "
Y Y -* Y
DO 140488 conftofntia;
f.\
Report L~ -s: Appendix B Page 11 of 12
,
Table 2 (Continued)
Ethylene Oxide Dominant Lethal Mutogenicity Inhalation Test in Rats individual Fertility Results for Males
*
Male An. No.
2863 2795 2731 2735 4810 4842 4795 2746 2740 2794
Sucessfully Mated One or Two Females during Mating Weeks
12
3
10 ppm Ethylene Oxide
YY Y Y?
-Y Y YY Y ?Y
YY YY YY
YY YY
2741
2722 2768 2799 2812 4803 4818 4822 2903 2838
Cl ppm Y
-
-
Y ?
Y
?
? Y
Y
Y Y
-
Y
-
-
-
Y Y Y
DO l4P489 r nc* Ti. rL^\rc, kINj tl ti r^i .
i, .
I
Appendix B
TV:;- i: of i;
Table 2 (Continued)
Ethylene Oxide Dominant Lethal Mutogenicity Inhalation Test In Rats Individual Fertility Results for Males
Male An. No.
4838 4788 2862 2883 4775 2807 2819 2829 2830 2711
Sucessfully Mated One or Two Females during Mating Weeks
12
3
CII ppm
Y YY
YY --
YY -Y -Y -Y ---
Y Y
Y
Y
Y ?
2857 2793 2818 2848 2775 2745 2774 2791 4844 2738
Positive Control (TEM, 0.25 mg/kg)
Y-Y Y. YY YY YY ---Y
?
Y -
Y Y Y -
Y -
^Question mark indicates that male was mated with a female that also was mated with a different male. Since the female did. not throw a vaginal plug (but
was pregnant), it is not known which male impregnated the female.
1 ~ -- --~~ ~ i
iu
r
DO 142490 CONFIDENTIAL