RTI-908; Project 0209189.000 Final Report



### Table of Contents Page

QUALITY ASSURANCE STATEMENT ........................................................................... iii GLP Compliance Statement ............................................................................................ v

SUMMARY....................................................................................................................... 1

INTRODUCTION ............................................................................................................. 1

MATERIALS AND METHODS ......................................................................................... 2 Designation of Responsibilities.................................................................................. 2

Test Material............................................................................................................... 3 Animals and Husbandry ............................................................................................. 4 Table A. Study Schedule........................................................................................... 4 Study Design .............................................................................................................. 7 Table B. Number of Animals Assigned to Study Groups........................................... 7

Statistics.................................................................................................................... . 10 Storage of Records .................................................................................................... 10

Personnel ................................................................................................................... 1 1

RESULTS ........................................................................................................................ 11 Test Chamber Analyses ............................................................................................. 11 Maternal Findings....................................................................................................... 12 Uterine and Embryofetal Findings .............................................................................. 13

DISCUSSION................................................................................................................... 1 4

CONCLUSION ................................................................................................................. 16

REFERENCES ................................................................................................................ 17

List of Protocol Deviations ............................................................................................... 19

Appendices Appendix I: Inhalation Report Appendix II: Individual Animal Data Tables Appendix III: Protocol and 2 Amendments



### iRTI-908; Project 0209189.000 Final Report



### List of Tables Page Table 1 Chamber Monitoring Results.......................................................................... 20

Table 2 Summary and Statistical Analysis of the Maternal Body Weights, Weight Changes, Organ Weights and Relative Organ Weights................................. 21

Table 3 Summary and Statistical Analysis of the Maternal Feed Consumption.......... 25

Table 4 Summary of the Maternal Clinical Observations ............................................ 28 Table 5 Summary and Statistical Analysis of Ovarian Corpora Lutea, Uterine Contents, Live Fetal Sex, and Live Fetal Body Weight .................................. 29

Table 6 Summary and Statistical Analysis of Malformations and Variations............... 32

Table 7 Summary of Morphological Abnormalities in CD-1 Mouse Fetuses: Listing by Defect Type.................................................................................... 33

Table 8 External Malformations in Control CD-1® Mouse Litters From Studies Performed for Governmental Clients at RTI From 1997 to 2002.................... 34



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iv**RT1-908; Project** **0209189.000 Final** **Report GLP** **COMPLIANCE STATEMENT** **This study** **was conducted** **in accordance** **with CFR** **Title 40,** **Vol. 16,** **Chapter 1,** **Subpart F,** **Paragraph 79.60** **(U.S. EPA,** **1994), and** **performed according** **to the** **protocol (and** **two amendments)** **and following** **the SOPs** **of HLS** **and RTI.** **This study** **complied with** **all appropriate** **parts of** **the USDA** **Animal Welfare** **Act regulations:** **9 CFR** **Parts 1** **and 2** **Final Rules,** **Federal Register,** **Vol. 54,** **No. 168,** **August 31,1989,** **pp. 36112-36163,** **effective October** **30,1989, and** **9 CFR** **Part 3** **Animal Welfare** **Standards; Final** **Rule, Federal** **Register, Volume** **55, No.** **32, February** **15,1991, pp.** **6426-6505, effective** **March 18,** **1991, and** **U.S. EPA** **TSCA GLPs** **(U.S. EPA,** **1989). It** **was the** **Sponsor's responsibility** **to maintain** **the method** **of synthesis,** **fabrication, or** **derivation of** **the test** **fuel, and** **this was** **not completed** **at the** **time of** **the study** **conduct but** **has been** **completed since** **and is** **on file** **with the** **Sponsor.**

# Roclfelle**W. Tyl,** **Ph.D., DABt/** **Date Study** **Director**

# iomasM.**Gray,^ /** **pate 'Sponsor's** **Repr**RTI-908; Project 0209189.000 Final Report



### SUMMARY The purpose of this range-finding study was to determine a tolerable exposure level of Gasoline MTBE Vapor Condensate (G/MTBE) in pr egnant CD-1 mice in order to identify an appropriate high exposure level for a subsequent developmenta l toxicity study. A previous developmental toxicity study conduc ted at another laboratory dem onstrated that pregnant mice tolerated exposures of G/MTBE vapor condensate up to 20,000 mg/m 3 

### on gestation days (gd) 5 through 16; this study provided suggestive evidence of an increase (not exposure-related) in midline defects among the offspring (EMBSI, 2009) . In the present range-finding study, groups of pregnant CD-1 female mice were exposed to 0 or 30,000 mg/m 3 

### of G/MTBE vapor condensate for 6 hours per day on gd 5 through 10. This 6-day exposure regimen was selected since exposing the dams to a higher concentration durin g the critical period of susceptibility for midline effects would maximize the opportunity to confirm whether G/MTBE causes an increase

in midline defects. In this range-finding study, th ere were no deaths or moribund animals in either group of pregnant mice. The only clinical sign observed among the dams exposed to 30,000 mg/m 3 

### of G/MTBE was lethargy. Embryofetal survival, gr owth, and development were not significantly affected by exposure to 30,000 mg/m 3 

### of G/MTBE. Based on the results of this range-finding study, the pregnant CD-1 females and their offs pring could tolerate inhalation exposure to 30,000 mg/m 3 

### on gd 5 through 10. It was therefore recommended that the definitive developmental toxicity study of G/MTBE should proceed with 25 plug-positive females/group at 0, 2,000, 10,000, and 20,000 mg/m 3 

### target concentrati on, with exposures on gd 5 through 16, and 40 plug-positive females at 30,000 mg/m 3 

### target concentration, with exposures on gd 5 through

10.

INTRODUCTION

G/MTBE is a fraction of the most volatile components of a gasoline containing methyl tertiary butyl ether (MTBE) that was specially fo rmulated to meet the specifications of the U.S. Alternative Tier 2 testing re quirements (U.S. EPA, 1998a). To mimic real world human exposures, G/MTBE was produced by a method designed to produce a test substance reasonably similar in composition to the vapor produced in a vehicle fuel tank head space near the maximum in use temperature of 130°F (Daughtrey et al., 2004).

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### Developmental toxicity evalua tions of G/MTBE by inhalati on exposure to rats and one other species were mandated by a final notificat ion letter issued under the Alternative Tier 2 testing provision of the fuels and fuel additive s health effects testi ng regulation (U.S. EPA, 1998b). The conduct and design of the studies were further proscribed by special and general EPA guidelines (U.S. EPA, 1994 and 1998c). One of the developmental studies conducted involved whole-body i nhalation exposure of timed-pregnant CD-1® mice for at least 6 hours/da y to baseline gasoline vapor condensate, containing approximately 25.5% MTBE, at ta rget concentrations of 0, 2000, 10,000, and 20,000 mg/m 3 

### of G/MTBE (the last is 50% of the lower explosive lim it) on gd 5 through 16 (EMBSI, 2009). This study was conducted to confirm and extend the findings observed in that earlier study, in which suggestive evidence of an increase (not exposure-related) in midline defects was observed among the offspring. The purpose of this range-finding study was to determine a tolerable exposure level of G/MTBE in pregnant CD-1 mice in order to id entify an appropriate high exposure level for a subsequent developmental toxicity study. In the present range-fi nding study, groups of pregnant CD-1 female mice were exposed to 0 or 30,000 mg/m 3 

### of G/MTBE for 6 hours per day on gd 5 through 10. This 6-day exposure regime n was selected since exposing the dams to a higher concentration during the critical peri od of susceptibility for midline effects would maximize the opportunity to confirm whether G/MT BE causes an increase in midline defects. Fetal malformations of specific interest in this study, ventral wall closur e defects, are formed early in the embryonic period of gesta tion, gd 7-9 in the mouse (e.g., Rugh, 1968).

MATERIALS AND METHODS Designation of Responsibilities 

### RTI International was responsible for study design, protocol generation, setting up mating and assignment of plug-positive study females, necropsy of the maternal and fetal animals on gd 17, generation of summary and individual data tabl es, and study draft and final reports (with RTI QA oversight). RTI’s Quality Assurance Unit pe rformed a prestudy on-site inspection, reviewed the protocol and any amendments, and monitore d all phases of the study in which RTI personnel participated. Huntingdon Life Sciences was respons ible for receipt of the test material, prestudy and study generation and analyses of the test vapo rs, receipt, quarantine, and housing of the test

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### females and breeder males, setting up the mati ng and assignment of the study females, in-life observations, loading and unloading study females in to and out of chambers, and submission of interim and final inhalation reports. The Quality Assurance Unit of HLS reviewed the protocol and monitored the facilities, equipment, personne l, methods, practices, records, raw data, draft and final inhalation reports, and controls used in this study to assure that they were in conformance with company standard operating pr ocedures and the referenced Good Laboratory Practice (GLP) regulations.

Test Material 

### The test material, G/MTBE (MRD- 00-713; “API 211BG with MTBE Vapor Condensate”), was a colorless liquid and identified by the s upplier (Chevron Global Technology Services Company (CRTC, Richmond, CA) as Lot/Batch Number API 00-02. Methods of synthesis, fabrication, or derivation were doc umented by the Sponsor and located at API. Information on identity, strength, purity, a nd composition of G/MTBE was provided by the Sponsor and documented in the raw data and in this final report (Appendix III, protocol attachment). Two separate types of chemical analysis we re performed on the test material. CRTC conducted proprietary characteri zation of all 120 com ponents. ExxonMobil Bi omedical Sciences, Inc. (EMBSI) conducted a GLP-compliant character ization of 18 representative hydrocarbons and MTBE. In the CRTC method, the we ight percent of each of the components wa s measured, whereas in the EMBSI method, the relative amount of each of the representative com ponents was measured on area-percent basis. EMBSI developed their method for API to shar e with CRTC and the la boratories performing toxicological studies on 2 11(b) testing program test substances. The objective of the method was to

monitor and docum ent the chemical and com positional stability of G/ MTBE from manufacture through transporta tion, storage, and animal ex posure using a standard method that all users could perform.

The test material was stable an d stored under ambient conditions in an outside solvent shed except when in use in the inhala tion laboratory. The test substa nce was handled as a flammable liquid. Detailed information on chemical handli ng is provided in the MSDS attached to the protocol (Appendix III).

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### Animals and Husbandry



### The test animals were Caesarean-ori ginated Virus Antibody Free (VAF) Crl:CD-1 ® (ICR) BR outbred albino mice s upplied by Charles River Laborato ries, Inc., Raleigh, NC. The use of live animals was requested by the Spons or and required by U.S. EPA OPPTS Testing Guidelines (U.S. EPA, 1998c). Alternative test systems are not available for the assessment of chemical effects on prenatal mammalian develo pment. The Charles River CD-1® mouse has been the subject of choice on developmental toxicology contracts at RTI since 1976. Large historical databases for reproduc tive performance and prevalence of spontaneous malformations in control mice are available from studies conducted at RTI (currently based on over 288 control litters and over 3600 fetuses [Table 8]). T hus, although the in-life study husbandry, exposures and observations occurred at HLS and could vary fr om that at RTI, the fetal evaluation criteria

should be consistent. The actual dates of all major phases of the study are presented in Table A.

Table A. Study Schedule

Event Dates

Animals arrive at HLS: August 31, 2004 Quarantine (14 days): August 31 – September 14, 2004 Animals paired: September 16-18, 2004 Dates of gd 0: September 17-19, 2004 TSCA experimental start date: September 22, 2004 Exposure dates (gd 5 through 10): September 22-29, 2004 Scheduled termination (gd 17) October 4-6, 2004 TSCA experimental termination date: October 6, 2004

Submission of draft data on test atmospheres to Sponsor: October 6, 2004 (within 1 week after the last exposure date, September 29, 2004



### Fifty (50) nulliparous female mice were orde red for this range-finding study. One extra female was received with the shipment. One hundred (100) male mice, 9-11 weeks old, and of the same strain and from the same supplier, were also ordered. The males from Charles River

Laboratories were not from the same litters as th e females. One female that would have been over the designated weight range on gd 0 (35 g) was excluded prior to mating. Fifty (50) male

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### mice were used to generate timed-mated animals for this study, and all 100 of the males were used to generate timed-mated animals for the s ubsequent definitive developmental toxicity study which will require the mating of 170 female mi ce to generate 140 plug-positive females. Acclimating all 100 male mice assured that they were the same age for this range-finding study and subsequent definitive study, a nd were exposed to the same environmental conditions, etc.

Female mice were 7-9 weeks old at arrival and were 9-11 weeks of age and 20-35 g in weight on gd 0. Fifty females were required to generate 20 plug-positive females in 2 to 3 consecutive days; 20 plug-positive females (10 per group and 2 groups) were required to supply the minimum number (based on EPA’s guidance; e.g., OPPTS 870.3600; U.S. EPA, 1996; for inhalation, range-finding, developm ental toxicity studies) of pr egnant animals to assess the maternal and embryofetal tolera nce of the highest c oncentration, possibly to be used in the subsequent developmental toxicity study. During an approximately 14-day quarantine and acclimation period at the HLS testing facility, animals were checked fo r viability twice daily. Prior to study assignment, all animals were examined to ascertain suitability for study. The HLS veterinarian formally released these animals for use by signature and date. Males and females were individually housed in stainless steel suspended cages with wire mesh floors a nd fronts, except for the mating period when 1 male and 1 female were housed together. Du ring cohabitation, male and female mice were housed in polycarbonate “shoebox” cages with st ainless steel lids and Alpha-Dri® bedding (Shepherd Specialty Papers, Watertown, TN). Each cage was fitted to secure a glass feeder jar

with a stainless steel lid. Clean feed jars and fresh feed were provided at least weekly for periods when feed consumption was not being recorded a nd at each interval when feed consumption was recorded. After cessation of exposures began on gd 11, a stainless steel, perforated insert was placed on the wire-mesh floor of the stainless steel suspended cage of each female and 1 Nestlet® (Ancare, Bellmore, NY) added to each cage until scheduled sacr ifice on gd 17. Feed (PMI 5002 Certified M eal) was available ad libitum , except during the daily 6-hour inhalation periods. Analytical certification of batches of feed provided by the manufacturer is maintained on file at the HLS testing facility, and there were no known contaminants found in the feed. Facility water (supplied by Elizabethtown Wa ter Company, Westfiel d, NJ) was available ad

libitum via the automatic watering system or wate r bottles (during mati ng), except during the daily 6-hour inhalation periods. Water anal yses were conducted by Elizabethtown Water

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### Company to assure that water met standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CFR Part 141). Water analyses provided by the supplier are maintained on file at the HLS testing facility. There were no known contaminants that interfered with the objectives of this study. At all times , animals were housed, handled, and used according to the National Research Council Guide (NRC, 1996). A 12-hour light/dark cycle was provided via au tomatic timer. Temperature and relative humidity were monitored in accordance with Testi ng Facility SOPs to ensure that the desired range of 18 to 26°C for temperature and 30 to 70 % relative humidity was maintained to the maximum extent possible (NRC, 1996). The actual ranges were 21 o 

### C - 22 o 

### C and 43% - 52%, respectively. Each animal was assigned a temporary iden tification number (designated on each cage) upon receipt. During the second week of the quarantine/acclimation period, the 51 females

received were tail tatt ooed with consecutive numbers, 1 th rough 51. The 100 males were also tail tattooed with consecutive numbers, 1 th rough 100. After selection for use on the study, mating, indication of copulation, and assignment to either of the 2 groups, each female was ear tagged with a number assigned by the HLS testing f acility. This number, plus the study number, comprised the unique animal identification for eac h animal. Each cage was provided with a cage card that was color coded for exposure level id entification and containe d the study and animal

numbers. It was anticipated that the concentration employed would not resu lt in irritation or corrosion to the respiratory tract of the test animals (since prev ious studies at close to 30,000 mg/m 3 

### did not report irritation or co rrosion to the maternal respir atory tracts (e.g., Bevan et al., 1997; EMBSI, 2009). Animals were not subjected to undue pain or distress. All procedures used in this study were designed to avoid discomfort, distress, and pain to the animals. The HLS

IACUC Protocol Review Subcommittee and the RTI IACUC reviewed th e protocol and found it to be in compliance with appropriate animal welfare regulations (9 CFR Parts 1 and 2 Final Rules, Federal Register , Vol. 54, No. 168, August 31, 1989, pp. 36112-36163, effective October 30, 1989, and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register , Volume 55, No. 32, February 15, 1991, pp. 6426-6505, effective March 18, 1991). Immediately prior to pairing, each female was weighed and subjected to a clinical examination. For breeding, 1 male with 1 fema le pairing was employed since other pairing

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### patterns (e.g., 1 male with 2 females) may ha ve resulted in an unacceptable number of plugpositive, nonpregnant females and/or sire eff ects. Individual females were placed in polycarbonate “shoebox” cages with stainle ss steel lids with singly-housed males. On the following morning and each morning thereafter, the females were examined for the presence of a vaginal or dropped copulation plug (Hafez, 1970). The day on which copulation plugs were found was designated as gd 0. Plug-positive females (dams) were individually housed until scheduled sacrifice on gd 17. Plug-negative females were retained in the same male's cage and checked for plugs on successive mornings until insemination occurred or the treatment groups were filled, whichever came first. RTI staff were present during the initial day of mating (September 17, 2004) to confirm expertise of HLS staff to detect vaginal copulation plugs and then set up the exposure schedule, based on the gd 0 dates. HLS staff continued to evaluate females for vaginal copulation plugs until both groups were filled and then completed the exposure schedule. When all treatment groups were filled, the remaining plug-negative and plug-positive females were sacr ificed by asphyxiation with CO 2 . The males were retained for the definitive developmental toxicity study. The fate of all an imals was fully documented in the study records.

Study Design 

### This preliminary study was conducted with 1 treatment group and 1 vehicle control group, each comprised of 10 plug-positive (presumed pregnant) female mice (Table B).

Table B. Number of Animals Assigned to Study Groups

Group No. No. Animals Exposed No. Days Exposed Exposure Period (gd) Target Exposure Concentration (mg/m 3 )

1 10 6 5 through 10 0 2 10 6 5 through 10 30,000



### The 30,000 mg/m 3 

### concentration was chosen to determ ine the tolerance of the dams and conceptuses to the highest exposure concentratio n to be possibly selected for the definitive toxicity study. The test substance was administered as a vapor in the breathing air of the animals. The test atmosphere was generated by an appropriate proc edure determined during prestudy trials and

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### described in the Inhalation Report (Appendix I). Th e prestudy trials were performed (at least two 6-hour periods) to evaluate the optimal set of conditions and equipment to generate a stable atmosphere at the target exposure levels and ma intain uniform conditions throughout the exposure

chambers. The whole-bo dy exposure chambers ea ch had a volume of ap proximately 1000 liters. The chambers were operated at a mini mum flow rate of 200 liters per minute. The fi nal airflow was set to provide at least 1 air change in 5 minutes (12 air changes/hour) and a T 99 

### equilibrium time of

at most 23 minutes. This chambe r size and airflow rate was consid ered adequate to maintain the oxygen level at least 19% and the animal loading fa ctor below 5%. At the end of each daily 6-hour exposure, all animals remained in the chamber for a minimum of the T 99 

### equilibrium tim e. During this time, the chamber wa s operated at approximately the same flow rate using clean air only. A nominal exposure concentra tion was calculated. The flow of air through the chamber was monitored using appropriate calibrated equipmen t. The test substance consumed during the exposure was divided by the total volume of air passing throug h the chamber (volumetric flow rate times total exposure time) to give the nominal concentration. During each 6-hour exposure, measurements of airborne concentrations were performed in the animals’ breathing zone at least 4 times using an appropriate sampling procedure and IR analytical procedure. Airborne test material co ncentrations were within +/- 10% of the target concentration. One sample per chamber during the trials period and the treatment period was analyzed by gas chromatography to characterize at least 10 major components (comprising at least 80% by weight of the te st substance) to show test substanc e stability and compar ison between the neat liquid test substance and the vaporized test atmosphe res. During the treatment period, particle size determinations were performed once per cham ber using a TSI Aerodyna mic Particle Sizer to confirm the absence of particulate test subs tance condensate in th e exposure atmosphere. Chamber temperature, humidity, airflow ra te, and static pressu re were monitored continuously and recorded every 30 minutes duri ng exposure. Chamber temperature and relative humidity were maintained, to the maximum exte nt possible, between 20 to 24°C and 40 to 60%, respectively. Chamber oxygen levels (maintained at least 19%) were measured pretest and at the beginning, middle, and end of the exposure period of the study. Air samples were taken in the vapor generation area pretest and at the beginning, middle, and e nd of the exposure period of the study. Light (maintained approximately 30 foot-candles at 1.0 meter above the floor) and noise levels (maintained below 85 decibels) in the expo sure room were measured pretest and at the

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### beginning, middle, and end of the exposure peri od of the study. The minimum frequency of chamber activity during the treatment period is summarized below:

Activity Frequency/Chamber Measured test substance concentration 4X/day Measured test substance characterization 1X Particle size 1X Temperature 13X/day Relative humidity 13X/day Airflow rate 13X/day Static pressure 13X/day Nominal test substance concentration (exc luding the air control chamber) 1X/day Rotation pattern of exposure cages 1X/day Loading/unloading verification 1X/day



### Plug-positive female mice (dams) were as signed to treatment groups by a stratified randomization method designed to provide unifor m mean body weights between the 2 exposure groups on gd 0. Females were exposed to G/MT BE or air 6 hours per day from gd 5 through 10. For each daily exposure, females were transferred to inhalation cages, and the cages were moved into the appropriate chambers for exposure. Following each daily exposure, females were transferred back to home caging for feed cons umption measurements overnight. Dams at 0 mg/m 3 

### were also exposed to air in the cham bers for 6 hours per day on gd 5 through 10.

Clinical observations of all animals were ma de once daily on gd 0 through 4 (prior to the exposure period), on gd 11 through 17 (after the exposure period, pr ior to necropsy), and twice daily, prior to and immediately after each dail y exposure, throughout the exposure period (gd 5 through 10). In addition, during each da ily exposure period, animals were observed at least once during each exposure. This was routinely performed near the middle of each exposure. Dams were weighed in the mornings (prior to exposures for those days that exposures occurred) on gd 0, 5, 6, 7, 8, 9, 10, 12, 14, 16, and 17. Maternal weight gains were calculated for gd 0-5 (pre-exposure period), gd 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-14, 14-16, 16-17, gd 5-10 (exposure period), gd 10-17 (postexposure pe riod), and gd 0-17 (gestational period). Maternal feed consumption was evaluated in the mornings from gd 0-5 (pre-exposure period), gd 5-6, 6-7, 7-8, 8- 9, 9-10, 10-12, 12-14, 14-16, 16-17, 5- 10 (exposure period), gd 10-17 (postexposure period), and gd 0-17 (gestation period).

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### On gd 17, approximately 1 to 11⁄2 days before expected parturition, all maternal animals were killed by CO 2 asphyxiation by RTI staff. The thorac ic and abdominal cavities and organs were examined, and pregnancy status was c onfirmed by uterine examination. Uteri that presented no visible implantation sites were stai ned with ammonium sulfide (10%) in order to visualize any implantation sites th at may have undergone very ear ly resorption (Salewski, 1964). At sacrifice, the body, liver, uter us, paired adrenal glands, and paired kidneys of each plugpositive female were weighed. Ovarian corpora lu tea were counted and uterine contents (i.e., number of implantation sites, early and late resorptions, dead fetuse s, live fetuses) recorded. Live fetuses were counted, weighed, sexed externally, and examined externally for gross malformations (including cleft palate) and variations by RTI staff. Each fetus was killed by intraperitoneal injection of s odium pentobarbital, dissected lo ngitudinally, and the thoracic and

abdominal viscera removed intact and retained in dividually in labeled vials in buffered neutral 10% formalin for possible subsequent visceral ex amination. The fetal carcasses were blanched, skinned, and retained in individually labeled scintillation vials in 70% ethanol for possible subsequent double staining (alizarin Red S and alcian blue) and skel etal evaluation. All maternal organs and carcasses were destroyed by incineration.

Statistics 

### The unit of comparison was the dam or th e litter, as appropriate. The single treatment group was compared by RTI staff to the concurrent c ontrol group using the Student’s t-Test (SAS® Releases 8.0 and 8.2; SAS® Institute 1999a,b,c,d,e; 2000; 2001). Litter derived percentage data were arcsine transformed and then analyzed using ANOVA (Analysis of Variance) (SAS® In stitute 1999a,b,c,d,e; 2000; 2001) weighted according to litter size. For frequency data, su ch as percent litters with resorptions, the Chi-square test was used.

Storage of Records



### All data documenting experimental detail s and study procedures and observations were

recorded and maintained as raw data. At th e completion of the study, all reports, raw data, preserved specimens, and retained samples will be maintained in RTI’s secure archives for a period of 1 year after submission of the signed final report. The Sponsor will be contacted in order to determine the final disposition of these materials.

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### Personnel 

### This study was conducted at HLS (Mr. G.M. Hoffman, Principal Investigator; Animal Research Facility Veterinarian, Dr. Teresa S. Kusz nir; Animal Research F acility Director, Mr. I. Vanterpool; Necropsy Laboratory Supervisor, Ms. G.E. Baxter; Inhalation Laboratory Supervisor, Mr. S. Cracknell; Associate Director of Formulation Chemistry Services, Ms. K. Saladdin; Quality Assurance, Ms. N.S. Iacono) u nder contract to Research Triangle Institute. Dr. R.W. Tyl of RTI served as Study Di rector. RTI Reproductive and Developmental Toxicology personnel included Ms. M.C. Marr (L aboratory Supervisor), Ms. C.B. Myers (Reproductive Toxicity Study Supervisor and Data Analyst), and Mr. W.P. Ross (Neurotoxicology Supervisor). RTI Quality Assurance personnel were Ms. D.A. Drissel (Manager), Ms. C.A. Ingalls, Ms. S.C. Wade, and Ms. M.M. Oh. Dr. Tyl and Ms. Marr went to HLS for the fi rst day of pairing and the first gd 0 to train HLS staff on detection of vaginal plugs/sperm and on distributing the plug/sperm positive females into the two groups by stratified ra ndomization. Ms. Marr, Mr. Ross, and Ms. Wade went to HLS to perform the term necropsy and fetal evaluations. Ms. Wade (the RTI QA study auditor) accompanied the staff to HLS for the necropsies. The final report was prepared by Dr. R.W. Tyl and Ms. M.C. Marr, with assistance from Ms. C.B. Myers for statistical analyses and gene ration of tables, and by Mr. T.W. Wiley for data entry. Ms. M.C. Marr was responsib le for all transfer of custody procedures for fetal viscera, fetal carcasses, data generated at HLS by HLS staff from HLS to RTI, and for archiving the study records.

RESULTS Test Chamber Analyses Table 1, Appendix I 

### Prestudy chamber distribution analyses show ed that the test substance was evenly distributed within the chamber. Prestudy a nd on-study chamber monitoring showed that the chamber oxygen levels were 20%. The analytically measured exposure levels of the airborne test subs tance were reasonably close to the targeted exposure level. The meas ured and nominal concentrations varied somewhat (less than 10%), but reas onably, from the expected 1:1 ratio for this type of vapor exposure.

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### Chamber environmental conditions averaged 21.5o C temperature and 46.6% relative humidity. Mean particle size distribution measurements fo r the exposures indicated that the atmospheres were essentially vapor only, as expected, since there was no s ubstantial difference between the

test substance chamber and the air contro l chamber for particle size distribution. Analysis of the major components in the neat test substance and the test atmospheres showed a reasonably close concordance between th e neat test substance and the vaporized test

substance. These data demonstrated that the test animals were exposed, as expected, to all of the major components of the test subs tance in their proper proportions . The data were consistent between the prestudy and on-study analyses, indica ting stability of the te st substance and the atmosphere generation techniques.

Maternal Findings Tables 2, 3, 4, Appendix II



### The test substance had no effect on matern al survival, body weights, body weight gains, or food consumption. No maternal animals die d, delivered early, were euthanized moribund, or were removed during the course of the study. Maternal body weights were statistically equivalent between the 2 groups throughout ge station; on gd 0, 5-10, 12, 14, 16, and 17 for inlife, and on gd 17 at sacrifice. Maternal body we ight change was similarly unaffected between the 2 groups throughout gestation, including intervals gd 0-5 (pre-e xposure period); 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-14, 14-16, 16-17, and 5-10 (exposure period); gd 10-17 (postexposure period); gd 0-17 (gestation); a nd corrected gestational body we ight change (gd 0-17 minus gravid interim weight) (Table 2). Maternal feed consumption in g/day and g/kg body weight/day was equivalent across both groups for all in tervals during gestation, although the mean consumption values at 30,000 mg/m 3 

### in g/day and g/kg/day were s lightly (but not statistically significantly) lower for gd 5 through gd 9 during the exposure period (Table 3). Some maternal toxicity (e.g., “lethargy (nar cosis)”) was noted in females in the 30,000 ppm chamber, particularly on the first days of exposure, as described below. There were no maternal clinical signs before or after the da ily, 6-hour exposure periods (gd 5 through 10; Table 4). Observations taken during each daily exposu re period indicated “most” (defined by HLS as 51-99% of the animals exhibiting a given observation) of the females at 30,000 mg/m 3 

### exhibited “lethargy (narcosis)” on the fi rst chamber exposure day (all an imals at gd 5), with the

12RTI-908; Project 0209189.000 Final Report



### approximate incidence dropping to “some” on cham ber exposure days 2 through 4 and 6 (defined by HLS as 21-50% of the animals exhibiting a given observation), and then “few” observed on chamber exposure days 7 and 8 (defined by HLS as < 20% of the animals exhibiting a given observation), and no animals on gd 9 or 10, as the exposure period progressed. These observations suggest that the initial responsiven ess attenuated over time.

The test substance had no effect on the absolute or relative weights of the gravid uterus and maternal liver, paired adrena l glands, and paired kidneys. Th ey were equivalent between the 2 groups (Table 2). In this small study, the test substance ha d no apparent effect on pregnancy or postimplantation loss. Of the 10 plug-positive females in each of the 2 groups (0 and 30,000 mg/m 3 

### ), 1 female in each group was nonpregnant, so the pregnancy rate was 90% in both groups. Two litters at 0 mg/m 3 

### and 1 litter at 30,000 mg/m 3 

### were fully resorbed, so the numbers (and percentage) of live litters were 7 of 9 (77.8%) at 0 mg/m 3 

### and 8 of 9 (88.9%) at 30,000 mg/m 3



### . Overall, the combined pregnancy rate was 90.0% (18/20), and the combined percentage of pregnant females with live litters was 83.3% (15/18) . The higher incidence of resorptions in the control group is obviously not an indica tion of test substance effects.

Uterine and Embryofetal Findings Tables 5, 6, 7, Appendix II 

### Of the 9 dams pregnant in each group, ther e were no differences between groups for the number of ovarian corpora lutea per dam (a measure of eggs ovulat ed), uterine implantation sites per litter (a measure of zygotes implanted), or percent preimplantation lo ss/litter (the difference between ovarian corpora lutea a nd uterine implant sites). In this small study, the test substance had no effect on the number or percentage of resorptions/litter, late fetal deaths per litter, nonli ve implants (resorptions plus late fetal deaths) per litter, or litters w ith adversely affected (nonlive plus ma lformed) implants. The number of resorptions/litter were nonsignificantly lower at 30,000 mg/m 3 

### , due to 2 females at 0 mg/m 3 

### and 1 female at 30,000 mg/m 3 

### with full litter resorption. RTI’s hi storical control range, based on 288 CD-1 mouse litters, for full litter resorption, is 0.0-20.0% per study, with the overall mean of 2.8%. In this study, the percentage of litters w ith resorptions was significantly lower at 30,000 mg/m 3 

### than at 0 mg/m 3 

### ; all 9 litters at 0 mg/m 3 

### had 1 or more resorptions, whereas only 4 of 9

13RTI-908; Project 0209189.000 Final Report



### litters at 30,000 mg/m 3 

### had 1 or more resorptions. The number and percentage of late fetal deaths per litter and litters with late fetal deat hs were statistically e quivalent between the 2 groups. The number and percenta ge of nonlive implants per litter were statistica lly equivalent but clearly lower at 30,000 mg/m 3 

### due to the lower number of re sorptions and fully resorbed litters at 30,000 mg/m 3 

### . The number (nonsignificant) and per centage (significant) of litters with 1 or more nonlive implants were lower at 30,000 mg/m 3 

### , respectively, due to fewer resorptions and fully resorbed litters. The number and pe rcentage of adversely affected (nonlive plus malformed) implants and litters with 1 or more adversely affect ed implants were nonsignificantly lower at 30,000 mg/m 3 

### . In this study, with small numbers of litters/group and only 2 groups, there is no apparent risk to embryofetal survival at 30,000 mg/m 3 

### . A larger study would be required to definitively confirm this finding (Table 5). In this study, the test substance had no eff ect on the number of live fetuses per litter, the percentage of male fetuses per litter, the number of male and female fetuses per litter, or fetal body weight/litter, with sexes combined or se parately. For the 7 live litters at 0 mg/m 3 

### and the 8 live litters at 30,000 mg/m 3 

### , the data were all equivalent between the 2 groups (Table 5). Also in this study, the test substance ha d no impact on external malformations or variations. There were clearly no differences in fetal findings be tween the 2 groups. There were 71 live fetuses examined in the 7 litters at 0 mg/m 3 

### and 94 fetuses in th e 8 litters at 30,000 mg/m 3 

### . There was 1 fetus (in 1 litter) in each group with an external malfor mation (cleft palate), with no fetuses in either group with an external variation (Tables 6 and 7).

DISCUSSION The objective of this study wa s to determine whether mouse dams and conceptuses could tolerate 30,000 mg/m 3 

### of G/MTBE on gd 6 through 10. The results of this study indicate both dams and conceptuses tolerated su ch exposures, and that 30,000 mg/m 3 

### can be used in a guideline study to maximize the likelihood of causi ng and detecting ventral wall closure deficits should they occur. Among the dams, there we re no deaths or moribund animals, no clinical signs before or after exposures and only “lethargy (narcosis) ” during the 30,000 mg/m 3 

### exposures, with an incidence high initially a nd dropping slowly over time. This lethargy was anticipated since narcosis was observed in CD-1 ® 

### female mice exposed to pure MTBE at 8000 ppm during the exposure periods (Bev an et al., 1997). “Lethargy” and “narcosis” are considered

14RTI-908; Project 0209189.000 Final Report



### interchangeable terms. Dorland’s Illustrated Medical Dictionary (27 th 

### Edition, 1988, W.B. Saunders Co., Philadelphia) defines lethargy as “abnormal drowsiness or st upor” and narcosis as “nonspecific and reversible depression of the fu nction of the central nervous system marked by stupor.” Among the conceptuses, embryofetal survival, growth, and development were not adversely affected. The only fetal external malformation observed in this study was cleft palate, observed in 1 fetus each at 0 and 30,000 mg/m 3 

### . Final palatal shelf fusion is completed on gd 14-15 in the mouse (Rugh, 1968), but methanol-indu ced cleft palate has an earlier peak sensitivity of gd 7-8 (Rogers and Mole, 1997). However in this study, cleft palate is likely a spontaneous

malformation since it was observed with equal frequency in the 0 and 30,000 mg/m 3 

### groups (1 fetus in each), and it is one of the most co mmon malformations observed in RTI’s historical

control database for CD-1 ® 

### mouse developmental toxicity stud ies. Whether (or not) continued exposure to G/MTBE vapor through gd 16 would resu lt in higher incidences of cleft palate (versus the control group) was clea rly not assessed in this study, but the presence of maternal toxicity at this concentration provides some support for the supposition (also supported in the

literature that maternal stress results in increase d adrenal corticosteroid production, which in turn causes fetal cleft palate; Carmich ael et al., 2007; Senda et al., 2005; Pradat et al., 2003). RTI

historical control data on 3641 control CD-1 mouse fetuses from 288 dams, from 16 governmental studies from 1997-2002, indicated cleft palate was present in 18 control fetuses from 11 control litters from 8 (50 %) of the studies, with an overal l litter inciden ce of 3.8% and fetal incidence of 0.50 (ra nge 0.0-2.51%) (Table 8). The last finding (or lack thereof) in this small study is the absence of any other fetal external malformations (for discussion on cleft pa late, see above). There was no evidence of any effect on ventral midline closure. Although this was a small study, there were 71 fetuses examined at 0 mg/m 3 

### and 94 fetuses examined at 30,000 mg/m 3 

### G/MTBE (a concentration higher than any of those employed in the 2009 EMBS I study). The results of this range-finding study, although not definitive, lend credence to a tentative interpretation that the fetal findings observed in the EMBSI study may not be relate d to test substance from exposure. The differences between the EMBSI (2009) study an d this study are the source of the CD-1 ® 

### mice (Charles River, Portage, MI, at EMBSI and Ch arles River, Raleigh, NC, for the present study), duration of exposure (EMBSI employed gd 5 th rough 17 with necropsy on gd 18, the present

15RTI-908; Project 0209189.000 Final Report



### study employed gd 5 through 10 with necropsy on gd 17), the number of dams/group (25 in the EMBSI study and 10 in the present study), and th e exposure concentrations (EBMSI employed 4 groups [0, 2000, 10,000, and 20,000 mg/m 3 

### ] and the present study had 2 groups [0 and 30,000 mg/m 3 

### ]). Both studies employed 6-hour daily ex posures. Since the fetal malformations of concern are both failures of ventral midline body wall fusion, which takes place on gd 7-9 in the mouse (Rugh, 1968), the different durations ar e not germane since both studies employed exposure periods that encompassed the period of time when ventral midline body wall fusion

occurs in the mouse.

CONCLUSION The results of this range-fi nding tolerance study indicate that pregnant CD-1 mice (and their conceptuses) tole rated inhalation exposure of G/MTBE to 30,000 mg/m 3 

### for 6 hours/day on gd 5 through 10. Therefore, the results of this study support a de sign for the subsequent robust study with 25 plug-positive females/group at 0, 2000, 10,000, and 20,000 mg/m 3 

### , with exposures on gd 5 through 16, and 40 plug-positive female s at 30,000 mg/m3, with exposures on gd 5 through 10 (with scheduled necr opsy of all groups on gd 17).

16RTI-908; Project 0209189.000 Final Report



### REFERENCES

Bevan, C., R.W. Tyl, T.L. Neeper-Bradley, L.C. Fisher, R.D. Panson, J.F. Douglas, and L.S. Andrews (1997). Developmental toxicity evaluati on of methyl tertiary- butyl either (MTBE) by inhalation in mice and rabbits. J. Appl. Toxicol. 17(1), 521-529.

Carmichael, S.L., G.M. Shaw, C. Ma, M.M. Werler, S.A. Rasmussen and E.J. Cammer (2007). Maternal corticosteroid us e and orofacial clefts. Am. J. Obstet. Gynecol. 197(6), 585, e1-7.

Daughtrey, W.C., D.M. Burnett, P. Podhasky, M. Henley, and R.D. Wh ite (2004). Inhalation Toxicity of Gasoline & Fuel Oxygena tes: Test Sample Preparation. The Toxicologist (Abstract 711).

ExxonMobil Biomedical Sciences, Inc. (EMBSI) (2009). Whole-Body Inhalation Developmental Toxicity Study in Mice With Gasoline With MTBE Vapor Condensate (MRD00-713). Study Director, Mr. G.W. Trimmer, EMBSI. Performe d for the American Petroleum Institute, Washington, DC. DRAFT final re port, Project No. 171334M, by EMBSI, Annandale, NJ.

Hafez, E.S.E. (Ed.) (1970). Reproduction and Breeding Techniques for Laboratory Animals

. Lea and Febiger, Philadelphia, PA.

NRC (1996). Guide for the Care and Use of Laboratory Animals . Institute of Laboratory Animal Resources, Commission on Life Scienc es, National Research Council. National Academy Press, National Institu tes of Health. Revised 1996.

Pradat, P., E. Robert-Gransia, G.L. DiTanna, A. Rosario, A. Lisi, and P. Mastroiacovo (2003). First trimester exposure to cortic osteroids and orofacial clefts. Birth Defects Res. A. Clin. Mol. Teratol. 67(12), 968-970.

Rogers, J.M., and M.L. Mole (1997). Critical periods of sensitivity to the developmental toxicity of inhaled metha nol in the CD-1 mouse. Teratology 55(6), 364-372.

Rugh, R. (1968). The Mouse: Its Reproduction and Development. Chapter 3, Normal Development of the Mouse , Chapter 4, Chronology of Development , and Chapter 5, Organogeny

. Burgess Publishing Company, Minneapolis, MN.

Salewski, E. (1964). Färbem ethode zum makroskopischen Nach weis von Implantationsstellen am Uterus der Ratte. Naunyn-Schmie debergs Arch. Exp. Pathol. Pharmakol. 247, 367.

SAS Institute Inc. (1999a). SAS® Language Reference: Concepts , Version 8, Cary, NC: SAS Institute Inc. 554 pp.

SAS Institute Inc. (1999b). SAS/STAT® Users’ Guide , Version 8, Cary, NC: SAS Institute Inc. 3884 pp.

SAS Institute Inc. (1999c). SAS® Language Reference: Dictionary , Version 8, Cary, NC: SAS Institute Inc. 1244 pp.

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### SAS Institute Inc. (1999d). SAS® Procedures Guide , Version 8, Cary, NC: SAS Institute Inc. 1643 pp.

SAS Institute Inc. (1999e). SAS® Companion for the Microsoft Windows Environment , Version 8, Cary, NC: SAS Institute Inc. 562 pp.

SAS Institute Inc. (2000). SAS/STAT® Software: Changes and Enhancements, Release 8.1

, Cary, NC: SAS Institute Inc. 554 pp.

SAS Institute Inc. (2001). SAS/STAT® Software: Changes and Enhancements, Release 8.2

, Cary, NC: SAS Institute Inc. 343 pp.

Senda, T., N. Natsume, J. Kuno, T. Toyoda, and K. Shimozato (2005). Rate of occurrence of dexamethasone-induced cleft palate affected by uterine environment in the mouse. Plast. Reconstr. Surg. 115(4), 1208-1210.

U.S. EPA (1989). U.S. Environmental Protec tion Agency; Toxic Substances Control Act (TSCA), Good Laboratory Practi ce Standards; Final Rule. Federal Register 54, 34034-34050, August 17, 1989 (40-CFR-792).

U.S. EPA (1994). U.S. Environmental Protec tion Agency. Good laborat ory practices (GLP) standards for inhalation exposur e health effects testing. - Federal Register 59 (122) , 33119- 33124, June 27, 1994 (40 CFR, part 79, subpart F, §79.60).

U.S. EPA (1996). U.S. Environmental Protec tion Agency; Prevention, Pesticides and Toxic Substances (OPPTS), Health Effects Test Guideline OPPTS 870.3600, Inhalation Developmental Toxicity Study (Public Draft, June 1996).

U.S. EPA (1998a). U.S. Environmental Protection Agency. Final Notification of Health Effects Testing Requirements for Baseline Gasolin e and Oxygenated Nonbaseline Gasolines and Approval of Alternative Emissions Generator. Federal Register 63 (236), 67877-67877, December 9, 1998 (40 CFR, part 79, subpart F).

U.S. EPA (1998b). U.S. Environmental Protectio n Agency. Letter from M.T. Oge, Director, Office of Mobil Sources, to C. J. Henry, Ph.D ., Director, Health and Environmental Sciences Department, American Petroleum Institute, November 2, 1998.

U.S. EPA (1998c). U.S. Environmental Protec tion Agency; Prevention, Pesticides and Toxic Substances (OPPTS), Health Effects Test Gu idelines, OPPTS 870.3700, Prenatal Developmental Toxicity Study (August, 1998).

18RTI-908; Project 0209189.000 Final Report



### LIST OF PROTOCOL DEVIATIONS

Study: Mi04-HLS1 Ma ster Protocol No.: RTI-908 HLS Study Code: 04-4262

HLS

1. Due to technician error, the gestational day 1 and 2 clinical observati ons were not recorded for Animal Nos. 1511-1513 or 2511-2513 on September 18, 2004. Animal Nos. 1511-1517 and 2511-2517 did not have clinical obser vations recorded on September 19, 2004.

2. On Exposure Day 2 (September 23, 2004), Sample No. 2008 (in Group 2) was more than 10% (35,000 mg/m 3 

### ) above the target (30,000 mg/m 3 

### ).

3. All pretest animals in Groups 1 and 2 were tattooed during the third week of quarantine/ acclimation instead of the second week.

4. The study protocol, Section 4.0 Statistics ( p. 12), inadvertently did not indicate how frequency data would be analyzed. The followi ng text was added to th e “Statistics” section of this final report and in a Memorandum to th e Study Record: “For frequency data, such as percent litters with resorptions, the Chi-square test was used.”

In the Study Director’s profe ssional opinion, these protocol devi ations did not affect the study design, performance, or interpretation and are presented for completeness.

19RTI-908; Project 0209189.000 Final Report

Table 1. Chamber Monitoring Results (page 1 of 1)

Target Chamber Concentrations: 0 mg/m 3 30,000 mg/m 3

Mean analytical chamber concentrations of G/MTBE (mg/m 3 ) a 0 d 30,688 ± 1,595

Percent of target concentration NA 102.29%

Particle Size Determinations: b

MMAD ( μ m) 1.317 3.667

GSD 2.303 2.315

TMC (mg/m 3 ) 4.41 x 10 -3 1.02 x 10 -2

Temperature ( ° C) c 21.0 ± 0.0 22.0 ± 0.0

Relative humidity (%) c 46.6 ± 1.1 46.6 ± 0.7

a Mean of 4 assays measured by infrared spectroscopy

b Measured once

c Measured 13 times

d LOQ = 433 mg/m 3 MMAD = mass median aerodynamic diameter GSD = geometric standard deviation TMC = total mass concentration (measure of aerosol concentration) NA = not applicable



### 20RTI-908; Project 0209189.000 Final Report

Table 2. Summary and Statistical Analysis of t he Maternal Body Weights, Weight Changes, Organ Weights and Relative Organ Weights (page 1 of 4)

G/MTBE (mg/m 3 , inhaled) 0 30,000 SUBJECTS (No. Dams): No. on Study 10 10 No. Removed 0 0 No. Dead or Euthanized 0 0 No. Nonpregnant 1 1 No. (%) Pregnant at Scheduled Sacrifice 9 (90.0) 9 (90.0) No. (%) with 100% Resorptions 2 (22.2) 1 (11.1)

Maternal Body Weight (gd 0) (g) a 26.6 26.9

+ 0.5 + 0.4 N=9 N=9

Maternal Body Weight (gd 5) (g) a 27.8 27.9

+ 0.7 + 0.3 N=9 N=9

Maternal Body Weight (gd 6) (g) a 28.3 28.1

+ 0.8 + 0.4 N=9 N=9

Maternal Body Weight (gd 7) (g) a 28.8 28.5

+ 0.7 + 0.4 N=9 N=9

Maternal Body Weight (gd 8) (g) a 29.4 29.0

+ 0.8 + 0.4 N=9 N=9

Maternal Body Weight (gd 9) (g) a 29.7 29.3

+ 0.9 + 0.5 N=9 N=9

Maternal Body Weight (gd 10) (g) a 30.4 30.1

+ 1.1 + 0.5 N=9 N=9

Maternal Body Weight (gd 12) (g) a 33.0 33.2

+ 1.5 + 0.9 N=9 N=9

Maternal Body Weight (gd 14) (g) a 36.2 37.5

+ 2.1 + 1.3 N=9 N=9



### 21RTI-908; Project 0209189.000 Final Report

Table 2. Summary and Statistical Analysis of t he Maternal Body Weights, Weight Changes, Organ Weights and Relative Organ Weights (page 2 of 4)

G/MTBE (mg/m 3 , inhaled) 0 30000

Maternal Body Weight (gd 16) (g) a 40.9 43.4

+ 2.9 + 2.1 N=9 N=9

Maternal Body Weight (gd 17) (g) a 43.0 46.1

+ 3.3 + 2.4 N=9 N=9

Maternal Body Weight (gd 17 at sacrifice) (g) a 42.11 45.42

+ 3.21 + 2.40 N=9 N=9

Maternal Body Weight Change (gd 0 to 5) (g) a 1.2 1.0

+ 0.5 + 0.2 N=9 N=9

Maternal Body Weight Change (gd 5 to 6) (g) a 0.5 0.1

+ 0.1 + 0.2 N=9 N=9

Maternal Body Weight Change (gd 6 to 7) (g) a 0.5 0.4

+ 0.1 + 0.1 N=9 N=9

Maternal Body Weight Change (gd 7 to 8) (g) a 0.5 0.5

+ 0.2 + 0.2 N=9 N=9

Maternal Body Weight Change (gd 8 to 9) (g) a 0.4 0.3

+ 0.2 + 0.1 N=9 N=9

Maternal Body Weight Change (gd 9 to 10) (g) a 0.7 0.8

+ 0.2 + 0.2 N=9 N=9

Maternal Body Weight Change (gd 10 to 12) (g) a 2.5 3.2

+ 0.5 + 0.5 N=9 N=9



### 22RTI-908; Project 0209189.000 Final Report

Table 2. Summary and Statistical Analysis of t he Maternal Body Weights, Weight Changes, Organ Weights and Relative Organ Weights (page 3 of 4)

G/MTBE (mg/m 3 , inhaled) 0 30000

Maternal Body Weight Change (gd 12 to 14) (g) a 3.2 4.3

+ 0.7 + 0.5 N=9 N=9

Maternal Body Weight Change (gd 14 to 16) (g) a 4.7 5.9

+ 0.8 + 0.8 N=9 N=9

Maternal Body Weight Change (gd 16 to 17) (g) a 2.0 2.7

+ 0.4 + 0.4 N=9 N=9

Maternal Body Weight Change (gd 5 to 10) (g) a 2.7 2.2

+ 0.5 + 0.4 N=9 N=9

Maternal Body Weight Change (gd 10 to 17) (g) a 12.5 16.0

+ 2.3 + 2.0 N=9 N=9

Maternal Body Weight Change (gestation) (g) a 15.5 18.5

+ 3.0 + 2.2 N=9 N=9

Maternal Body Weight Change (corrected) (g) a,b 3.58 2.95

+ 0.91 + 0.42 N=9 N=9

Gravid Uterine Weight (g) a 11.9289 15.5566

+ 2.3183 + 2.0903 N=9 N=9



### 23RTI-908; Project 0209189.000 Final Report

Table 2. Summary and Statistical Analysis of t he Maternal Body Weights, Weight Changes, Organ Weights and Relative Organ Weights (page 4 of 4)

G/MTBE (mg/m 3 , inhaled) 0 30000

Maternal Liver Weight (g) a 2.2892 2.3848

+ 0.1507 + 0.0963 N=9 N=9

Maternal Paired Adrenal Gland Weight (g) a 0.0116 0.0103

+ 0.0008 + 0.0011 N=9 N=9

Maternal Paired Kidney Weight (g) a 0.4221 0.4303

+ 0.0194 + 0.0130 N=9 N=9

Relative Maternal Liver Weight (% sacrifice weight) a 5.4895 5.2984

+ 0.1371 + 0.1390 N=9 N=9

Relative Maternal Paired Adrenal Gland Weight (% sacrifice weight) a 0.0283 0.0234

+ 0.0017 + 0.0028 N=9 N=9

Relative Maternal Paired Kidney Weight (% sacrifice weight) a 1.0364 0.9770

+ 0.0630 + 0.0786 N=9 N=9

a Includes all pregnant dams until terminal sacrifice on gd 17. Reported as the mean + S.E.M.;

gd=gestational day.

b Weight change during gestation (gd 17 sacrifice weight minus gd 0 weight) minus gravid uterine weight.



### 24RTI-908; Project 0209189.000 Final Report

Table 3. Summary and Statistical Analysis of t he Maternal Feed Consumption (page 1 of 3)

G/MTBE (mg/m 3 , inhaled) 0 30000 No. of Pregnant Dams 9 9

Maternal Feed Consumption (gd 0 to 5) (g/day) a 5.4 5.4

+ 0.3 + 0.3 N=7 b N=8 b

Maternal Feed Consumption (gd 5 to 6) (g/day) a 6.6 5.2

+ 1.1 + 0.2 N=9 N=8 b

Maternal Feed Consumption (gd 6 to 7) (g/day) a 6.1 5.5

+ 0.3 + 0.2 N=8 b N=9

Maternal Feed Consumption (gd 7 to 8) (g/day) a 6.2 5.5

+ 0.6 + 0.2 N=7 b N=9

Maternal Feed Consumption (gd 8 to 9) (g/day) a 6.5 5.7

+ 0.6 + 0.1 N=9 N=9

Maternal Feed Consumption (gd 9 to 10) (g/day) a 5.7 5.6

+ 0.3 + 0.2 N=8 b N=9

Maternal Feed Consumption (gd 10 to 12) (g/day) a 5.9 5.9

+ 0.4 + 0.2 N=9 N=9

Maternal Feed Consumption (gd 12 to 14) (g/day) a 6.6 6.9

+ 0.6 + 0.3 N=9 N=9

Maternal Feed Consumption (gd 14 to 16) (g/day) a 7.2 7.3

+ 0.7 + 0.3 N=8 c N=9

Maternal Feed Consumption (gd 16 to 17) (g/day) a 7.1 7.3

+ 0.5 + 0.4 N=9 N=9



### 25RTI-908; Project 0209189.000 Final Report

Table 3. Summary and Statistical Analysis of t he Maternal Feed Consumption (page 2 of 3)

G/MTBE (mg/m 3 , inhaled) 0 30000

Maternal Feed Consumption (gd 5 to 10) (g/day) a 5.8 5.4

+ 0.4 + 0.1 N=7 d N=8 d

Maternal Feed Consumption (gd 10 to 17) (g/day) a 6.6 6.8

+ 0.5 + 0.3 N=8 d N=9

Maternal Feed Consumption (gd 0 to 17) (g/day) a 5.5 5.8

+ 0.5 + 0.2 N=5 d N=7 d

Relative Maternal Feed Consumption (gd 0 to 5) (g/kg/day) a 198.3 196.3

+ 9.0 + 9.5 N=7 b N=8 b

Relative Maternal Feed Consumption (gd 5 to 6) (g/kg/day) a 234.6 184.4

+ 35.0 + 7.3 N=9 N=8 b

Relative Maternal Feed Consumption (gd 6 to 7) (g/kg/day) a 213.0 193.0

+ 8.1 + 5.4 N=8 b N=9

Relative Maternal Feed Consumption (gd 7 to 8) (g/kg/day) a 216.3 193.4

+ 19.8 + 7.2 N=7 b N=9

Relative Maternal Feed Consumption (gd 8 to 9) (g/kg/day) a 216.8 195.6

+ 15.1 + 5.3 N=9 N=9 Relative Maternal Feed Consumption (gd 9 to 10) (g/kg/day) a 190.5 189.6

+ 7.5 + 5.5 N=8 b N=9

Relative Maternal Feed Consumption (gd 10 to 12) (g/kg/day) a 186.7 185.3

+ 8.8 + 5.3 N=9 N=9



### 26RTI-908; Project 0209189.000 Final Report

Table 3. Summary and Statistical Analysis of t he Maternal Feed Consumption (page 3 of 3)

G/MTBE (mg/m 3 , inhaled) 0 30000

Relative Maternal Feed Consumption (gd 12 to 14) (g/kg/day) a 189.2 193.6

+ 13.1 + 5.6 N=9 N=9 Relative Maternal Feed Consumption (gd 14 to 16) (g/kg/day) a 189.5 180.2

+ 10.5 + 4.8 N=8 c N=9

Relative Maternal Feed Consumption (gd 16 to 17) (g/kg/day) a 171.5 164.0

+ 6.8 + 5.4 N=9 N=9 Relative Maternal Feed Consumption (gd 5 to 10) (g/kg/day) a 202.6 189.8

+ 10.3 + 3.6 N=7 d N=8 d

Relative Maternal Feed Consumption (gd 10 to 17) (g/kg/day) a 183.7 177.9

+ 8.6 + 4.1 N=8 d N=9

Relative Maternal Feed Consumption (gd 0 to 17) (g/kg/day) a 179.4 180.1

+ 4.9 + 4.7 N=5 d N=7 d

a Includes all pregnant dams until terminal sacrifice on gd 17. Reported as the mean + S.E.M.; gd =

gestational day.

b Decrease in N is due to one or more feeders spilling, and therefore an accurate feed weight could not be

obtained.

c Decrease in N is due to the feed being contaminated with feces and/or urine, and therefore the feed

weight was excluded.

d Decrease in N is due to interim feed consumption value(s) for one or more dams being missing, and

therefore the overall feed consumpt ion value could not be calculated.



### 27RTI-908; Project 0209189.000 Final Report

Table 4. Summary of the Maternal Clinical Observations (page 1 of 1)

G/MTBE (mg/m 3 , inhaled) Observation 0 30000 Prior to Exposures: a

Normal 10 10 During Exposures: b

Normal 10 0-8 e Lethargy (narcosis) 0 10-2 e Red vaginal discharge (Female No. 1518 on gd 9 postexposure) c 1 0

After Exposures: b

Normal 10 10 After Exposure Period d

Normal 10 10

a The daily clinical observations were recorded prio r to the exposure period on gd 0-5 and on the days of

treatment (gd 5 through 10) prior to the daily exposures.

b These clinical observations were recorded for gd 5 through 10 only.

c Female No. 1518 (at 0 mg/m 3 ) had all resorptions at scheduled necropsy on gd 17.

d These clinical observations were recorded for gd 11 through 17. e Numbers are presented as incidence on first ex posure day (initial number) and incidence on last

exposure day (end number).



### 28RTI-908; Project 0209189.000 Final Report

Table 5. Summary and Statistical Analysis of Ovaria n Corpora Lutea, Uterine Contents, Live Fetal Sex, and Live Fetal Body Weight (page 1 of 3)

G/MTBE (mg/m 3 , inhaled) 0 30000

A LL L ITTERS a 9 9

No. Corpora Lutea per Dam b 11.56 12.56

+ 1.76 + 1.37 N=9 N=9

No. Implantation Sites per Litter b 11.78 12.33

+ 0.57 + 0.44 N=9 N=9

Percent Preimplantation Loss per Litter b 9.84 8.16

+ 3.84 + 3.05 N=9 N=9

No. Resorptions per Litter b 3.67 1.67

+ 1.41 + 1.30 N=9 N=9

Percent Resorptions per Litter b 32.72 13.84

+ 12.92 + 10.85 N=9 N=9

No. Litters with Resorptions 9 4

% Litters with Resorptions 100.00 44.44 ££

No. Late Fetal Deaths per Litter b 0.22 0.22

+ 0.22 + 0.15 N=9 N=9

Percent Late Fetal Deaths per Litter b 1.71 2.04

+ 1.71 + 1.35 N=9 N=9

No. Litters with Late Fetal Deaths 1 2

% Litters with Late Fetal Deaths 11.11 22.22



### 29RTI-908; Project 0209189.000 Final Report

Table 5. Summary and Statistical Analysis of Ovaria n Corpora Lutea, Uterine Contents, Live Fetal Sex, and Live Fetal Body Weight (page 2 of 3)

G/MTBE (mg/m 3 , inhaled) 0 30000

No. Nonlive Implants per Litter b,c 3.89 1.89

+ 1.38 + 1.29 N=9 N=9

Percent Nonlive Implants per Litter b,c 34.43 15.88

+ 12.61 + 10.70 N=9 N=9

No. Litters with Nonlive Implants c 9 5

% Litters with Nonlive Implants c 100.00 55.56 £

No. Litters with 100% Nonlive Implants c 2 1

% Litters with 100% Nonlive Implants c 22.22 11.11

No. Adversely Affected Implants per Litter b,d 4.00 2.00

+ 1.35 + 1.27 N=9 N=9

Percent Adversely Affected Implants per Litter b,d 35.29 16.73

+ 12.41 + 10.57 N=9 N=9 No. Litters with Adversely Affected Implants d 9 6 % Litters with Adversely Affected Implants d 100.00 66.67

L IVE L ITTERS e : 7 8

No. Live Fetuses per Litter b 10.14 11.75

+ 0.74 + 0.65 N=7 N=8



### 30RTI-908; Project 0209189.000 Final Report

Table 5. Summary and Statistical Analysis of Ovaria n Corpora Lutea, Uterine Contents, Live Fetal Sex, and Live Fetal Body Weight (page 3 of 3)

G/MTBE (mg/m 3 , inhaled) 0 30000

Percent Male Fetuses per Litter b 52.34 44.10

+ 4.33 + 4.72 N=7 N=8

No. Male Fetuses per Litter b 5.29 5.13

+ 0.52 + 0.55 N=7 N=8

No. Female Fetuses per Litter b 4.86 6.63

+ 0.51 + 0.75 N=7 N=8

Average Fetal Body Weight (g) per Litter b 0.9697 0.9920

+ 0.0380 + 0.0440 N=7 N=8 Average Male Fetal Body Weight (g) per Litter b 0.9875 1.0177

+ 0.0412 + 0.0478 N=7 N=8 Average Female Fetal Body Weight (g) per Litter b 0.9424 0.9735

+ 0.0304 + 0.0392 N=7 N=8

a Includes all dams pregnant at terminal sacrifice on gd 20; litter size = no. implantation sites per dam.

b Reported as the mean + S.E.M.

c Nonlive = late fetal deaths plus resorptions.

d Adversely affected = nonlive plus malformed.

e Includes only dams with live fetuses; litter size = no. live fetuses per dam.

£ p<0.05; Chi-Square Test.

££ p<0.01; Chi-Square Test.



### 31RTI-908; Project 0209189.000 Final Report

Table 6. Summary and Statistical Analysis of Malformations and Variations (page 1 of 1)

G/MTBE (mg/m 3 , inhaled) 0 30000

No. Fetuses Examined a 71 94

No. Litters Examined b 7 8

No. Fetuses with External Malformations per Litter c, d 0.14 0.13

+ 0.14 + 0.13 N=7 N=8

Percent Fetuses with Exter nal Malformations per Litter c,d

1.19 0.96

+ 1.19 + 0.96 N=7 N=8

No. Fetuses with External Malformations d 1 1

% Fetuses with External Malformations d 1.40 1.06

No. Litters with External Malformations e 1 1

% Litters with External Malformations e 14.29 12.50

No. Fetuses with External Variations per Litter c,d 0.00 0.00

+ 0.00 + 0.00 N=7 N=8

Percent Fetuses with Exter nal Variations per Litter c,d 0.00 0.00

+ 0.00 + 0.00 N=7 N=8

No. Fetuses with External Variations d 0 0

% Fetuses with External Variations d 0.00 0.00

No. Litters with External Variations e 0 0

% Litters with External Variations e 0.00 0.00

a Only live fetuses were examined for malformations and variations. b Includes only litters with live fetuses.

c Reported as the mean + S.E.M. d Fetuses with one or more malformations or variations.

e Litters with one or more fetuses with malformations or variations.



### 32RTI-908; Project 0209189.000 Final Report

Table 7. Summary of Morphological Abnormalities in CD-1 Mouse Fetuses: Listing by Defect Type a (page 1 of 1)

G/MTBE (mg/m 3 , inhaled) 0 30000

EXTERNAL MALFORMATIONS

Total No. of Fetuses Examined for External Malformations b 71 94

No. of Fetuses with External Malformations c 1 1 % Fetuses with External Malformations 1.4% 1.1% Total No. of Litters Examin ed for External Malformations d 7 8

No. of Litters with External Malformations e 1 1 % Litters with External Malformations 14.3% 12.5%

Cleft Palate 1(1) 1(1)

EXTERNAL VARIATIONS

Total No. of Fetuses Examined for External Variations b 71 94

No. of Fetuses with External Variations c 0 0 % Fetuses with External Variations 0.0% 0.0% Total No. of Litters Examined for External Variations d 7 8

No. of Litters with External Variations e 0 0 % Litters with External Variations 0.0% 0.0%

a A single fetus may be represented more than once in listing individual defects. Data are presented as

the number of fetuses (number of litters).

b Only live fetuses were examined.

c Fetuses with one or more malformations/variations.

d Includes only litters with live fetuses.

e Litters with one or more malformed/variant fetuses.



### 33RTI-908; Project 0209189.000 Final Report



### 34 Table 8. External Malformations in Control CD-1® Mouse Litters From Studies Performed for Governmental Clients at RTI From 1997 to 2002 (page 1 of 1)

Control Group With Gastroschisis b Study Code Year Study Type a No. Dams No. Fetuses No. Fetuses No. Litters Incidence of External Malformations No. Fetuses (No. Litters)

A 2002 RF 10 138 0 0 1 (1) cleft palate

B 2002 RF 9 123 0 0 1 (1) exencephaly

C 1999 RF 15 184 0 0 0

D 1999 RF 15 200 0 0 0

E 2000 RF 14 172 0 0 0

F 1999 RF 15 187 0 0 0

G 2000 RF 12 165 0 0 0

H 1999 D 24 302 0 0 0

I 2000 D 24 326 0 0 2 (1) cleft palate

J 1999 D 23 291 0 0 0

K 1997 D 22 271 0 0 1 (1) cleft palate

L 2000 D 23 283 0 0 1 (1) exencephaly

M 2001 D 24 270 0 0 1 (1) cleft palate

N 2002 D 20 254 0 0 6 (4) cleft palate

O 2002 D 22 279 0 0 7 (3) cleft palate

P 2000 D 16 196 0 0 0

TOTAL 288 3641 0 0 18 (11) cleft palate 2 (2) exencephaly

a RF = range-finding study D = definitive study

b There were no fetuses with ectopia cordis

# Appendix**I:**

# Inhalation**Report**

# Huntingdon**Life Sciences,** **Inc. Study** **No. 04-4262** 

# Appendix**II:**

# Individual**Animal Data** **Tables Table** **of Contents** **A-1 Individual** **Maternal Body** **Weights and** **Organ Weights** **(g) 1** **A-2 Individual** **Maternal Feed** **Consumption (g/day)** **2 A-3** **Individual Embryo/Fetal** **Data 3** Appendix II

1Table A-1. Individual Maternal Body Weights and Organ Weights (g) (page 1 of 1)Gestational DayDoseaDam ID056789101214161717bGravidUterusWeightLiverWeightPairedAdrenalGlandsWeightPairedKidneyWeight0 1511 24.7 29.0 29.5 30.0 31.532.1 33.4 36.2 39.1 44.4 46.145.60 11.9625 2.6173 0.0150 0.47081512 27.7 28.3 28.6 29.0 30.0 30.1 30.8 34.338.9 44.8 47.5 46.26 15.7460 2.4840 0.0146 0.38921513 27.7 29.2 30.2 30.4 30.4 30.8 32.1 35.239.2 45.6 47.9 46.89 15.9233 2.4676 0.0118 0.49991514 25.6 26.2 27.3 27.2 27.7 28.1 28.2 30.735.1 40.7 42.8 41.94 12.5632 2.5244 0.0123 0.42411515 26.6 26.6 27.3 27.9 27.5 27.3 27.0 26.9 27.5 28.5 28.2 27.410.1131 1.6844 0.0089 0.38741516c1517 27.4 29.2 29.6 30.5 30.9 32.5 34.0 37.441.9 48.0 50.9 49.88 17.3224 2.4481 0.0126 0.47081518 24.6 23.8 23.7 24.5 25.1 24.8 24.7 24.7 23.9 24.4 24.7 24.340.1135 1.3532 0.0085 0.30471519 26.1 26.6 26.7 28.0 27.9 28.4 29.8 33.437.7 42.7 45.9 45.26 16.5531 2.3760 0.0101 0.41681520 29.0 31.0 31.6 31.9 33.2 33.4 33.8 37.842.4 49.3 52.6 51.39 17.0628 2.6478 0.0109 0.435030000 2511 25.3 26.8 26.0 26.2 26.9 27.4 28.7 33.0 38.3 45.1 48.5 48.10 18.8171 2.5123 0.0132 0.48652512 26.8 28.3 27.5 27.9 29.3 29.5 29.8 32.237.7 43.3 46.3 45.56 14.9403 2.3680 0.0116 0.37812513c2514 26.2 26.7 27.1 27.1 28.2 28.5 29.3 31.735.5 42.0 44.9 44.03 13.8178 2.3036 0.0110 0.43672515 26.5 27.3 27.7 28.4 28.5 28.9 29.3 32.736.8 43.2 46.1 45.58 17.4442 2.3534 0.0033 0.39822516 27.0 28.3 29.4 29.6 30.4 30.9 32.7 36.943.2 50.5 53.0 52.75 21.2817 2.5755 0.0116 0.47592517 29.5 29.3 29.9 30.1 30.3 31.2 31.7 35.539.7 48.4 51.3 50.28 19.8939 2.5127 0.0087 0.46272518 25.6 27.5 27.5 27.7 27.8 27.4 27.6 27.7 28.5 28.4 28.2 27.700.1236 1.6867 0.0099 0.43762519 27.4 28.5 28.7 29.3 30.1 30.4 31.4 35.639.4 46.3 50.0 49.09 18.2453 2.4512 0.0093 0.39422520 27.9 28.6 28.8 29.8 29.5 29.6 30.3 33.938.8 43.4 46.9 45.70 15.4457 2.6996 0.0145 0.4027aMg/m3of gasoline MTBE vapor condensate.bBody weight at sacrifice.cFemale was not pregnant.Appendix II

2Table A-2. Individual Maternal Feed Consumption (g/day) (page 1 of 1)Gestational DaysDoseaDam ID 0-5 5-6 6-7 7-8 8-9 9-1010-12 12-14 14-16 16-17 5-10 10-17 0-170 1511 6.0 7.5 7.2 .b8.8 5.8 6.9 6.6 9.9 8.7 .c7.9 .c1512 4.9 4.9 5.0 5.4 5.1 5.15.3 6.5 7.4 7.1 5.1 6.5 5.61513 .b15.0 .b.b7.9 .b5.9 8.4 7.9 7.9 .c7.4 .c1514 .b6.0 5.8 8.4 7.6 6.3 7.3 9.1 8.7 8.1 6.8 8.3 .c1515 4.7 4.7 5.3 4.7 4.4 4.84.5 4.6 5.5 5.8 4.8 5.0 4.81516d1517 5.8 5.4 6.3 8.1 6.7 6.9 6.1 6.3 .e8.0 6.7 .c.c1518 5.5 4.3 5.1 4.6 4.2 4.34.4 3.7 3.9 4.0 4.5 4.0 4.61519 4.5 5.6 6.1 4.9 5.8 6.25.8 6.3 6.4 6.4 5.7 6.2 5.61520 6.3 6.4 7.6 7.5 7.6 6.27.3 8.0 8.1 8.2 7.1 7.8 7.230000 2511 .b4.5 4.8 5.9 6.1 6.1 6.1 8.4 8.0 8.3 5.5 7.6 .c2512 5.2 4.2 5.0 5.7 5.4 4.95.3 6.3 6.5 6.7 5.0 6.1 5.52513d2514 4.1 5.3 4.6 5.2 5.7 5.15.6 6.1 6.6 7.0 5.2 6.2 5.32515 5.1 4.8 5.7 5.2 5.5 5.25.1 6.7 6.9 6.6 5.3 6.2 5.62516 6.5 .b6.6 6.5 5.3 6.5 6.7 7.5 8.1 8.4 .c7.5 .c2517 5.5 6.5 6.0 5.8 6.2 6.15.9 7.5 7.9 8.0 6.1 7.2 6.42518 5.9 5.3 5.0 5.8 5.8 5.35.8 5.3 5.9 4.7 5.4 5.5 5.62519 6.2 5.1 5.8 5.3 5.9 6.26.8 6.9 8.4 7.3 5.7 7.3 6.52520 5.0 5.5 5.7 4.5 5.3 5.35.7 7.2 7.4 9.0 5.3 7.1 5.9aMg/m3of gasoline MTBE vapor condensate.bFeed spilled; therefore an accurate feed weight could not be obtained.cInterim feed consumption value(s) are missing and thereforethe overall feed consumption value could not be calculated.dFemale was not pregnant.eFeed was contaminated; thereforethe feed weight was excluded.Appendix II

3 Table A-3. Individual Embryo/Fetal Data (page 1 of 5)

Implant Dam Posi- Fetus Defect f Dose a ID# NCL b # Type c tion d # Sex Wt. e Exam Type Description

0 1511 11 1 A L 1 M 1.2080 2 E L 3 A L 2 M 1.1416 4 A L 3 M 1.1652 5 E L 6 A L 4 F 1.1544 7 E L 8 A R 5 M 1.1947 9 A R 6 M 1.2229 10 A R 7 F 0.8638 1512 13 1 A L 1 F 1.0607 2 A L 2 F 0.8947 3 A L 3 F 0.9114 4 A L 4 M 0.6561 5 A L 5 M 0.9103 6 A L 6 M 0.8906 7 A L 7 F 0.9512 8 A R 8 M 0.9243 9 A R 9 M 1.0387 10 E R 11 A R 10 F 0.9565 12 A R 11 M 1.0578 1513 18 1 A L 1 F 0.8099 2 E L 3 A L 2 M 0.9897 4 A L 3 M 0.9108 5 A L 4 F 0.8661 6 A L 5 M 0.8298 7 A R 6 F 0.7425 8 F R 0.2490 9 A R 7 F 0.7755 10 A R 8 F 0.7934 11 A R 9 M 0.8145 12 A R 10 M 0.8377 13 A R 11 M 0.8503 14 A R 12 F 0.8192 1514 13 1 A L 1 F 0.8836 2 A L 2 M 0.9409 3 A L 3 F 0.7931 4 A L 4 M 0.8536 5 E L 6 A L 5 F 0.9164 7 A R 6 F 0.9430 8 A R 7 M 0.9992 9 A R 8 F 0.9298 1515 0 1 I L 2 I L 3 I L 4 I L 5 I R 6 I R 7 I R 8 I R 9 I R 10 I R 11 I R 12 I R 1516 . gAppendix II

4 Table A-3. Individual Embryo/Fetal Data (page 2 of 5)

Implant Dam Posi- Fetus Defect f Dose a ID# NCL b # Type c tion d # Sex Wt. e Exam Type Description

0 1517 13 1 A L 1 F 1.0828 2 A L 2 F 1.0061 3 A L 3 M 1.1616 4 A L 4 F 1.0813 5 A R 5 F 0.9930 6 A R 6 M 1.0533 External Malformation Cleft Palate 7 A R 7 M 0.9497 8 A R 8 M 1.1190 9 E R 10 A R 9 M 0.9991 11 A R 10 M 1.0107 12 A R 11 F 1.0039 13 A R 12 M 1.0375 1518 7 1 I L 2 I L 3 I L 4 I L 5 I L 6 I L 7 I L 8 I R 9 I R 10 I R 1519 13 1 A L 1 F 0.8887 2 D L 0.7886 3 A L 2 F 0.8797 4 A L 3 F 0.8849 5 E L 6 A L 4 F 0.9113 7 A L 5 M 0.8804 8 D L 0.7497 9 A R 6 M 0.9570 10 A R 7 F 0.9628 11 A R 8 M 0.9181 12 A R 9 F 0.9405 13 A R 10 M 0.9491 1520 16 1 A L 1 M 0.9749 2 A L 2 M 1.1073 3 E L 4 A L 3 M 1.0296 5 A L 4 M 1.0548 6 A R 5 M 1.1481 7 A R 6 F 0.9627 8 A R 7 F 1.0874 9 E R 10 A R 8 M 0.9004 11 A R 9 F 1.0246 12 A R 10 F 0.8967 13 A R 11 F 0.9971Appendix II

5 Table A-3. Individual Embryo/Fetal Data (page 3 of 5)

Implant Dam Posi- Fetus Defect f Dose a ID# NCL b # Type c tion d # Sex Wt. e Exam Type Description

30000 2511 16 1 A L 1 F 0.9097 2 A L 2 F 0.8902 3 A L 3 F 1.0038 4 A L 4 M 1.0807 5 A L 5 M 1.0045 6 A L 6 F 0.9964 7 A R 7 F 1.1017 8 A R 8 F 0.9516 9 A R 9 F 1.0171 10 A R 10 F 0.9881 11 A R 11 F 0.9327 12 A R 12 M 0.9888 13 A R 13 F 0.8993 2512 13 1 A L 1 M 0.8966 2 A L 2 F 0.9912 3 A R 3 F 0.9295 4 A R 4 M 0.9734 5 A R 5 F 0.9657 6 E R . 7 A R 6 M 0.9817 8 A R 7 F 0.9658 9 A R 8 M 0.8802 10 A R 9 M 1.0423 11 A R 10 F 0.9900 2513 . g 2514 11 1 A L 1 M 0.9541 2 A L 2 F 0.7654 3 A R 3 M 0.9092 4 D R . 0.5424 5 A R 4 F 0.8117 6 A R 5 F 0.8227 7 A R 6 F 0.9987 8 A R 7 F 0.9737 9 A R 8 M 0.9013 10 A R 9 F 0.8918 2515 17 1 A L 1 F 0.9424 2 A L 2 M 0.9124 3 A L 3 M 0.8148 External Malformation Cleft Palate 4 A L 4 F 0.8965 5 A L 5 F 0.8518 6 A L 6 F 0.8904 7 A L 7 F 0.9606 8 A R 8 F 0.9293 9 A R 9 M 0.9842 10 A R 10 M 0.9846 11 A R 11 M 0.9588 12 A R 12 F 0.9191 13 A R 13 F 0.9280Appendix II

6 Table A-3. Individual Embryo/Fetal Data (page 4 of 5)

Implant Dam Posi- Fetus Defect f Dose a ID# NCL b # Type c tion d # Sex Wt. e Exam Type Description

30000 2516 14 1 A L 1 M 1.3223 2 L L 3 A L 2 M 1.2995 4 A L 3 F 1.1934 5 A L 4 M 1.4107 6 A L 5 M 1.3818 7 A R 6 M 1.3251 8 A R 7 F 1.2660 9 A R 8 F 1.2325 10 A R 9 M 1.2527 11 A R 10 F 1.2141 12 A R 11 F 1.2527 13 A R 12 F 1.2403 14 A R 13 M 1.3281 2517 15 1 A L 1 F 0.9643 2 A L 2 F 1.0539 3 A L 3 F 0.9751 4 A L 4 M 1.1039 5 A L 5 M 1.0307 6 A L 6 M 1.0258 7 A L 7 M 1.0202 8 A R 8 F 0.9284 9 A R 9 F 0.9127 10 A R 10 F 0.8877 11 A R 11 F 0.9235 12 A R 12 F 0.9446 13 A R 13 M 1.0347 14 A R 14 F 0.9547 2518 3 1 I L 2 I L 3 I L 4 I L 5 I L 6 I L 7 I L 8 I L 9 I R 10 I R 11 I R 12 I R 2519 12 1 A L 1 F 0.9621 2 A L 2 F 0.9309 3 A L 3 M 1.0104 4 A L 4 M 1.0717 5 A L 5 F 0.9861 6 A R 6 M 0.9635 7 A R 7 M 1.0321 8 A R 8 M 0.9550 9 A R 9 F 0.9994 10 A R 10 F 1.0005 11 A R 11 M 1.0335 12 A R 12 M 0.9762Appendix II

7 Table A-3. Individual Embryo/Fetal Data (page 5 of 5)

Implant Dam Posi- Fetus Defect f Dose a ID# NCL b # Type c tion d # Sex Wt. e Exam Type Description

30000 2520 12 1 A L 1 F 0.8464 2 A L 2 M 0.8999 3 A L 3 F 0.9263 4 A L 4 M 0.9872 5 A L 5 F 1.0089 6 A R 6 M 0.9040 7 A R 7 M 0.8767 8 D R . 0.4609 9 A R 8 M 0.9586 10 A R 9 F 0.8164 11 M R . . 12 A R 10 M 0.9416

a Mg/m 3 of gasoline MTBE vapor condensate. b Number of corpora lutea.

c Implant type codes are as follows: A - Live Fetus; D - Dead Fetus; F - Full Resorption; L - Late Resorption; M -

Middle Resorption; E - Early Resorption and I - Implantation Site. d Position refers to uterine horn (R - right, L - left).

e Weight is in grams. f Absence of entries under "Exam," "Type," and "Description " for "Defect" indicates no external malformation or

variation observed for that fetus. g Female was not pregnant.