EPA/635/R-14/333
                                                                Preliminary Materials
                                                                    www.epa.gov/iris
 Preliminary Materials for the Integrated Risk Information System (IRIS)
            Toxicological Review of Diisobutyl Phthalate (DIBP)
                              (CASRN No. 84-69-5)
                                  September 2014
                                      NOTICE

This document is comprised of preliminary materials.  This information is distributed solely for
the purpose of pre-dissemination review under applicable information quality guidelines. It has
not been formally disseminated by EPA.  It does not represent and should not be construed to
represent any Agency determination or policy. It is being circulated for review of its technical
accuracy and science policy implications.
                      National Center for Environmental Assessment
                          Office of Research and Development
                         U.S. Environmental Protection Agency
                                  Washington, DC

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               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

                                      DISCLAIMER

       This document is comprised of preliminary materials for review purposes only. This
information is distributed solely for the purpose of pre-dissemination review under applicable
information quality guidelines. It has not been formally disseminated by EPA. It does not represent
and should not be construed to represent any Agency determination or policy. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
          This document is a draft for review purposes only and does not constitute Agency policy.
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              Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
CONTENTS
PREFACE	viii
1.  INTRODUCTION	1-1
   1.1. DIBP IN THE ENVIRONMENT	1-1
       1.1.1. Production and Use	1-1
       1.1.2. Environmental Fate	1-2
       1.1.3. Human Exposure Pathways	1-2
   1.2. SCOPE OF THE ASSESSMENT	1-3
2.  METHODS FOR IDENTIFYING AND SELECTING STUDIES	2-1
   2.1. DRAFT LITERATURE SEARCH AND SCREENING STRATEGY	2-1
   2.2. SELECTION OF CRITICAL STUDIES IN EARLY STAGES OF DRAFT DEVELOPMENT	2-14
       2.2.1. General Approach	2-14
       2.2.2. Exclusion of Studies	2-15
   2.3. STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE FUTURE EVALUATION AND
       SYNTHESIS OF THE CRITICAL EPIDEMIOLOGICAL STUDIES FOR DIBP	2-16
   2.4. STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE FUTURE EVALUATION AND
       SYNTHESIS OF THE CRITICAL EXPERIMENTAL STUDIES FOR DIBP	2-30
3.  PRELIMINARY EVIDENCE TABLES AND EXPOSURE-RESPONSE ARRAYS	3-1
   3.1. DATA EXTRACTION FOR EPIDEMIOLOGICAL AND ANIMAL STUDIES:  PREPARATION OF
       PRELIMINARY EVIDENCE TABLES	3-1
   3.2. EPIDEMIOLOGICAL STUDIES	3-2
       3.2.1. Sexual Differentiation Measures	3-2
       3.2.2. Male Reproductive Effects in Humans	3-4
       3.2.3. Male Pubertal  Development in Humans	3-7
       3.2.4. Female Pubertal Development in Humans	3-9
       3.2.5. Female Reproductive Effects in Humans	3-12
       3.2.6. Pregnancy Outcomes in Humans	3-15
       3.2.7. Immune Effects in Humans	3-18
       3.2.8. Neurodevelopmental Effects in Humans	3-25
       3.2.9. Thyroid Hormone Effects in Humans	3-30
       3.2.10.Obesity and Metabolic Effects in  Humans	3-31
       3.2.11. Male Reproductive Effects	3-62

          This document is a draft for review purposes only and does not constitute Agency policy.
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               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
       3.2.12. Female Reproductive Effects	3-73
       3.2.13. Liver Effects	3-79
       3.2.14. Kidney Effects	3-83
       3.2.15. Hematopoietic Effects	3-87
       3.2.16.Other Effects	3-90
    3.4. PRELIMINARY MECHANISTIC INFORMATION FOR DIBP	3-94
4.   REFERENCES	4-1
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                Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate


TABLES

Table 2-1. Database search strategy for DIBP	2-2
Table 2-2. Summary of additional search strategies for DIBP	2-3
Table 2-3. Inclusion criteria used to identify animal studies of health-related endpoints,
              supporting data, or secondary literature	2-8
Table 2-4. Summary of search terms: targeted epidemiology search	2-9
Table 2-5. Inclusion criteria used to identify epidemiology studies of health-related endpoints	2-11
Table 2-6. Summary of additional search strategies for epidemiology studies of phthalate
              exposure in relation to health-related endpoints	2-12
Table 2-7. Primary source epidemiological studies examining health effects of DIBP	2-12
Table 2-8. General and outcome-specific considerations for DIBP study evaluation	2-28
Table 2-9. Questions and relevant experimental information for the evaluation of experimental
              animal studies	2-31
Table 3-1. Evidence pertaining to DIBP and sexual differentiation effects in humans	3-2
Table 3-2. Evidence pertaining to DIBP and semen parameters or infertility in adult men or
              couples	3-4
Table 3-3. Evidence pertaining to DIBP and reproductive hormones in adult men	3-6
Table 3-4. Evidence pertaining to DIBP and the timing of male puberty or sex hormones in boys	3-7
Table 3-5. Evidence pertaining to DIBP and timing of female puberty or sex hormones in girls	3-9
Table 3-6. Evidence pertaining to DIBP and reproductive hormones in adult women	3-12
Table 3-7. Evidence pertaining to DIBP and gynecological conditions in humans	3-13
Table 3-8. Evidence pertaining to DIBP and pregnancy outcomes in humans	3-15
Table 3-9. Evidence pertaining to DIBP and allergy/immune effects in humans	3-18
Table 3-10. Evidence pertaining to DIBP and asthma/wheezing and hypersensitivity in humans	3-22
Table 3-11. Evidence pertaining to DIBP and neurodevelopmental effects in humans	3-25
Table 3-12. Evidence pertaining to DIBP and thyroid hormones in humans	3-30
Table 3-13. Evidence pertaining to DIBP and obesity in humans	3-31
Table 3-14. Evidence pertaining to DIBP and diabetes/insulin resistance in humans	3-36
Table 3-15. Evidence pertaining to DIBP and cardiovascular disease risk factors in humans	3-40
Table 3-16. Evidence pertaining to DIBP and cancer in humans	3-43
Table 3-17. Evidence pertaining to developmental effects in animals following oral exposure to
              DIBP	3-44
Table 3-18. Evidence pertaining to male reproductive effects in animals following oral exposure
              to DIBP	3-62
Table 3-19. Evidence pertaining to female reproductive effects in animals following oral
              exposure to DIBP	3-73
Table 3-20. Evidence pertaining to hepatic effects in animals following oral exposure to DIBP	3-79
Table 3-21. Evidence pertaining to renal effects in animals following oral  exposure to DIBP	3-83
Table 3-22. Evidence pertaining to hematopoietic effects in animals following oral exposure to
              DIBP	3-87
Table 3-23. Evidence pertaining to other toxicity effects in animals following oral exposure to
              DIBP	3-90
Table 3-24. Summary of mechanistic outcomes evaluated following DIBP or MIBP
              administration	3-95
           This document is a draft for review purposes only and does not constitute Agency policy.
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                Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate


FIGURES

Figure 1-1. Chemical structure of DIBP	1-1
Figure 2-1. Literature search approach for DIBP	2-7
Figure 3-1. Exposure-response array of effects on developmental growth and survival following
              developmental oral exposure to DIBP	3-59
Figure 3-2. Exposure-response array of effects on postnatal and adult body weight following
              developmental oral exposure to DIBP	3-60
Figure 3-3. Exposure-response array of effects on fetal morphological developmental following
              developmental oral exposure to DIBP	3-61
Figure 3-4. Exposure-response array of effects on male reproductive development following
              developmental oral exposure to DIBP	3-70
Figure 3-5. Exposure-response array of effects on fetal testosterone (T) following
              developmental oral exposure to DIBP	3-71
Figure 3-6. Exposure-response array of male reproductive effects following oral exposure to
              DIBP	3-72
Figure 3-7. Exposure-response array of female reproductive effects, maternal weight and
              toxicity, following oral exposure to DIBP	3-77
Figure 3-8. Exposure-response array of female reproductive effects, fertility and fetal survival,
              following oral exposure to DIBP	3-78
Figure 3-9. Exposure-response array of liver effects following oral exposure to DIBP	3-82
Figure 3-10.  Exposure-response array of kidney effects following oral exposure to DIBP	3-86
Figure 3-11.  Exposure-response array of hematopoeitic effects following oral exposure to DIBP	3-89
Figure 3-12.  Exposure-response array of effects on other toxicities following oral exposure to
              DIBP	3-93
Figure 3-13.  Summary of in vivo and in vitro mechanistic data for DIBP and MIBP by mechanistic
              category	3-96
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                Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
ABBREVIATIONS
AGO     anogenital distance                         IQR
aOR     adjusted odds ratio                         IRIS
BASC-PRS Behavior Assessment System for              Koc
         Children—Parent Rating Scales               LDL
BBP     butyl benzyl phthalate                       LH
BMI     body mass index                           LMW
BP       blood pressure                             LOD
BPA     bisphenolA                               LOQ
BRIEF    Behavior Rating Inventory of Executive        MBzP
         Function                                  MBP
BW      body weight                               MCPP
CASRN   Chemical Abstracts Service Registry           MDI
         Number                                  MEHP
CHAP    Chronic Hazard Advisory Panel               MEP
CI       confidence interval                         MHBP
CPSC     Consumer Product Safety Commission         MIBP
DBF     dibutyl phthalate                           MMP
DEP     di-ethyl phthalate                          MOA
DEHP    di(2-ethylhexyl)phthalate                    MOINP
DHEAS   dehydroepiandrosterone                    MRI
DIBP     diisobutyl phthalate                         NCEA
DINP     diisononyl phthalate
DnBP    dibutyl phthalate                           NHANES
DNA     deoxyribonucleic acid
DPP     dipentyl phthalate                          NHS
DXA     dual energy x-ray absorptiometry             NRC
EPA     Environmental Protection Agency             OR
FBG     fasting blood glucose                        ORD
FDA     Food and Drug Administration               PAH
FSH     follicle stimulating hormone                 PCO
GD       gestational day                             PCOS
HbAlc   glycosolated hemoglobin                    PDI
HCG     human chorionic gonadotropin               PND
HDL     high-density lipoprotein                     PPS
HERO    Health and Environmental Research           PVC
         Online                                   RBC
Hgb     hemoglobin                               SD
HOMA   homeostatic model assessment               SE
HOMA-IR homeostatic model assessment of             SHBG
         insulin resistance                          T3
HOME    Health Outcomes and Measures of the         T4
         Environment                              TSH
IgE       immunoglobulin E                          VO
ICC       intra-class correlation coefficient             VOC
IM-GSM  grey scale media of the intima media          WBC
         complex                                  WHO
IMT     intima media thickness
interquartile range
Integrated Risk Information System
partition coefficient
low-density lipoprotein
luteinizing hormone
low molecular weight
level of detection
level of quantification
mono-benzyl phthalate
monobutyl phthalate
mono-(3-carboxypropyl) phthalate
mental delay index
mono-(2-ethylhexyl) phthalate
monoethyl phthalate
mono-3-(3-carboxypropyl)phthalate
monoisobutyl phthalate
monomethyl phthalate
mode of action
oxo-(mono-oxoisononyl) phthalate
magnetic resonance imaging
National Center for Environmental
Assessment
National Health and Nutrition
Examination Survey
Nurses' Health Study
National Research Council
odds ratio
Office of Research and Development
polycyclic aromatic hydrocarbon
polycystic ovarian morphology
polycystic ovarian syndrome
psychomotor delay index
postnatal day
preputial separation
polyvinyl chloride
red blood cell
standard deviation
standard error
sex-hormone binding globulin
triiodothyronine
thyroxine
thyroid stimulating hormone
vaginal opening
volatile organic compound
white blood cell
World Health Organization
          This document is a draft for review purposes only and does not constitute Agency policy.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1
 2
PREFACE
 3          This draft document presents preliminary materials for an assessment of diisobutyl
 4    phthalate (DIBP) prepared by the U.S. Environmental Protection Agency's (EPA's) Integrated Risk
 5    Information System (IRIS) Program. These preliminary materials include a planning and scoping
 6    summary, information on the approaches used to identify pertinent literature, results of the
 7    literature search, approaches for selection of studies for hazard identification, presentation of
 8    critical studies in evidence tables and exposure-response arrays, and mechanistic information for
 9    DIBP. This material is being released for public review and comment prior to a public meeting,
10    providing an opportunity for the IRIS Program to engage  in early discussions with stakeholders and
11    the public on data that may be used to identify adverse health effects and characterize dose-
12    response relationships.
13          The planning and scoping summary includes information on the uses of DIBP, occurrence of
14    DIBP in the environment, and the rationale and scope for the development of the assessment  This
15    information is responsive to recommendations in the 2009 National Research Council (NRC) report
16    Science and Decisions: Advancing Risk Assessment [NRC. 2009] related to planning and scoping in
17    the risk assessment process.
18          The preliminary materials are also responsive to the 2011 NRC report Review of the
19    Environmental Protection Agency's Draft IRIS Assessment of Formaldehyde [NRC, 2011].  The IRIS
20    Program's implementation of the NRC recommendations  is following a phased approach that is
21    consistent with the NRC's "Roadmap for Revision" as described in Chapter 7 of the formaldehyde
22    review report The NRC stated that "the committee recognizes that the changes suggested would
23    involve a multi-year process and extensive effort by the staff of the National Center for
24    Environmental Assessment and input and review by the EPA Science Advisory Board and others."
25    Phase 1 of implementation has focused on a subset of the short-term recommendations, such as
26    editing and streamlining documents, increasing transparency and clarity, and using more tables,
27    figures, and appendices to present information and data in assessments. Phase 1 also focused on
28    assessments near the end of the development process and close to final posting. Phase  2 of
29    implementation is focused on assessments that are in the beginning stages of assessment
30    development The IRIS DIBP assessment is in Phase 2 and represents a significant advancement in
31    implementing the NRC recommendations. In the development of this assessment, many of the
32    recommendations are being implemented in full, while others are being implemented in part
33    Achieving full and robust implementation of certain recommendations will be an evolving process
34    with input and feedback from the public, stakeholders,  and independent external peer review.
35    Phase 3 of implementation will incorporate the longer-term recommendations  made by the NRC,
36    including the development of a standardized approach  to describe the strength of evidence for
37    noncancer  effects.
                This document is a draft for review purposes only and does not constitute Agency policy.
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1           In May 2014, the NRC released their report reviewing the IRIS assessment development
 2    process. As part of this review, the NRC reviewed current methods for evidence-based reviews and
 3    made several recommendations with respect to integrating scientific evidence for chemical hazard
 4    and dose-response assessments.  In their report, the NRC states that EPA should continue to
 5    improve its evidence-integration process incrementally and enhance the transparency of its
 6    process. The committee did not offer a preference but suggests that EPA consider which approach
 7    best fits its plans for the IRIS process. The NRC recommendations will inform the IRIS Program's
 8    efforts in this area going forward. This effort is included in Phase 3 of EPA's implementation plan.
 9           The literature search strategy, which describes the processes for identifying scientific
10    literature, screening studies for consideration, and identifying primary sources of health effects
11    data, is responsive to NRC recommendations regarding the development of a systematic and
12    transparent approach for identifying the primary literature for analysis. The preliminary materials
13    also describe EPA's approach for the selection of critical studies to  be included in the evidence
14    tables, as well as the approach for evaluating methodological features of studies that will be
15    considered in the overall evaluation and synthesis of evidence for each health effect. The
16    development of these materials is in response  to the NRC recommendation to thoroughly evaluate
17    critical studies with standardized approaches that are formulated and based on the type of research
18    (e.g., observational epidemiology or animal bioassays). In addition, NRC recommendations for
19    standardized presentation of key study data are addressed by the development of the preliminary
20    evidence tables and preliminary exposure-response arrays for primary health effect information.
21           EPA welcomes all comments on the preliminary materials in this document, including the
22    following:

23       •   the clarity and transparency of the materials;

24       •   the approach for identifying pertinent  studies;

25       •   the selection of critical studies  for data extraction to preliminary evidence tables and
26           exposure-response arrays;

27       •   any methodological considerations that could affect the interpretation of or confidence in
28           study results; and

29       •   any additional studies published or nearing publication that may provide data for the
30           evaluation of human health hazard or dose-response relationships.

31           The preliminary evidence tables and exposure-response arrays should be regarded solely as
32    representing the data on each endpoint that have been identified as a result of the draft literature
33    search strategy. They do not reflect any conclusions as to hazard identification or dose-response
34    assessment.
35           After obtaining public input and conducting additional study evaluation and data
36    integration, EPA will revise these materials to  support the hazard identification and dose-response
37    assessment in a draft Toxicological Review that will be made available for public comment

                This document is a draft for review purposes only and does not constitute Agency policy.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1
 2
1.    INTRODUCTION
 3          This introduction contains a planning and scoping summary for the Integrated Risk
 4   Information System (IRIS) assessment of diisobutyl phthalate (DIBP). The planning and scoping
 5   summary includes information on the properties, sources, and uses of DIBP, occurrence and fate of
 6   DIBP in the environment, potential for human exposure, and the rationale for the development of
 7   this assessment

 8   1.1.  DIBP IN THE ENVIRONMENT

 9   1.1.1.  Production and Use
10          DIBP (Figure 1-1) is used as a plasticizer (HSDB. 2013) in a wide range of materials
11   including polyvinyl chloride (PVC) formulations; paints; lacquers; varnish; paper, pulp and board
12   industry; as a softener; in viscosity adjustment; nail polish; cosmetics; lubricants; carpets; clothing
13   treatments; rubber dentistry settings; as a fuel stabilizer; as a concrete additive; explosive
14   materials; and printing inks. DIBP has also been classified by the Food and Drug Administration
15   (FDA) as an indirect food additive through its use as a component of adhesives. Because DIBP has
16   similar properties to di-n-butyl phthalate (DBF), it can be used as a substitute for DBF (HSDB.
17   2013). Approximately 500,000 pounds were manufactured in the United  States in 2012
18   (http://www.epa.gov/oppt/cdr/index.html). In July 2014, the Consumer  Product Safety
19   Commission's (CPSC) Chronic Hazard Advisory Panel (CHAP) recommended that DIBP be
20   permanently banned from use in children's toys and child care articles at levels greater than 0.1%
21   fCHAP. 20141
22
                                             H;C
23
24          Figure 1-1. Chemical structure of DIBP (HSDB. 2013).
               This document is a draft for review purposes only and does not constitute Agency policy.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    1.1.2. Environmental Fate
 2          If released to air, DIBP will exist in both the vapor and particulate phases in the atmosphere.
 3    Vapor-phase DIBP will be photolytically degraded with a half-life of about 1.2 days, and particulate-
 4    phase DIBP will be removed from the atmosphere by wet or dry deposition [HSDB. 2013). In soil,
 5    DIBP is expected to have low mobility due to a moderately high organic carbon partition coefficient
 6    (Koc). Biodegradation in aerobic soil and water is expected to occur over days or weeks. Anaerobic
 7    biodegradation rates are expected to be slower. Volatilization from moist soil or water is expected
 8    to be an important fate process for DIBP, but volatilization from dry soil is not expected.  If released
 9    into water, DIBP is expected to adsorb to sediments and solids, and volatilization from water
10    surfaces is expected to be an important process. An estimated bioconcentration factor of 240
11    suggests that there is a potential for the chemical to concentrate in aquatic organisms, but
12    metabolism in the organisms can reduce accumulation [HSDB. 2013). As noted by Wormuth et al.
13    [2006]. the majority of phthalates that are found in the environment come from their slow releases
14    from plastics and other phthalate-containing articles.  Certain waste streams, sludges, and
15    industrially contaminated sites, however, may contain higher levels of phthalates than other sites.

16    1.1.3. Human Exposure Pathways
17          The routes by which humans are exposed to phthalates and the  magnitude of individual
18    phthalate exposures have changed over time as the quantities and uses  of the various phthalates
19    have changed. Human exposure to phthalates occurs mainly in occupational or household settings
20    because they are used and released from products in the home environment.  Environmental
21    concentrations of phthalates are typically the highest in house dust, and they may be present in
22    food due to the use of phthalates in packaging and food preparation materials. For most phthalates,
23    food ingestion is the dominant pathway of exposure, with dust exposures (ingestion and dermal
24    contact) and inhalation also being important in some circumstances. Infant and toddler exposures
25    occur due to teething and playing with plastic toys that contain phthalates [Wormuth etal.. 2006).
26          The presence of parent phthalates or their metabolites in a body matrix, such as blood or
27    urine, provides evidence of exposure to that chemical.  The predominant metabolite of DIBP in
28    humans is monoisobutyl phthalate (MIBP). Zotaetal.  [2014] evaluated the prevalence and
29    temporal trends of MIBP in urine samples collected as  part of the National Health and Nutrition
30    Examination Survey [NHANES] conducted between 2001 and 2010. MIBP was found in 72% of the
31    samples in the 2001-2002 cycle and 96% of the samples in the 2009-2010 cycle, and increased in
32    concentration over time, starting at about 2.4 ng/mL in the 2001-2002  cycle, and rising to about
33    7.8 ng/mL in the 2009-2010 cycle.
34          Intake exposures can be estimated on a pathway-basis by combining exposure media
35    concentrations and contact rates. Using this approach, Clark etal. [2011] estimated median intakes
36    of DIBP for various lifestages as defined by the authors: between 0.75 and 1.0 [ig/kg-day for teens
37    (12-19 years of age] and adults (20-70 years of age], based on ingestion of food, drinking water,
38    dust/soil, and inhalation of air; and between 1.3 and 2.6 [ig/kg-day for infants (0-0.5 years of age],
39    toddlers (ages 0.5-4 years of age], and children (5-11  years of age]. The exposure was found to be
                This document is a draft for review purposes only and does not constitute Agency policy.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    dominated by food, with inhalation of indoor air also important. The intakes determined by Clark
 2    etal. [2011] were higher than those found by Wormuth etal. [2006], who determined intakes for
 3    these age ranges at about <0.5 [ig/kg-day.  Clark etal. [2011] attributed this difference to use of
 4    higher food concentrations in the estimates.
 5          Wittassek et al. [2011] reported median intakes of DIBP in the range of 0.1-1.7 |ig/kg-day
 6    based on a literature survey of urinary biomonitoring data and intake estimates provided therein.
 7    Their review included a single study in the United States of a cohort of pregnant woman that found
 8    median intakes at 0.1 [ig/kg-day. Three other studies from Germany had median intakes ranging
 9    from 1.1 to 1.7 [ig/kg-day. Qianetal. [2014] used NHANES 2007-2008 and found a median intake
10    of 0.2 [ig/kg-day and a 95th percentile intake of 0.9 [ig/kg-day. Christensen et al. [2014] combined
11    the data from NHANES 2005-2008 and found similar results to Qianetal. [2014], with a median
12    over that time span of 0.2 [ig/kg-day and a 95th percentile intake of 0.8 [ig/kg-day.

13    1.2.   SCOPE OF THE ASSESSMENT
14          The National Research Council [NRC] has recommended that, "[Cumulative risk assessment
15    based on common adverse outcomes is a feasible and physiologically relevant approach to the
16    evaluation of the multiplicity of human exposures and directly reflects EPA's mission to protect
17    human health" [NRC. 2008. pll]. They envisioned facilitating the process by "defining the groups
18    of agents that should be included for a given outcome" [NRC. 2008. p!2]. In humans, the NRC cited
19    results from NHANES that demonstrate exposure to multiple phthalates in most people [NRC. 2008.
20    p23-25]. A recent review of human exposure to eight phthalates estimated that indoor air
21    contributed to approximately 25% of DIBP exposure in children [CHAP, 2014, Appendix El, p35].
22    The unique exposure scenarios and potential sensitivities of children contribute to  the need for an
23    assessment of phthalate toxicity. This IRIS assessment will help to inform EPA programs and
24    regions of the potentially unique vulnerabilities of children to DIBP exposure and enable future
25    cumulative risk assessments that assess effects on human health outcomes that might be associated
26    with DIBP and other phthalates. There is currently no IRIS assessment of DIBP.
27
               This document is a draft for review purposes only and does not constitute Agency policy.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1

 2    2.    METHODS FOR IDENTIFYING AND  SELECTING

 3    STUDIES	


 4          The NRCf20111 recommended that the U.S. Environmental Protection Agency (EPA)
 5    develop a detailed search strategy utilizing a graphical display documenting how initial search
 6    findings are narrowed to the final studies that are selected for further evaluation on the basis of
 7    inclusion and exclusion criteria.  Following these recommendations, a literature search and
 8    screening strategy was applied to identify literature related to characterizing the health effects of
 9    diisobutyl phthalate (DIBP). This strategy consisted of a search of online scientific databases and
10    other sources, casting a wide net in order to identify all potentially pertinent studies.  In subsequent
11    steps, references were screened to exclude papers not pertinent to an assessment of the health
12    effects of DIBP, and remaining references were sorted into categories for further evaluation.
13    Section 2.1 describes the literature search and screening strategy in detail. The NRG [2011] further
14    recommended that after studies are identified for review by utilizing a transparent search strategy,
15    the next step is to summarize the details and findings of the most pertinent studies in the evidence
16    tables. The NRC suggested that such tables should provide a link to the references, and include
17    details of the study population, methods, and key findings. This approach provides for a systematic
18    and concise presentation  of the evidence. The NRC also  recommended that the methods and
19    findings should then be evaluated with a standardized approach.  The approach that was outlined
20    identified standard issues for the evaluation of epidemiological and experimental animal studies.
21    Section 2.2 describes the approach taken for DIBP for selecting studies to be included in the
22    preliminary evidence tables and exposure-response arrays. Section 3 presents the selected studies
23    in preliminary evidence tables and exposure-response arrays, arranged by health effect

24    2.1.   DRAFT LITERATURE SEARCH AND SCREENING STRATEGY
25          The literature search for DIBP was conducted in four online scientific databases (PubMed,
26    Web of Science, Toxline, and Toxic Substances Control Act Test Submissions (TSCATS2)) in
27    February of 2013; the search was repeated in March of 2014. This document is complete through
28    March 2014. Additional updates will be performed at regular (e.g., 6-month) intervals. The
29    detailed search approach, including the search strings and number of citations identified per
30    database, is presented in Table 2-1.  The search strings and search terms described for DIBP
31    captured studies using the parent compound  and metabolites (i.e., the active metabolite,
32    monoisobutyl phthalate [MIBP]). This search of online databases identified 504 citations (after
33    electronically eliminating duplicates). The computerized database searches were also
34    supplemented by a manual search of citations from other regulatory documents (Table 2-2);
               This document is a draft for review purposes only and does not constitute Agency policy.
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                      Preliminary Materials for the IRIS Toxicological Review of Diisobutyl Phthalate
1
2
3
343citations were obtained using these additional search strategies.  In total, 809 citations were
identified using online scientific databases and additional search strategies.


        Table 2-1.  Database search strategy for DIBP
         Database
       (search date)
                                                      Keywords3
      PubMed
      03/2014
      02/2013
                  dibp OR (mibp AND phthalate) OR "diisobutylphthalate" OR "di-isobutyl phthalate" OR "84-69-
                  5" OR "diisobutyl phthalate" OR "di(i-butyl)phthalate" OR "di-iso-butyl phthalate" OR "isobutyl
                  phthalate" OR "phthalic acid diisobutyl ester" OR ("diisobutyl ester" AND phthalate) OR "1,2-
                  benzenedicarboxylic acid bis(2-methylpropyl) ester" OR "1,2-benzenedicarboxylic acid 1,2-
                  bis(2-methylpropyl) ester" OR "monoisobutyl phthalate"  OR "mono(i-butyl)phthalate" OR
                  "mono-iso-butyl phthalate" OR "phthalic acid monoisobutyl ester" OR "1,2-
                  benzenedicarboxylic acid, mono(2-methylpropyl) ester" OR "2-[(2-
                  methylpropoxy)carbonyl]benzoic acid" OR "1,2-benzenedicarboxylic acid, mono(2-
                  methylpropyl) ester (9CI)" OR "isobutyl hydrogen phthalate" OR "1,2-benzenedicarboxylic acid
                  l-(2-methylpropyl) ester"
      Web of Science
      03/2014
      02/2013
                 TS=dibp OR (TS=mibp AND TS=phthalate) OR TS="diisobutylphthalate" OR TS="di-isobutyl
                 phthalate" ORTS="84-69-5" ORTS="diisobutyl phthalate" ORTS="di(i-butyl)phthalate" OR
                 TS="di-iso-butyl phthalate" ORTS="isobutyl phthalate" ORTS="phthalicacid diisobutyl ester"
                 OR (TS="diisobutyl ester" AND TS=phthalate) OR TS="l,2-benzenedicarboxylic acid bis(2-
                 methylpropyl) ester" ORTS="l,2-benzenedicarboxylicacid l,2-bis(2-methylpropyl) ester" OR
                 TS="monoisobutyl phthalate" ORTS="mono(i-butyl)phthalate" ORTS="mono-iso-butyl
                 phthalate" ORTS="phthalicacid monoisobutyl ester" ORTS="l,2-benzenedicarboxylicacid,
                 mono(2-methylpropyl) ester" OR TS="2-[(2-methylpropoxy)carbonyl]benzoic acid" OR
                 TS="l,2-benzenedicarboxylic acid, mono(2-methylpropyl) ester (9CI)" OR TS="isobutyl
                 hydrogen phthalate" ORTS="l,2-benzenedicarboxylicacid l-(2-methylpropyl) ester"
      Toxline
      03/2014
      02/2013
                 Split into 4 separate search strings:

                 @TERM+@rn+84-69-5

                 @AND+mibp+phthalate

                 @AND+"diisobutyl ester"+phthalate

                 @OR+(dibp+"diisobutylphthalate"+"di-isobutyl+phthalate"+"diisobutyl+phthalate"+"di(i-
                 butyl)phthalate"+"di-iso-
                 butyl+phthalate"+"isobutyl+phthalate"+"phthalic+acid+diisobutyl+ester"+"l,2-
                 benzenedicarboxylic+acid+bis(2-methylpropyl)+ester"+"l,2-benzenedicarboxylic+acid+l,2-
                 bis(2-methylpropyl)+ester"+"monoisobutyl+phthalate"+"mono(i-butyl)phthalate"+"mono-iso-
                 butyl+phthalate"+"phthalic+acid+monoisobutyl+ester"+"l,2-
                 benzenedicarboxylic+acid,+mono(2-methylpropyl)+ester"+"2-[(2-
                 methylpropoxy)carbonyl]benzoic+acid"+"l,2-benzenedicarboxylic+acid,+mono(2-
                 methylpropyl)+ester+(9CI)"+"isobutyl+hydrogen+phthalate"+"l,2-
                 benzenedicarboxylic+acid+l-(2-methylpropyl)+ester")
      TSCATS2
      03/2014
                  (2000-) 84-69-5
5
6
7
aThe search strings and search terms described above captured studies using the parent compound and the
 metabolite MIBP.
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                 Preliminary Materials for the IRIS Toxicological Review of Diisobutyl Phthalate

       Table 2-2. Summary of additional search strategies for DIBP
   System used
       Selected key reference(s) or sources
  Date
    Additional
    references
     identified
Manual search of
citations from
regulatory
documents
CPSC (2010). Toxicity Review for Diisobutyl phthalate
(DIBP). Bethesda, MD: Consumer Product Safety
Commission.
3/2014
9 citations added
Web of Science,
forward search
Hannas et al. (2011). Dose-response assessment of fetal
testosterone production and gene expression levels in
rat testes following in utero exposure to diethylhexyl
phthalate, diisobutyl phthalate, diisoheptyl phthalate,
and diisononyl phthalate. Toxicol Sci. 123(1):206-16.

Saillenfait et al. (2008). Diisobutyl phthalate impairs the
androgen-dependent reproductive development of the
male rat. Reprod Toxicol. 26(2):107-15.

Rayet al. (2012). Ovarian development in Wistar rat
treated prenatally with single dose diisobutyl phthalate.
Bratisl Lek Listy. 113(10):577-82.

Kleinsasser et al. (2001b).  Genotoxicity of di-butyl-
phthalate and di-iso-butyl-phthalate in human
lymphocytes and mucosal cells. Teratog Carcinog
Mutagen. 21(3):189-96.
3/2014
2 citations added
                                                                         3/2014
                                                                         3/2014
                                                                         3/2014
         1 citation added
         0 citations added
         1 citation added
Web of Science,
backward search
Hannas et al. (2011). Dose-response assessment of fetal
testosterone production and gene expression levels in
rat testes following in utero exposure to diethylhexyl
phthalate, diisobutyl phthalate, diisoheptyl phthalate,
and diisononyl phthalate. Toxicol Sci. 123(1):206-16.

Saillenfait et al. (2008). Diisobutyl phthalate impairs the
androgen-dependent reproductive development of the
male rat. Reprod Toxicol. 26(2):107-15.

Rayet al. (2012). Ovarian development in Wistar rat
treated prenatally with single dose diisobutyl phthalate.
Bratisl Lek Listy. 113(10):577-82.

Kleinsasser et al. (2001b).  Genotoxicity of di-butyl-
phthalate and di-iso-butyl-phthalate in human
lymphocytes and mucosal cells. Teratog Carcinog
Mutagen. 21(3):189-96.
3/2014
1 citation added
                                                                         3/2014
                                                                         3/2014
                                                                         3/2014
         1 citation added
         4 citations added
         2 citations added
Snowball search
DIBP references in previous assessment or previously
added to the HERO project page
4/2014
45 citations added
Background Check
Searched a combination of CASRNs and synonyms on the
following databases:

ACGIH (http://www.acgih.org/home.htm)

ATSDR (http://www.atsdr.cdc.gov/substances/index.asp)

CalEPA Office of Environmental Health Hazard Assessment
(http://www.oehha.ca.gov/risk.html)
2/2013,
update
3/2014
17 citations added
           This document is a draft for review purposes only and does not constitute Agency policy,
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              Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
System used
Selected key reference(s) or sources
Date
Additional
references
identified
                       OEM HA Toxicity Criteria Database
                       (http://www.oehha.ca.gov/tcdb/index.asp)
                       Biomonitoring California-Priority Chemicals
                       (http://www.oehha.ca.gov/multimedia/biomon/pdf/Priori
                       tvChemsCurrent.pdf)
                       Biomonitoring California-Designated Chemicals
                       (http://www.oehha.ca.gov/multimedia/biomon/pdf/Desig
                       natedChemCurrent.pdf)
                       Cal/Ecotox database
                       (http://www.oehha.ca.gov/scripts/cal ecotox/CHEMLIST.
                       ASP)
                       OEHHA Fact Sheets
                       (http://www.oehha.ca.gov/public  info/facts/index.html)
                       Non-cancer health effects Table (RELs) and Cancer
                       Potency Factors (Appendix A and Appendix B)
                       (http://www.oehha.ca.gov/air/hot spots/index.html)

                  CPSC (http://www.cpsc.gov)

                  eChemPortal
                  (http://www.echemportal.Org/echemportal/participant/page.a
                  ction?pagelD=9)
                   Environment Canada - Search entire site if not found below:
                   (http://www.ec.gc.ca/default.asp?lang=En&n=ECD35C36)

                    Toxic Substances Managed under CEPA
                    (http://www.ec.gc.ca/toxiques-
                    toxics/Default.asp?lang=En&n=98E80CC6-l)
                    Screening Assessment reports
                    Risk Management reports
                    Final Assessments (http://www.ec.gc.ca/lcpe-
                    cepa/default.asp?lang=En&xml=09F567A7-BlEE-lFEE-73DB-
                    8AE6C1EB7658)
                    Draft Assessments (http://www.ec.gc.ca/lcpe-
                    cepa/default.asp?lang=En&xml=6892C255-5597-C162-95FC-
                    4B905320F8C9)

                   EPA Acute Exposure Guideline Levels
                   (http://www.epa.gov/oppt/aegl/pubs/chemlist.htm)

                   EPA - IRISTrack/New Assessments and Reviews

                   EPA NSCEP (http://www.epa.gov/ncepihom/)

                   EPA RfD/RfC and CRAVE meeting notes

                   EPA Science Inventory (http://cfpub.epa.gov/si/)

                   FDA (http://www.fda.gov/)

                   Federal Docket (www.regulations.gov)

                   Health Canada First Priority List Assessments (http://www.hc-
                   sc.gc.ca/ewh-semt/pubs/contaminants/psll-lspl/index-


                   Health Canada Second Priority List Assessments
                   (http://www.hc-sc.gc.ca/ewh-semt/pubs/contaminants/psl2-
                   Isp2/index-eng.php)
                   IARC (http://monographs.iarc.fr/htdig/search.html)
        This document is a draft for review purposes only and does not constitute Agency policy.
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
System used

Selected key reference(s) or sources
ITER (TERA database)
(http://iter.ctcnet.net/publicurl/pub search list.cfm)
NAP - Search Site (http://www.nap.edu/)
NRC - AEGLs via NAP search for "Acute Exposure Guideline
Level" and the chemical
NCI (http://www.cancer.gov)
National Institute for Environmental Health Sciences (NIEHS)
http://www.niehs.nih.gov/
NICNAS (PEC only covered by eChemPortal)
(http://www.nicnas.gov.au/industrv/aics/search.asp)
NIOSH (http://www.cdc.gov/niosh/topics/)
NIOSHTIC2 (http://www2a.cdc.gov/nioshtic-2/)
NTP - RoC, status, results, and management reports
(http://ntpsearch.niehs.nih.gov/auerv.html)
OSHA
(http://www.osha.gov/dts/chemicalsampling/toc/toc chemsa
mp.html)
RTECS http://www.ccohs.ca/search.html
Date

Additional
references
identified

 1
 2           These citations were screened using the title, abstract, and in limited instances, full text for
 3    pertinence to examining the health effects of DIBP exposure.  The citations were then screened
 4    using inclusion criteria (Table 2-3) describing specific information to help identify primary source
 5    health effect data and mechanistic and/or genotoxic data, as well as resources useful in preparation
 6    of the DIBP package. The process for screening the literature search is described below and is
 7    shown graphically in Figure 2-1:

 8       •   31 references were identified as animal studies with health effects data and were
 9           considered for data extraction to evidence tables and exposure-response arrays.

10       •   54 references were identified as supporting studies; of these, 12 were toxicokinetic studies
11           and 43 were mechanistic and genotoxicity studies.

12       •   97 references were identified as secondary literature (e.g., reviews and editorials, risk
13           assessments, meta analyses, and regulatory documents); these references were kept as
14           additional resources for development of the Toxicological Review.

15       •   632 references were excluded because these studies did not include primary source data
16           evaluating DIBP in relation to any kind of toxicity or health endpoint, and did not provide
17           either supporting information (e.g., toxicokinetic or mechanistic/genotoxicity data) or
18           secondary literature information (see Figure 2-1 and Table 2-3 for inclusion categories and
19           criteria).
                This document is a draft for review purposes only and does not constitute Agency policy,
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1           Note that some studies were identified as belonging to multiple categories. As a result, the
 2    total number of studies in a given category may be less than the sum of the individual studies listed
 3    in subcategories. For example, the category "Studies with Supporting Data" included one study that
 4    contained information relevant to both the toxicokinetics and mechanistic and/or genotoxicity
 5    subcategories.
 6           Among the studies identified in the DIBP literature searches, there were a number of
 7    foreign language studies.  Based on a review of the English titles and, when available, English
 8    abstracts, two of the foreign language articles, Maetal. [2013b] and lijo [1975], were tagged as
 9    toxicity studies and four foreign language articles, Ma etal. [2010], Maetal. [2013c], Kleinsasser et
10    al. [1999], and Kleinsasser et al. [2001a], were tagged as mechanistic and genotoxicity studies. The
11    other foreign language articles were excluded (tagged to Excluded: No primary data on toxic
12    effects]. Maetal. [2013b] is a report of neurotoxicological effects after DIBP exposure. With the
13    exception of one study [University of Rochester. 1954] that assessed brain weight, the Ma et al.
14    [2013b] article was the only available neurotoxicological study; this article was translated into
15    English (certified translation, Maetal., 2013a]. The remaining five foreign language articles
16    [above], tagged to toxicity studies or mechanistic and genotoxicity studies, have not yet been
17    translated or considered for inclusion in either evidence or mechanistic tables.  These studies  will
18    be further evaluated and considered during the development of the draft assessment of the
19    available evidence of DIBP-induced health effects.
20
                This document is a draft for review purposes only and does not constitute Agency policy.
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                        Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1

2
3
                               Database Searches
                       (see Table 2-1 for keywords and limits)
                   PubMed
                    n = 243
                              Web of
                              Science
                              n = 310
  Toxline
(incl.TSCATS)
                       (After duplicates removed electronically)
                                     n=504
                Additional Search
                   Strategies
                 (see Table 2-2 for
                methods and results)
                    n = 343
                                                                    Phthalates - Epidemiological
                                                                           Studies Search
                                                                  (see Table 2-4 for keywords and limits)
                                                                             Primary Source Human Data
                                                                                      n = 185
                                                                           (See Table 2-5 for Inclusion Criteria)
                                Combined Dataset
                            (After all duplicates removed)
                                     n=809
                         Manual Screening For Pertinence
                            (Title/Abstract/Full Text)
                        (see Table 2-3 for inclusion criteria)
              Excluded: No Primary Data on Toxic Effects
              (n=632)
              14  Abstract Only
              187  Not Chemical Specific
              66  Manufacture/use
              23  Chemical Treatment/Disposal/Remediation
              12  Use in sample prep or assay
              59  Measurement Methods
              16  Miscellaneous
              14  Ecosystem Effect
              179  Exposure levels
              52  Fate and Transport
              25  Chemical/physical properties
              9   Mixtures only
              Other Studies:

              Studies with Supporting Data (n=54)
              12 Toxicokinetics
              43 Mechanistic and Genotoxicrty Studies

              Secondary Literature (n=97)
              49  Reviews/editorials
              26  Regulator,' documents
              27  Risk assessments
              1  Meta analyses
                                                                                          selection of studies
                                                                                          that include DIBP
                                                 Animal Primary
                                                  Source Health
                                                  Effects Studies
                                                      (n = 31)
                               Human Primary
                                Source Health
                                Effects Studies
                                    (n = 52)

                             (See Table 2-7 for a listing
                                 of DIBP-specific
                              epidemiological studies)
Note: Studies containing multiple information categories were sorted into multiple tags.  For this reason, the
 subcategory numbers do not always add up to the category total.
              Figure 2-1.  Literature search approach for DIBP.

                  This document is a draft for review purposes only and does not constitute Agency policy.
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
 2           Table 2-3. Inclusion criteria used to identify animal studies of health-related
 3           endpoints, supporting data, or secondary literature
                                            Inclusion criteria3
              Did the study evaluate effects of DIBP or its metabolites known to be formed in humans?
              Did the study evaluate effects in a tissue (organ) or cells derived from a tissue (organ)?
              Did the study evaluate cellular, biochemical or molecular effects relevant to any mode of action?
                                                   or
              Does the study include information from other agencies, risk assessments, or reviews that would aid in
              the development of a toxicological review of DIBP?
 4
 5    alf the answer is "no" to any of these criteria questions, the study was placed under "No Primary Data on Toxic
 6     Effects."
 7
 8           Thirty-six human studies were also identified from the initial literature search using the
 9    search strings presented in Table 2-1. However, work being done concurrently on the development
10    of other phthalate preliminary materials revealed that this set of DIBP epidemiology studies was
11    incomplete. Epidemiology studies frequently examine multiple compounds (e.g., metabolites of
12    several different phthalates).  The indexing terms and abstracts may not include a comprehensive
13    list of all of the specific phthalates examined, resulting in the inappropriate exclusion of studies and
14    the potential for introduction of bias in the selection process.  Specifically, "negative" studies (i.e.,
15    studies that did not demonstrate an association between exposure and disease) are potentially
16    more likely to be missed than "positive" studies. This issue did not arise in the search process for
17    experimental (animal toxicology) studies, for which the test compound is virtually always identified
18    through search terms or key word searches of abstracts.
19           Another issue encountered in the development of the search and screening process for the
20    phthalate epidemiology studies relates to  the duplication of efforts involved in the development of
21    EPA's health assessments for several individual phthalates (e.g., dibutyl phthalate [DBF], DIBP,
22    butyl benzyl phthalate [BBP],  di(2-ethylhexyl)phthalate  [DEHP], di-ethyl phthalate [DEP],
23    diisononyl phthalate [DINP], and dipentyl phthalate [DPP]). In contrast to animal toxicology
24    studies, most of the epidemiology studies  examine more than one phthalate, resulting in
25    considerable overlap in the sets of studies identified using individual-phthalate search terms. Full
26    text screening of the same studies identified in multiple searches results is an inefficient use of
27    resources.
28           For these reasons, EPA developed a process for identifying epidemiological studies
29    evaluating phthalates by performing a single broad search to create a listing of epidemiological
30    studies of all phthalates mentioned above, from which the selection of studies examining potential
31    health effects of an individual phthalate could be drawn. This list records each of the phthalates
32    included in the study, based on information in the methods section of the paper, and the outcome(s)
                This document is a draft for review purposes only and does not constitute Agency policy.
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

1    examined. This literature search for epidemiological studies examining phthalates in relation to
2    health-related endpoints (from which the DIBP studies were drawn) was conducted in PubMed,
3    Web of Science, and ToxNet databases in June 2013, using keywords and limits described in
4    Table 2-4; the search was updated in December 2013 and in June 2014. For this search, "phthalate"
5    (and related terms) rather than names of specific phthalates was used as the foundation of the
6    search, along with terms designed specifically to identify epidemiological studies. These terms
7    were based on terms used in previously identified epidemiology studies of six different phthalates.
            Table 2-4. Summary of search terms: targeted epidemiology search
           Database,
          search date
                    Terms
           Hits
     June 2013 search
      PubMed
      06/2013
      No date restriction
(phthalate OR phthalates OR phthalic acid) AND
(human OR case-control OR pregnancy OR cohort OR
workers OR children OR survey)
      Imported: 2,505
After duplicates deleted: 2,482
      Web of Science
      06/2013
      No date restriction
(TS="phthalic acid" ORTS="phthalate" OR
TS="phthalates") AND (TS="humans" ORTS="human"
ORTS="case-control" ORTS="pregnancy" OR
TS="cohort" ORTS="workers" ORTS="child" OR
TS="children" OR TS="survey")
      Imported: 1,840
After duplicates deleted: 1,836
      ToxNet
      06/2013
      No date restriction
(phthalate OR phthalates OR phthalic acid) AND
(human OR case-control OR pregnancy OR cohort OR
workers OR children OR survey)
      Imported: 2,505
After duplicates deleted: 2,426
      Merged
      Reference Set
Merged dataset, with duplicates eliminated through
electronic screen
          4,127
                          Epidemiology articles meeting inclusion criteria
                                                          127
     December 2013
     search
PubMed
Web of Science
ToxNet
Merged Reference Set
Additional epidemiology articles meeting inclusion
criteria
           155
           249
           114
           350
            22
     June 2014
     search
PubMed
Web of Science
ToxNet (was not searched because no articles have
been found solely through this source in all the
previous searches)
Merged Reference Set
Additional epidemiology articles meeting inclusion
criteria
           184
           409
            0
                                                                                    494

                                                                                    24
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1           More than 4,000 citations were identified through this search. These were then screened
 2    using inclusion criteria describing specific population (i.e., human), exposure measures,
 3    comparison, and health effects (Table 2-5). Note that other studies obtained in the search, for
 4    example mechanistic and pharmacokinetic studies, are excluded from consideration with respect to
 5    the specific objective of this search (i.e., identification of epidemiology studies), but could be
 6    included in other steps in the assessment. Duplicate citations of the same article were excluded,
 7    and articles written in a language other than English were retained for subsequent review.  Earlier
 8    analyses that are updated in a subsequent paper (e.g., with a larger sample size) are not included as
 9    a primary paper, but may be used as background material regarding study methods.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
                This document is a draft for review purposes only and does not constitute Agency policy.
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

1            Table 2-5. Inclusion criteria used to identify epidemiology studies of health-
2            related endpoints
                                                Inclusion criteria
              Is the study population humans?
                                                      and
              Is exposure to one or more phthalate (parent compound or metabolite(s)a...
              - measured in air, dust, or biological tissue?
              - based on  knowledge of industrial hygiene (occupational settings)?
              - based on  knowledge of specific contamination sites or accidental exposure?
                                                      and
              Does the study compare a health effect in higher versus lower or no exposure?
                                                      and
              Does the study include a measure of one or more primary health effect endpoints relating to...
              - sexual differentiation measures (e.g., male genital malformations, anogenital distance, gender-related
              play behavior)
              - male reproductive effects (e.g., steroidal and gonadotropin hormone levels, measures of male-
              mediated infertility)?
              - female reproductive effects (e.g., steroidal and gonadotropin hormone levels, measures of female-
              mediated infertility, gynecological conditions)?
              - pregnancy outcomes (e.g., birth weight, gestation age)?
              - puberty (male and female) (e.g., timing of development, precocious puberty, gynecomastia)?
              - neurodevelopment  (infants and children) (e.g., standardized tests of reflexes, behavior, and
              intelligence)?
              - thyroid effects (e.g., thyroid stimulating hormone and thyroid hormones, subclinical and clinical thyroid
              disease)?
              - immune system effects (e.g., asthma, allergies, immunoglobulin E (IgE) levels, skin prick tests)?
              - pulmonary function (e.g., standardized test of lung volume, diffusing capacity)?
              - neurological effects (adults) (e.g., peripheral neuropathy, vision or hearing or other sensory tests)?
              - liver effects  (e.g., cholestasis, biomarkers of liver function)?
              - kidney effects (e.g.,  end stage renal disease, biomarkers of kidney function)?
              - diabetes and measures of insulin resistance?
              - obesity (and other measures of adiposity)?
              - cardiovascular disease (cause-specific incidence or mortality)?
              - cardiovascular risk factors (e.g., triglyceride and lipid levels, blood pressure or hypertension)?
              - cancer (cause-specific incidence or mortality)?
                                                       or
              Does the study include a measure of one or more secondary health effect endpoints (to be considered
              within context of mechanistic evidence) relating to...
              - oxidative  stress?
              - inflammation?
              -gene expression?
3
4    aFor DIBP, the primary metabolite of interest is MIBP.
5
6            One hundred and seventy-three epidemiological studies examining one or more phthalates
7    in relation to one or more endpoints were identified by the searches conducted through June 2014
                 This document is a draft for review purposes only and does not constitute Agency policy.
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
 1    (127 in the initial search, 22 in the December 2013 update, and 24 in the June 2014 update;
 2    Figure 2-1). Other strategies were also used to supplement this broad search for epidemiology
 3    studies of phthalates), resulting in the identification of 12 additional publications (Table 2-6), for a
 4    total of 185 epidemiological studies. From this set of all of the epidemiological studies examining
 5    any phthalate, 52 studies analyzed one or more health effects in relation to a measure of DIBP
 6    (Table 2-7).
 7
 8
       Table 2-6. Summary of additional search strategies for epidemiology studies
       of phthalate exposure in relation to health-related endpoints
Approach used
Testing and refinement of search terms based on terms used for the
identified articles within each category
Review of references cited in the identified list of epidemiology studies
("backward" search)
Electronic forward search through Web of Science of one to three
studies within each health endpoint category (early studies within each
category generally selected to maximize potential for citation in
subsequent publications)3
Date
performed
June 2014
July 2014
July 2014
Number of additional
citations identified
6
1
5
 9
10
11
12
13
14
15
16
aThe following studies were used to conduct the forward searches: Trasande et al. (2013b); James-Todd et al.
 (2012); Lind and Lind (2011); Boas et al. (2010); Cho et al. (2010); Engel et al. (2010); Lopez-Carrillo et al. (2010);
 Wolff etal. (2010); Adibietal. (2009); Chouetal. (2009); Hatch et al. (2008); Wolff etal. (2008); Meeker et al.
 (2007); Stahlhut et al. (2007); Mauser et al. (2006); Reddyet al. (2006); Jonssonetal. (2005); Swan et al. (2005);
 Bornehagetal. (2004); Hoppin etal. (2004); Aschengrau et al. (1998); Heineman etal. (1992); Nielsen etal.
 (1989); Nielsen etal. (1985).
17
18
       Table 2-7. Primary source epidemiological studies examining health effects of
       DIBP
Outcome category
Sexual differentiation measures
(Table 3-1)
Male reproductive (semen
parameters, infertility, and
hormones)
(Tables 3-2 and 3-3)
Male pubertal development
(Table 3-4)
Female pubertal development
(Table 3-5)
Reference3
Swan (2008)
Swan et al. (2010)

Buck Louis etal. (2014)
Joensen etal. (2012)
Kranvogl et al. (2014)
Mendiola etal. (2011)
Wirth et al. (2008)

Mieritzetal. (2012)
Mouritsen etal. (2013b)

Frederiksen et al. (2012)
Hart et al. (2013)
Lomenicketal. (2010)
Mouritsen etal. (2013b)
DIBP measure
MIBP (maternal urine)
MIBP (maternal urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (maternal urine)
MIBP (urine)
Sum MIBP +MBP (urine)3
Sum MIBP+ MBP (urine)3
MIBP (maternal serum)
MIBP (urine)
Sum MIBP+MBP(urine)3
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Outcome category
Female reproductive (infertility,
hormones, gynecological conditions)
(Tables 3-6 and 3-7)
Pregnancy outcomes (fetal growth,
preterm birth)
(Table 3-8)
Immune: allergy (rhinitis, eczema)
(Table 3-9)
Immune: asthma
(Table 3-10)
Neurodevelopment
(Table 3-11)
Thyroid
(Table 3-12)
Obesity
(Table 3-13)
Diabetes and insulin resistance
(Table 3-14)
Other cardiovascular disease risk
factors
(Table 3-15)
Reference3
Buck Louis etal. (2013)
Hart et al. (2013)
Sathyanarayana et al. (2014)
Upson etal. (2013)

Ferguson etal. (2014a)
Ferguson et al. (2014b)
Huang etal. (2014b)
Meeker et al. (2009)
Philippatetal. (2012)
Wolff etal. (2008)

Ait Bamai etal. (2014)
Bornehag et al. (2004)
Callesen et al. (2014a)
Callesen et al. (2014b)
Hoppinetal. (2013)
Sun etal. (2009)

Ait Bamai etal. (2014)
Bertelsen et al. (2013)
Callesen et al. (2014a)
Callesen et al. (2014b)
Hoppinetal. (2013)
Sun etal. (2009)

Braunetal. (2014)
Engel etal. (2010)
Kobrosly et al. (2014)
Tellez-Roio et al. (2013)
Whyatt et al. (2012)

Dirtu et al. (2013)
Meeker and Ferguson (2011)

Buseretal. (2014)
Dirtu et al. (2013)
Hart et al. (2013)
Kasper-Sonnenberg et al. (2012)
Lindetal. (2012a)
Olsen et al. (2012)
Svensson et al. (2011)
Teitelbaumetal. (2012)
Trasandeetal. (2013a)
Wang et al. (2013)

Huang etal. (2014a)
James-Todd etal. (2012)
Lindetal. (2012b)
Olsen et al. (2012)
Svensson et al. (2011)
Trasandeetal. (2013c)

Lind and Lind (2011)
Shiue (2014)
Trasandeetal. (2013b)
DIBP measure
MIBP (urine)
MIBP (maternal serum)
MIBP (maternal urine)
MIBP (urine)
MIBP (maternal urine)
MIBP (maternal urine)
DIBP (cord blood)
MIBP (maternal urine)
MIBP (maternal urine)
MIBP (maternal urine)
DIBP (dust)
DIBP (dust)
MIBP (urine)
DIBP (dust)
MIBP (urine)
DIBP (dust)
DIBP (dust)
MIBP (urine)
MIBP (urine)
DIBP (dust)
MIBP (urine)
DIBP (dust)
MIBP (maternal urine)
MIBP (maternal urine)
MIBP (maternal urine)
MIBP (maternal urine)
MIBP (maternal urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (maternal serum)
Sum MIBP + OH-MIBP (urine)
MIBP (serum)
MIBP (serum)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (urine)
MIBP (serum)
MIBP (serum)
MIBP (urine)
MIBP (urine)
MIBP (serum)
MIBP (urine)
MIBP (urine)
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Outcome category

Cancer
(Table 3-16)
Reference3
Olsen et al. (2012)

Lopez-Carrillo et al. (2010)

DIBP measure
MIBP (serum)
MIBP (urine)
 1
 2    Included in DIBP tables because in this population, at this time, MIBP concentrations were greater than
 3     monobutyl phthalate (MBP) concentrations.
 4
 5           The literature for both epidemiological and animal studies will be regularly monitored for
 6    the publication of new studies. The documentation and results for this supplementary search can
 7    be found on the Health and Environmental Research On-line (HERO) website1
 8    [http://hero.epa.gov/DIBP and http://hero.epa.gov/phthalates-humanstudies].

 9    2.2.   SELECTION OF CRITICAL STUDIES IN EARLY STAGES OF DRAFT
10    DEVELOPMENT

11    2.2.1.  General Approach
12           Each study retained following the literature search and screen was evaluated for aspects of
13    design, conduct, or reporting that could affect the interpretation of results and the overall
14    contribution to the synthesis of evidence for determination of hazard potential.  Much of the key
15    information for conducting this evaluation can generally be found in the study's methods section
16    and in how the study results are reported. Importantly, this evaluation does not consider study
17    results or, more specifically, the direction or magnitude of any reported effects.  For example,
18    standard issues for evaluation of experimental animal data identified by the NRC and adopted in
19    this approach include consideration of the species and sex of animals studied, dosing information
20    (dose spacing, dose duration, and route of exposure), endpoints considered, and the relevance of
21    the endpoints to the human endpoints of concern. Similarly, observational epidemiologic studies in
22    this approach for evaluation should consider the following:

23       •   Approach used to identify the study population and the potential for selection bias.

24       •   Study population characteristics and the generalizability of findings to other populations.
      iHERO is a database of scientific studies and other references used to develop EPA's risk assessments aimed
      at understanding the health and environmental effects of pollutants and chemicals. It is developed and
      managed in EPA's Office of Research and Development (ORD) by the National Center for Environmental
      Assessment (NCEA). The database includes more than 1,400,000 scientific articles from the peer-reviewed
      literature.  New studies are added continuously to HERO.

      Note: The HERO database will be regularly updated as additional references are identified during assessment
      development. Therefore, the numbers of references (by tag) displayed on the HERO webpage for DIBP may
      not match the numbers of references identified in Figure 2-1 (current through March 2014).
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 1       •   Approach used for exposure assessment and the potential for information bias, whether
 2           differential (nonrandom) or nondifferential (random).

 3       •   Approach used for outcome identification and any potential bias.

 4       •   Appropriateness of analytic methods used.

 5       •   Potential for confounding to have influenced the findings.

 6       •   Precision of estimates of effect

 7       •   Availability of an exposure metric that is used to model the severity of adverse response
 8           associated with a gradient of exposures.

 9           To facilitate the evaluation outlined above, evidence tables are constructed that
10    systematically summarize the important information from each study in a standardized tabular
11    format as recommended by the NRG [2011]. In general, the evidence tables include all studies that
12    inform the overall synthesis of evidence for hazard potential. At this early stage of study
13    evaluation, the goal is to be inclusive. Exclusion of studies may unnecessarily narrow subsequent
14    analyses by eliminating information that might later prove useful. Premature exclusion might also
15    give a false sense of the consistency of results across the database of studies by unknowingly
16    reducing the diversity of study results. However, there may be situations in which the initial review
17    of the available data will lead to a decision to focus on a particular set of health effects and to
18    exclude others from further evaluation.

19    2.2.2.  Exclusion of Studies
20           After the literature search was manually screened for pertinence, studies were excluded if
21    fundamental flaws were identified  in their design, conduct, or reporting. The DIBP experimental
22    animal database consists of studies designed to examine repeat-dose intraperitoneal or oral toxicity
23    (including subchronic and short-term duration studies) and endpoint-specific toxicities (including
24    reproductive and developmental toxicity). Four studies administered DIBP via the intraperitoneal
25    route of exposure. These studies were excluded from the DIBP evidence tables because the
26    intraperitoneal route of exposure is generally considered less relevant to human health exposure.
27    The remaining studies involved administration of DIBP in the diet or via gavage administration.
28    Acute studies are generally less pertinent for characterizing health hazards associated with chronic
29    exposure.  There was one acute study that was excluded from the evidence tables. Two BASF
30    reports identified in the literature searches could not be obtained and thus, could not be evaluated
31    for inclusion in the evidence tables  (BASF, 2003,1961). For these reasons, these studies are not
32    summarized in the preliminary evidence tables. Nevertheless, with the exception of the studies that
33    could not be obtained, the studies will still be evaluated  as possible sources of supporting health
34    effects information during assessment development. Experimental animal studies that were
35    sources of short-term, subchronic, or chronic health effects were evaluated for potential flaws in
36    their design, reporting, or conduct.  As a result,  one study, Maetal. (2013b) (English translation
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    cited as Maetal. [2013a]], was removed from consideration in the assessment because of
 2    incomplete description of experimental methods that leads to uncertainty in the results. Another
 3    study, Eastman Kodak [1978], a one-page data summary, was excluded because it does not provide
 4    detailed data reporting.
 5          The remaining studies are all sources of health effects data that may be used in the
 6    assessment The 20 studies summarized in the evidence tables are considered the "critical"
 7    studies from which the study methods and results are presented in preliminary evidence tables
 8    and exposure-response arrays (Section 3).  There were also a few cases of the same study data
 9    being contained in multiple reports; in those cases, the studies are listed together in the evidence
10    tables.

11    2.3. STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE
12         FUTURE  EVALUATION AND SYNTHESIS OF THE CRITICAL
13         EPIDEMIOLOGICAL STUDIES FOR DIBP
14          Several considerations will be used in EPA's evaluation of epidemiological studies of human
15    health effects of DIBP. These considerations include aspects of the study design affecting the
16    internal or external validity of the results (e.g., population characteristics and representativeness,
17    exposure and outcome measures, confounding, data analysis), focusing on specific types of bias
18    (e.g., selection bias; information bias due to exposure misclassification) and other considerations
19    that could otherwise influence or limit the interpretation of the data. A study is externally valid if
20    the study results for the study population can be extrapolated to external target populations. An
21    internally valid study is free from different types of biases, and is a prerequisite for generalizing
22    study results beyond the study population. These issues are outlined in the IRIS Preamble, and are
23    described below.

24    Study Population
25          Evaluation of study population characteristics (including key socio-demographic variables
26    and study inclusion criteria) can be used to evaluate external validity (i.e., generalizability) and to
27    facilitate comparison of results across different study populations. Some aspects of the selection
28    process may also affect the interval validity of a study, resulting in a biased effect estimate.
29          The general considerations for evaluating issues relating to the study population include
30    adequate documentation of participant recruitment, including eligibility criteria and participation
31    rates, missing data, and loss to follow-up. This information is used to evaluate internal study
32    validity related to selection bias. Different types of selection bias that may occur include the
33    healthy worker effect, differential loss to follow up, Berkson's bias (relating to selection of
34    participants in hospital-based case-control studies), and participation bias. It is important to note
35    that low participation rates, or differences in participation rates between exposed and non-exposed
36    groups or between cases and controls, is not evidence of selection bias. Rather, selection bias arises
37    from a differential pattern of participation with respect to both the exposure and the outcome, i.e.,
38    patterns of participation that would result in a biased effect estimate. An example of differential
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    participation would be when people with high levels of exposure and the outcome of interest are
 2    more likely to participate than people with low levels of exposure and the outcome.
 3          The available DIBP studies have generally examined metabolites from many different
 4    phthalates within the context of research on environmental exposures.  Most of these studies rely
 5    on objective exposure measures (e.g., biomonitoring data), some of which are collected prior to
 6    onset of the outcomes being examined (e.g., in the prospective pregnancy cohort studies). Study
 7    participants generally do not have knowledge of the study hypothesis or their exposure to DIBP and
 8    thus, knowledge of exposure or exposure level is unlikely to result in differential participation with
 9    respect to outcomes. These study features should minimize the potential for selection bias.
10    However, EPA will consider the possibility that a particular concern about the specific sources of
11    DIBP, in conjunction with knowledge of specific health outcomes, may motivate people to
12    participate in a study or to continue participation throughout a follow-up period. In the absence of
13    evidence that any of these scenarios is likely to occur in a study, EPA will not consider selection bias
14    as a limitation of a study.

15    Exposure Considerations
16          General considerations for evaluating exposure include: (1) identifying how exposure can
17    occur (e.g., exposure sources, routes, and media); (2) determining appropriate critical exposure
18    period(s) for the outcomes under study; (3) evaluating variability in the exposure metrics of
19    interest (e.g., temporal and spatial variability for environmental measures or inter-individual
20    variability for biomonitoring data) that can impact different types of exposure metrics (e.g.,
21    cumulative, average, or peak exposure); (4) determining if an appropriate analytical methodology
22    was employed (e.g., choice of biological matrix, sampling protocol, quantification approach);
23    (5) evaluating the choice of exposure surrogate evaluated (e.g., constituent chemical or
24    group/mixture); and (6) evaluating the classification of individuals into exposure categories. These
25    six considerations help determine the accuracy and precision of the exposure estimates, and the
26    likelihood of measurement error with respect to the exposure metrics used. Nondifferential
27    misclassification of exposure categories, for example, can also result from measurement error and
28    is expected to predominantly result in attenuated effect estimates (Blair etal., 2007).
29          Some common sources of exposure to DIBP include cosmetics, food, and food packaging
30    (Zota etal., 2014) with the primary route of exposure occurring through ingestion and some
31    exposure occurring via inhalation and dermal routes (see Section 1.1.3). Thus, exposure to DIBP is
32    typically from multiple sources, and occurs episodically on a daily basis. Exposure to DIBP may be
33    increasing; a recent study of the U.S. general population found that urinary concentrations of the
34    DIBP metabolite MIBP have increased over time and were 206% higher in 2009-2010 compared to
35    2001-2002 (Zota etal.. 2014).
36          Urine provides an integrated measure of phthalate exposure from all sources.
37    Measurement of DIBP metabolites, rather than the parent compound, is preferred because the
38    parent compound is metabolized very quickly and does not provide an accurate measure of
39    exposure. The simple monoester metabolite, monoisobutyl phthalate (MIBP) is the most commonly
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    measured DIBP metabolite in epidemiologic studies.  MIBP accounts for an estimated 70.3% of the
 2    urinary excretion of DIBP; this value is based on human data from a controlled dosing study of a
 3    single volunteer [Kochetal., 2012]. EPA considers the use of MIBP to be a good proxy for total
 4    DIBP exposure.
 5          Although urine measures are most commonly used in epidemiological studies  of phthalate
 6    exposure, measures in serum, semen, and breast milk have also been used. Studies examining DIBP
 7    metabolites in breast milk or serum have generally reported low levels of detection. One study in
 8    Taiwan reported that MIBP above the limit of detection was found in 33.3% of breast milk samples
 9    from 30 women. The detection rate in 30 cord blood samples in this study was 100%, but the
10    correlation between MIBP measured in cord blood and maternal urine was -0.11 (Pearson
11    correlation of log-transformed levels] [Linetal., 2011]. Hogberg et al. [2008] reported that few
12    breast milk (2 out of 42] or serum (3 out of 36] samples in a study in Sweden had detectable MIBP
13    concentrations. Another study conducted among 60 men ages 18-26 years found that 33.3% of
14    serum samples and 16.9% of seminal plasma samples had MIBP concentrations above the limit of
15    detection [Frederiksenetal., 2010]. The Spearman correlation coefficient between urine and
16    serum concentrations was 0.39; the correlation between urine and seminal plasma concentrations
17    was not calculated because of the low detection rate for the latter samples [Frederiksenetal.,
18    2010]. The lower detection rate in tissues other than urine reduces EPA's confidence  in DIBP
19    metabolite measures in these biological matrices.
20          Given their first-order kinetics with half-lives on the order of hours [3.9 hours for MIBP in
21    [Koch and Angerer. 2007]]. urinary phthalate metabolite concentrations peak shortly after
22    exposure. Thus, for single-time exposure scenarios (rather than multi-source, multiple time
23    exposure scenarios], urine sampled during this time of peak concentration could lead  to
24    overestimates of average daily intake, and conversely, measurements made after concentrations
25    have peaked and declined could lead to underestimates of intake. One study conducted among
26    139 pregnant women in Puerto Rico included measurement of MIBP found that specific gravity
27    adjusted concentrations were lower in samples collected from 9 am to noon (geometric mean 9.4]
28    compared with samples collected in early morning, early afternoon, or evening (geometric means
29    13-14] (Cantonwine etal., 2014]. Urinary measures of DIBP metabolite concentrations in
30    epidemiological studies are generally conducted using spot urine samples (i.e., collected at time of a
31    clinic or study examination visit] rather than at a specified time (e.g., first morning void] or in 24-
32    hour urine samples. Although the time of sample  collection described above may affect the
33    accuracy of an estimated intake for a single individual, studies of other phthalates (e.g., DEHP] have
34    demonstrated that on  a group level, spot urine samples provide a reasonable approximation of
35    concentrations that would have been observed using full-day urine samples (Christensenetal..
36    2014] and that a single spot sample was reliable in ranking subjects according to tertile of MIBP
37    (Teitelbaum etal., 2008]. Based on this information, EPA does not consider the reliance on spot
38    urine samples for exposure estimation (including ranking of individuals into different DIBP
39    categories] to be a major limitation for epidemiological studies. However because of the potential
40    for greater inaccuracy of estimates in the "tails" of the distribution, EPA will include additional
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    considerations (e.g., discussion of analysis of residuals, outliers) when evaluating analyses based on
 2    use of DIBP metabolites as continuous measures.
 3          Another potential limitation of measurement of DIBP metabolites in urine is the
 4    reproducibility of phthalate metabolite concentrations over time; that is, how well does a single
 5    measure reflect the key exposure metric (average, peak) for the critical exposure window of
 6    interest For many short-lived chemicals, considerable temporal variability in exposure level is
 7    expected, and thus, repeated measures in the critical exposure window are preferred over a single
 8    measurement Reproducibility is usually evaluated with the intraclass correlation coefficient (ICC),
 9    a measure of the 'between-individual' variance divided by the total variance (between and within
10    individuals).  A higher ICC indicates greater reproducibility (i.e., lower within-person variance). An
11    ICC of 0.51 for MIBP was reported in a study of 25 Hmong women ages 19-51 years with samples
12    collected 2-4 weeks apart (Pecketal.. 2010). In studies of reproducibility of measures during
13    pregnancy, Cantonwine etal. (2014) reported ICCs of 0.35 and 0.34 (unadjusted and specific gravity
14    adjusted) when comparing urine samples taken at approximately 18, 22, and 26 weeks of gestation.
15    ICCs of 0.36 and 0.38, respectively, were seen before pregnancy and in early pregnancy (Braun et
16    al., 2012), and an ICC of approximately 0.5 was seen over a 6-week period in the last trimester
17    (Adibi etal.. 2008). Among women participating in the Nurses' Health Study (NHS) (in 2000-2001
18    for NHS and in 1996-1999 for NHS II), the ICC for samples collected 1-3 years apart was 0.30 for all
19    samples, and was 0.29 for first-morning samples (Townsendetal.. 2013).  Data for children are
20    sparse, limiting the ability to examine this source of uncertainty in this population. One study
21    evaluated variability in children aged 6-10 years old over a 6-month period (Teitelbaum etal..
22    2008) and found a relatively low ICC (0.21 unadjusted, 0.28 creatinine-adjusted). The available
23    data highlight the value of repeated exposure measures collected during the appropriate critical
24    period for the outcome(s) under study. Based on these studies, however, EPA does not consider the
25    use of a single measurement to be a major limitation in studies in adults in which the measure of
26    exposure is closely aligned with the relevant window(s) of exposure, if known, for the effect under
27    study. EPA has greater uncertainty, however, about measurements taken outside of the relevant
28    time window (e.g., several years after diagnosis, or the difference between first and third trimesters
29    of pregnancy), and about measurements taken in children.
30          Some studies present analyses using a combined measure based on summation of MIBP and
31    monobutyl phthalate (MBP), as a measure of both DIBP and DBF, respectively. The relative
32    contribution of DIBP to this total has varied over time (as the use of DIBP has increased), and can
33    vary between populations (e.g., greater use of DIBP compared with DBF in some countries).  EPA
34    includes studies in the DIBP evidence tables using this summed exposure measure in situations in
35    which the concentration of MIBP is greater than that of MBP, but recognizes that this measure
36    introduces an additional source of exposure misclassification. Other studies present analyses using
37    a combined "low molecular weight" phthalate measure based on the summation of MIBP, MBP, and
38    monoethyl phthalate (MEP) (reflecting exposure to the parent compounds of DIBP, DBF, and DEP,
39    respectively). Because MIBP does not represent a major contributor to this summation
40    measurement, EPA has not included data from these studies in the DIBP evidence tables.
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 1           EPA will also consider the potential for differential misclassification of biomarker measures
 2    of exposure; for example, in situations in which a health outcome (e.g., diagnosis with diabetes or
 3    cancer) could lead to a behavioral change that results in a change in DIBP exposure. This type of
 4    scenario adds an additional challenge to the interpretation of the DIBP metabolites as valid
 5    measures of exposure in a relevant time window(s) with respect to disease development
 6           The distribution of exposure will also be considered in evaluating individual studies and
 7    when comparing results among groups  of studies.  One consideration is the contrast of exposure
 8    levels (i.e., the difference between "high" and "low"): a study with a very narrow contrast may not
 9    have sufficient variability to detect an effect that would be seen over a broader range. Another
10    consideration is the absolute level of exposure, as different effect estimates may be expected in
11    studies examining different exposure levels even if they had similar exposure contrasts.

12    Prim ary Outcom e Measures
13           The general considerations for evaluating issues relating to accuracy, reliability, and
14    biological relevance of outcomes include adequate  length of follow-up to evaluate the outcomes of
15    interest, and use of appropriate ascertainment methods to classify individuals with regard to the
16    outcome (e.g., high sensitivity and specificity). With respect to continuous measures, such as
17    hormone concentrations or semen parameters, EPA will consider, in addition to assessing whether
18    reported parameters are outside normal physiological range, evidence of smaller changes in the
19    distribution of a parameter that may represent an effect on a population level [e.g., as is the case for
20    early childhood exposure to lead and decrements in intelligence as measured by IQ (U.S. EPA.
21    2013).
22           Issues relating to assessment of the specific primary health effects are discussed below and
23    summarized in Table 2-8 at the end of Section 2.3.

24    Sexual differentiation
25           Cryptorchidism and hypospadias are two disorders of the development of the male
26    reproductive system.  Cryptorchidism, or undescended testes, can be present at birth  (congenital
27    Cryptorchidism) or can occur later during infancy and childhood (acquired Cryptorchidism).
28    Surgical correction (orchiopexy)  is recommended in cases of Cryptorchidism that do not resolve
29    during infancy because long-term complications include impaired sperm production and increased
30    risk of testicular cancer (Virtanen et al., 2007). Retractile testes can move back and forth between
31    the scrotum and the abdomen; this condition usually resolves by puberty and is not associated with
32    reproductive or other complications. Classification criteria for Cryptorchidism that involve
33    testicular positioning are commonly used in clinical research (Tohn Radcliffe Hospital
34    Cryptorchidism Study Group, 1988: Scorer, 1964).  EPA will consider the definition used and age
35    range in interpreting studies of Cryptorchidism or related outcomes.
36           In animal toxicology studies, anogenital distance (AGD) is a routine marker to assess
37    endocrine disruption; this marker has only recently been adapted for use in epidemiological
38    studies.  One study in adult men reported associations between decreased AGD and measures
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    relating to infertility [Eisenbergetal., 2011]: most studies have used this measure in infants,
 2    however, as a marker of endocrine environment during development. It is important to consider
 3    general size, in addition to sex, in the evaluation of AGD, for example by incorporating birth weight
 4    or length (e.g., calculation of "anogenital index" by dividing anogenital distance by weight). With
 5    regard to reproducibility of this measure, a low degree of between-observer variability was found
 6    using a standardized protocol and trained observers [Romano-Riqueraetal.. 2007: Salazar-
 7    Martinez etal.. 2004). Because of the importance of size and age in the interpretation of this
 8    measure, EPA has greater confidence in studies with measures taken at birth or over a narrow age
 9    range and lesser confidence in studies among a group spanning a larger age range.
10           Gender-related behaviors, as measured by the Pre-School Activities Inventory [Golombok
11    and Rust, 1993] or other scales, has been examined in relation to direct or indirect measures of
12    fetal testosterone levels, including studies of DIBP. This outcome measure has been examined in
13    studies of relatively rare genetic conditions (e.g., congenital adrenal hyperplasia and complete
14    androgen insensitivity syndrome], as well as in studies focusing on the normal variability seen in
15    the general population  (reviewed in Hines, 2006].  EPA will consider evidence pertaining to the
16    reliability and validity of the Pre-School Activities Inventory in its evaluation of studies using this
17    scale.

18    Male and female reproductive outcomes
19           The  DIBP literature includes studies of reproductive and gonadotropin hormone levels in
20    men and studies of semen parameters that can be indicative of reduced fertility. The details of the
21    laboratory procedures, including information on the basic methods, level of detection, and
22    coefficient of variation, are important considerations for hormone assays and measures of semen
23    parameters. The World Health Organization (WHO] laboratory methods for analysis of sperm
24    counts and semen parameters (see, for example. WHO. 1999] are generally recognized as standards
25    in this field.  EPA will consider studies that reference these methods, regardless of which revision
26    used, to be reliable measures.
27           Much of the focus of the research on male steroidal and gonadotropin hormones in the DIBP
28    database concerns testosterone.  One issue with respect to these measures is the estimation method
29    used for free testosterone. Based on the analysis by Vermeulen et al. (1999], EPA will consider
30    estimates based on total testosterone divided by immunoassay-derived sex-hormone binding
31    globulin (SHBG] levels to be most reliable.
32           The  DIBP literature also includes studies of reproductive hormones in women. In addition
33    to the general considerations regarding hormone assays noted above, timing within a menstrual
34    cycle for studies of pre- and peri-menopausal women, and timing with respect to gestational age for
35    studies of women during pregnancy, are also be an important considerations for interpretation of
36    reproductive hormone concentrations.
37           Another female reproductive outcome included in the DIBP literature is endometriosis.
38    Endometriosis can be symptomless, or can lead to surgical intervention; it is often diagnosed as
39    part of a work-up for infertility. Variability in clinical presentation and in access and use of health
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    care services present considerable challenges to conducting epidemiological studies of this
 2    condition [Holt and Weiss, 2000]. Confirmation of "case" and "control" status (i.e., presence or
 3    absence of endometriosis) by ultrasound or clinical evaluation is recommended to reduce outcome
 4    misclassification, and representation of the source population should be carefully considered.
 5           Infertility is generally defined clinically and for research purposes as the inability to
 6    conceive a clinically-recognized pregnancy after 12 months of intercourse of regular frequency
 7    without use of contraceptives. Fecundity or fecundability are terms for the capacity for
 8    reproduction. "Time to pregnancy" (i.e., the number of cycles of unprotected intercourse before
 9    conception) has been used as a measure of fecundability in studies of environmental and
10    occupational exposures (Bairdetal., 1986: Baird and Wilcox, 1985].  Time to pregnancy is a
11    measure of a couple's fecundability, incorporating effects that can be manifested through the male
12    or female (or both]. Considerations in time to pregnancy studies include the source of data (i.e.,
13    retrospective or prospective designs] and incorporation of information on "non-pregnancy
14    planners" (Weinberg et al.. 1994].

15    Timing of male and female puberty, and conditions of unusual pubertal development
16           Pubertal development in humans is often assessed using timing of peak height velocity
17    ("growth spurt"] and secondary markers of sexual development Secondary markers for females
18    include breast development (thelarche] and pubic hair development (pubarche], and age at first
19    period (menarche]. Secondary markers for males include gonadal development (gonadarche] and
20    pubic hair development, and age at first sperm  emission (spermarche].
21           Evaluation of breast, pubic hair, and gonadal development is frequently performed using
22    the Tanner stages (Marshall and Tanner, 1970,1969], which places the individual in one of five
23    stages, ranging from pre-pubertal (stage 1] to adult maturation (stage 5). However, the process of
24    this staging is not straightforward, and is most reliable when performed by trained personnel
25    (rather than by the individual or a parent, for example] (Slough etal.. 2013: Schlossberger et al..
26    1992: Espeland etal.. 1990]. Age at menarche is considered to more reliable when assessed via
27    self-report (Koprowskietal.. 2001], although reliability may decrease with increasing time since
28    menarche (Cooper et al., 2006]. Additionally, hormone levels may sometimes be used to evaluate
29    pubertal development  Individuals may vary widely in the timing of these developmental
30    milestones.
31           Several clinical syndromes are known to disrupt the timing and order of markers of
32    pubertal development  Considerations in the diagnosis of either precocious or delayed puberty
33    include the diagnostic criteria used and the source of the information (e.g., whether collected from
34    medical records or from self- or parental report]. For females, precocious puberty is usually
35    defined as the onset of puberty before the age of 8 years, while delayed puberty is usually defined
36    as the lack of pubertal development by the age  of 13 years (Marshall  and Tanner, 1969]:
37    corresponding ages in males are before the age of 9 years for precocious puberty and lack of
38    pubertal development by the age of 14 years for delayed puberty (Marshall and Tanner. 1970].
39    Clinical evaluation would involve hormone assays to distinguish between gonadotropin dependent
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    ("central"), gonadotropin independent ("peripheral"), or a combination of both (Traggiai and
 2    Stanhope, 2003) forms of these conditions.

 3    Pregnancy-related outcomes
 4           Infant birth weight and gestational age are two outcomes commonly used in reproductive
 5    epidemiology studies. EPA considers analyses of the various indices for both outcomes (fetal
 6    growth and gestational age) to be informative with respect to hazard identification, but will
 7    consider each separately as they address different issues.  Gestational duration can be measured as
 8    a continuous outcome or dichotomous outcome such as preterm birth.  Preterm births include
 9    infants delivered earlier than 37 gestational weeks, and those delivered earlier than 32 gestational
10    weeks are classified as very preterm births. Different measures of fetal growth restriction are often
11    examined in epidemiological studies. In addition to the continuous measure of birth weight,
12    another commonly used measure of fetal growth restriction is the categorical variable of low birth
13    weight (defined as <2,500 g). Small for gestational age (defined as birth weight less than the 10th
14    percentile for the gestational birth weight distribution) is considered a better measure of fetal
15    growth rate as it takes into consideration gestational duration, and would be preferred over a
16    measure of birth weight in a study that includes preterm births. Birth weight and gestational
17    duration can also be examined as continuous variables, often in analysis that excludes preterm or
18    low birth weight births, so that the focus of the analysis is on variability within the  "normal" range.
19           EPA considers birth weight obtained from medical records to be a reliable source as this is a
20    very accurate and precise measurement Although more prone to measurement error than birth
21    weight measures, gestational age can be estimated from several approaches.  Some of these include
22    ultrasonography, estimates based on date of last menstrual period based on maternal recall, or
23    from clinical examination based on antenatal or newborn assessments (which may include an
24    ultrasound).  Menstrual dating of gestational age dependent on maternal recall of the last menstrual
25    period can be subject to considerable measurement error in some cases, so ultrasonography-based
26    estimates may be considered more accurate (Savitzetal.. 2002: Taipale and Hiilesmaa. 2001).

27    Immune-related outcomes: allergy and asthma
28           Skin prick testing is a standard method for assessing atopy (allergic disease) used in some
29    epidemiologic studies. Other studies use an assessment protocol based on reported history of
30    symptoms (e.g., rhinitis, hay fever) or specific types of allergies. These can be considered
31    complementary types of measures: skin prick tests provide information on a defined set of
32    potential antigens to which a person may be exposed, and symptom-based evaluations provide
33    information on experiences of individuals and the variety of exposures they encounter. Studies
34    comparing questionnaire responses with skin prick tests in children have reported relatively high
35    specificity (89-96%) and positive predictive value (69-77%) for self-reported history of pollen or
36    pet dander allergy or for answers to a combination of questions incorporating itchy eyes with nasal
37    congestion in the absence of a cold or flu (Braun-Fahrlander etal.. 1997: Dotterudetal.. 1995).  The
38    validity was somewhat lower for a more restricted set of questions (nasal congestion in the absence
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    of a cold or flu; specificity 83%, positive predictive value 52%) [Braun-Fahrlander etal., 1997].
 2    Based on these data, EPA considers allergy history based only on rhinitis symptoms to have a
 3    greater likelihood of outcome misclassification compared with those based on a combination of
 4    symptoms.
 5           Epidemiologic studies of asthma typically use a questionnaire-based approach to define
 6    asthma based on symptoms relating to wheezing episodes or shortness of breath, reported history
 7    of asthma attacks, or use of asthma medication, usually for a period defined as "current" or in the
 8    past year. Much of this work is based upon the American Thoracic Society questionnaire [Ferris,
 9    1978] or subsequent instruments that built upon this work, including the International Society of
10    Arthritis and Allergies in Children Questionnaire and the European Community Respiratory Health
11    Survey. These questionnaire-based approaches have been found to have an adequate level of
12    specificity and positive predictive value for use in etiologic research [Ravault and Kauffmann. 2001:
13    Pekkanen and Pearce. 1999: Burneyetal.. 1989: Burney and Chinn. 1987]. EPA considers
14    outcomes defined over a recent time period (e.g., symptoms in the past 12 months] to be more
15    relevant within the context of concurrent exposure measurements compared with outcomes
16    defined over a lifetime (e.g., ever had asthma].

17    Neurodevelopment
18           With respect to neurodevelopmental outcomes, a major consideration is the assessment
19    tool(s] used by the study investigators; details of the assessment method, or references providing
20    this information, should be provided.  In addition, EPA also looks for discussion of (or reference to]
21    validation studies and the appropriateness of the tool for evaluation in the specific study population
22    (e.g., age range, language].

23    Thyroid
24           Thyroid-related endpoints examined in epidemiological studies of DIBP include thyroid
25    hormones (triiodothyronine, T3, and thyroxine, T4] and thyroid stimulating hormone (TSH] (or
26    thyrotropin] produced by the pituitary.
27           As with other hormone assays, the details of the laboratory procedures, including
28    information on the basic methods, limit of detection, and coefficient of variation, are important
29    considerations for the hormone assays. Thyroid hormones are generally measured in serum,
30    although they may also be measured in dried blood  spots, such as are collected from newborn
31    infants  in screening for congenital hypothyroidism.  Studies in older age groups have also shown a
32    very high correlation (r = 0.99] between thyroid hormone levels measured in dried blood spots and
33    levels in serum  (Hofman et al.. 2003].
34           With respect to thyroid hormones, time of day and season of sampling are two main
35    potential sources of variability. For example, serum TSH measured shortly after midnight may be
36    as much as twice as high as the value measured in late afternoon (Brabant etal., 1991: Weeke and
37    Gundersen. 1978]. The evidence with respect to seasonal variability is mixed (Plasqui etal.. 2003:
38    Nicolau etal.. 1992: Simonietal.. 1990: Behalletal.. 1984: Postmes etal..  1974] and this effect is
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    likely to be smaller than that of time of day.  The impact of these sources of variation will depend on
 2    whether they are also related to DIBP (i.e., whether DIBP levels vary diurnally or seasonally). If this
 3    is the case, failure to address these factors in the design or analysis could result in confounding of
 4    the observed association, with the direction of this bias determined by the direction of the
 5    association between these factors and DIBP. If this is not the case, the lack of consideration of time
 6    of day or seasonality would result in greater variability in the hormone measures, and would thus
 7    result in more imprecise (but not biased) estimates was located. EPA has not found studies
 8    examining seasonal variation in DIBP levels. With respect to variability relating to time of day, as
 9    noted previously, one study of 139 pregnant women in Puerto Rico reported lower concentrations
10    of specific gravity-adjusted MIBP in samples collected from 9 am to noon (geometric mean of 9.4)
11    compared with samples collected in early morning, early afternoon, or evening (geometric means of
12    13-14) (Cantonwine etal.. 2014). Based on these data, EPA has greater confidence in thyroid
13    hormone studies that consider time of sample collection in the analysis, but recognizes the limited
14    nature of the available data pertaining to this issue.

15    Obesity
16          Most of the studies of obesity measures in the DIBP database  are based on body mass index
17    (BMI, calculated as kg/m2) or waist circumference using measurements taken as part of the data
18    collection protocol. BMI is highly correlated with  body fat, and standardized cut-points have been
19    established for characterization of "normal" (BMI  between 18.5 and 24.9 kg/m2), "overweight"
20    (BMI between 25.0 and 29.9 kg/m2) and "obese" (BMI  > 30.0 kg/m2)  categories. Waist
21    circumference is also highly correlated with body fat, and is a more direct measure of abdominal
22    obesity.  EPA notes that use of self-reported weight (e.g., report of pre-pregnancy weight) would
23    not be considered to be as reliable as actual measurements.

24    Diabetes and measure of insulin resistance
25          In the DIBP database, diabetes has been assessed by a variety of biomarkers of glucose and
26    insulin and by self-report of diabetes diagnosis. Oral glucose tolerance testing and glycosolated
27    hemoglobin (HbAlc) are used clinically and in epidemiological research (Selvinetal., 2011). Self-
28    report of prevalent diabetes can have high sensitivity and specificity in comparison to diagnosed
29    diabetes based on validated medical record data (Oksanenetal., 2010: Leikauf and Federman,
30    2009). The biomarker-based classifications, however, offer an added advantage of being able to
31    include undiagnosed disease.  EPA will consider these points in assessing the reliability and validity
32    of the diabetes measures used in the studies. None of the currently available studies assessed
33    diabetes through cause of death data; sensitivity of diabetes assessed using cause of death data is
34    low, even if underlying and other contributing cause of death fields are included (Cheng etal.,
35    2008).
36          Insulin resistance, a marker of diabetes risk, can be measured using the homeostatic model
37    assessment (HOMA) method, a physiologically-based structural model, using fasting glucose and
38    insulin or C-peptide concentrations.  HOMA is a validated tool for the estimation of insulin
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    resistance in epidemiology studies, and requires a single measurement of fasting glucose and
 2    insulin [Wallace etal., 2004]. Although the mean of three samples taken at 5-minute intervals
 3    results in a more precise estimate, insulin resistance estimated using a single baseline
 4    measurement is well correlated with that using the mean of three measurements when used to
 5    estimate a group mean. Therefore, EPA does not consider the use of a single measurement as an
 6    input to the HOMA model to be a limitation.

 7    Cancer
 8          With respect to studies of cancer, EPA considers the source of the outcome data (e.g., cause
 9    of death data, hospital cancer registry data, hospital discharge data, histopathology reports) in its
10    evaluation of the accuracy of the data. An additional issue is the validity of mortality data as a
11    representation of cancer incidence; mortality data for cancer types with a high survival rate may
12    underrepresent disease incidence, require additional considerations with respect to determining
13    appropriate time windows of exposure, and may lead to biased risk estimates if survival is related
14    to exposure.

15    Confounding
16          The general considerations for evaluating issues relating to potential confounding include
17    consideration of which factors may be potential confounders (i.e., those which are strongly related
18    to both the exposure and the outcome under consideration, and are not intermediaries on a causal
19    pathway), adequate control for these potential confounders in the study design or analysis, and
20    where appropriate, quantification of the potential impact of mismeasured or unmeasured
21    confounders. Uncontrolled confounding by factors that are positively associated with both the
22    exposure (e.g., DIBP) and health endpoint of interest, and those that are inversely associated with
23    both exposure and health endpoint, will result in an upward bias of the effect estimate.
24    Confounding by factors that are positively associated with exposure and inversely associated with
25    the health endpoint (or vice versa) will result in a downward bias of the effect estimate.

26    Potential confounding by other phthalates
27          Few studies have reported results of analyses evaluating the correlation between MIBP and
28    metabolites of other phthalates. In an analysis conducted by EPA of 5,109 samples  from the
29    2003-2008 National Health and Nutrition Examination Survey (NHANES) participants aged >6
30    years, the pairwise Spearman correlation coefficient between MIBP and MEP (the primary
31    metabolite of DEP) was low (0.33). A more moderate correlation was seen with the DEHP
32    metabolites (correlations of approximately 0.5); higher correlations were seen with MBzP (the
33    primary metabolite of BBP, correlation coefficient = 0.58) and MBP (the primary metabolite of DBF;
34    correlation = 0.72). Similar or some what lower correlations were seen between MIBP and other
35    phthalate metabolites in a small study (n = 45) of men seen in an infertility clinic (Wirthetal.,
36    2008], in 319 pregnancy women (Whyattetal., 2012], and in 600 reproductive age women in a
37    study of endometriosis (Buck Louis etal.. 2013). EPA will evaluate the potential for confounding by
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    examining the similarity of the results seen with different metabolites. Thus, for example, lack of
 2    adjustment for mono-benzyl phthalate (MBzP) would not be considered a limitation in a study in
 3    which an association was seen with MIBP that was not seen with MBzP; however this lack of
 4    adjustment would be considered a limitation if an association of similar or higher magnitude was
 5    seen for both of metabolites.

 6    Potential confounding by demographic factors
 7           Age, race/ethnicity, and sex are considered important explanatory factors for most types of
 8    outcomes measured in epidemiological research. In NHANES 2009-2010 data, urinary MIBP levels
 9    decreased with age (geometric means of 13.2, 8.63, and 7.45 |ig/g-creatinine, respectively, in ages
10    6-11,12-19 and >20 years) [CDC, 2013]. Concentrations were lower levels in males compared
11    with females (geometric means of 6.99 and 9.05 |ig/g-creatinine, respectively, in males and
12    females), and variability by ethnicity was also observed, with lower levels in non-Hispanic whites
13    (geometric mean of 7.12 |ig/g-creatinine) compared with non-Hispanic blacks and Mexican
14    Americans (geometric means of 10.1 and 9.27 [ig/g-creatinine, respectively). EPA will consider
15    these differences in assessing the potential influence of demographic factors on observed effect
16    estimates for DIBP.

17    Potential confounding by other factors
18           Some of the health effects under consideration may have strong associations with other risk
19    factors. For example, smoking is associated with increased risk of low birth weight and preterm
20    births, and with infertility. Abstinence time is strongly related to sperm concentration measures.
21    In evaluating the potential for confounding by any of these factors, EPA will review evidence
22    pertaining to the strength and direction of its association with DIBP (or its metabolites).

23    Data An alysis
24           The general considerations for evaluating issues relating to data analysis include adequate
25    documentation of statistical assumptions and analytic approach (including addressing skewness of
26    exposure or outcome variable and shape of exposure-response), consideration of sample size and
27    statistical power, and use of appropriate statistical methods for the study design.
28           One other issue, specific to much of the DIBP literature, concerns the optimal approach to
29    addressing urinary volume or dilution in the analysis of spot urine or first morning void samples.
30    Options include use of creatinine- or specific gravity-adjusted metabolite concentrations, or use of
31    unadjusted concentrations. Although use of some kind of correction factor has been advocated for
32    studies of obesity (Goodman etal.. 2014). a simulation study reported that creatinine-adjusted
33    exposure measures may produce biased effect estimates for outcomes that are strongly related to
34    factors affecting creatinine levels, of which obesity is a prime example (Christensenetal., 2014).
35    EPA recognizes the lack of consensus at this time, as well as the need for continued research into
36    the potential bias introduced by different analytic approaches. Based on current understanding of
37    this issue, EPA prefers results using unadjusted concentration for outcomes strongly related to
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                   Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

1    creatinine levels; for other outcomes, EPA does not have a basis for preferring one type of analysis
2    over another.
3
4
Table 2-8. General and outcome-specific considerations for DIBP study
evaluation
General considerations
Study population
Exposure
Analysis
• Study population and setting: geographic area, site, time period, age and sex
distribution, other details as needed (may include race/ethnicity,
socioeconomic status)
• Recruitment process; exclusion and inclusion criteria, knowledge of study
hypothesis; knowledge of exposure and outcome
• Participation rates: total eligible; participation at each stage and for final
analysis group and denominators used to make these calculations
• Length of follow-up, loss to follow-up
• Comparability: participant characteristic data by group, data on non-
participants
• Biological matrix or target tissue/organ (e.g., urine, serum, semen, breast
milk)
• Level of detection (LOD) or level of quantitation (LOQ)
• Exposure distribution (e.g., central tendency, interquartile range), proportion

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                Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Steroidal and
gonadotropin
hormones (adults; sex-
specific)
  Measures
  Consideration of
  confounding
  Relevant exposure
  time window(s)
•   Type of assay
•   Sensitivity/detection limits, coefficient of variation; number of samples
    below LOD
•   Age, day or phase of menstrual cycle (if cycling)
•   Up to 6 months preceding hormone sample collection
Sperm parameters
  Measures
  Consideration of
  confounding

  Relevant exposure
  time window(s)
    Type of assay (e.g., WHO protocol)
•   Age, smoking, BMI, abstinence time (consider if these are related to
    exposure)
•   Up to 6 months preceding semen sample collection
Infertility
  Measures
  Consideration of
  confounding

  Relevant exposure
  timewindow(s)
•   Definition, source of data
•   Age, smoking, alcohol use, heavy metal exposure, radiation time (consider if
    these are related to exposure)
•   Time preceding and during attempt to become pregnant
Timing of puberty
  Measures
  Consideration of
  confounding

  Relevant exposure
  timewindow(s)
•   Source of data (e.g., self-report, physician assessment)
•   Age, sex, ethnicity, body size, nutritional status (consider if these are related
    to exposure)
•   In utero? Up to 12 months preceding transition from one stage to another
    stage?
Gestational age
  Measures

  Consideration of
  confounding

  Relevant exposure
  timewindow(s)
•   Source of data and estimation procedure (ultrasound; last menstrual period
    or clinical assessment)
•   Smoking, pregnancy complications, assisted reproduction technologies
    (consider if these are related to exposure)
•   In utero
Birth weight
  Measures
  Consideration of
  confounding

  Relevant exposure
  timewindow(s)
•   Source of data (e.g., medical records, birth certificate)
•   Gestational age, maternal age, ethnicity, nutritional intake, smoking,
    maternal height/BMI, (consider if these are related to exposure)
•   In utero
Immune - allergy and
asthma
  Measures
  Consideration of
  confounding
•   Number of allergens used in skin prick testing or allergen-specific IgE assay;
    sensitivity/specificity of specific questions used in history assessment
•   Age, family history (consider if these are related to exposure)
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
        Relevant exposure
        time window(s)
•   For current conditions (e.g., asthma in past 12 months): up to 12 months
    preceding outcome assessment
      Ne urob ehavioral

        Measures
        Consideration of
        confounding
        Relevant exposure
        time window(s)
•  Standardized assessment tool, validation studies for specific study
   population (e.g., age group, geographic location)

•  Blinding of assessor to exposure
•  Age, sex, socioeconomic status
•   In utero; early childhood
      Thyroid

        Measures
        Consideration of
        confounding

        Relevant exposure
        time window(s)
•  Assay used and evidence from validation studies, if available

•  Sensitivity/detection limits, coefficient of variation; number of samples
   below LOD

•  Time of day and season when samples for thyroid hormone (and TSH)
   collected
•  Age, sex, smoking, iodine, radiation exposure (consider if these are related
   to exposure)
•  Varies by lifestage (i.e., infants, children, adults)
      Obesity
        Measures
        Consideration of
        confounding

        Relevant exposure
        time window(s)
•  Source of data (e.g., measured or self-reported weight and height)
•  Age, sex, ethnicity, caloric intake, physical activity (consider if these are
   related to exposure)
•   Not established (likely to be more than one, including in utero)
      Diabetes and insulin
      resistance
        Measures


        Consideration of
        confounding
        Relevant exposure
        time window(s)
•  Source of data (e.g., biomarkers of insulin or glucose, medical records, self-
   report)
•  Age, sex, ethnicity
•   Not established (likely to be more than one, including in utero)
2    2.4. STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE
3         FUTURE EVALUATION AND SYNTHESIS OF THE CRITICAL
4         EXPERIMENTAL STUDIES FOR DIBP
5           Beyond the initial methodological screening described above in Section 2.2.2,
6    methodological aspects of a study's design, conduct, and reporting will be considered again in the
7    overall evaluation and synthesis of the pertinent data that will be developed for each health effect.
8    Some general questions that will be considered in evaluating experimental animal studies are
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
 1    presented in Table 2-9. These questions are, for the most part, broadly applicable to all
 2    experimental studies.
 3
 4
       Table 2-9. Questions and relevant experimental information for the
       evaluation of experimental animal studies
Methodological
feature
Test animal
Experimental setup
Exposure
Endpoint evaluation procedures
Outcomes, data, and reporting
Question(s) considered
Based on the endpoint(s) in question, are concerns raised regarding the
suitability of the species, strain, or sex of the test animals on study?
Are the timing, frequency and duration of exposure, as well as animal age
and experimental group allocation procedures/group size for each endpoint
evaluation, appropriate for the assessed endpoint(s)?
Are the exposure conditions and controls informative and reliable for the
endpoint(s) in question, and are they sufficiently specific to the compound
of interest?
Do the procedures used to evaluate the endpoint(s) in question conform to
established protocols, or are they biologically sound? Are they sensitive for
examination of the outcome(s) of interest?
Were data reported for all pre-specified endpoint(s) and study groups, or
were any data excluded from presentation/analyses?
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
Note: "Outcome" refers to findings from an evaluation (e.g., steatosis), whereas "endpoint" refers to the
 evaluation itself (e.g., liver histopathology).

       Evaluation of some specific methodological features identified in Table 2-9 such as
exposure, is likely to be relatively independent of outcome.  Other methodological features, in
particular those related to experimental setup and endpoint evaluation procedures, are generally
outcome specific (i.e., reproductive and developmental toxicity). In general, experimental animal
studies will be compared against traditional assay formats (e.g., those used in guideline studies),
with deviations from the protocol evaluated in light of how the deviations could alter interpretation
of the outcome in question.  A full evaluation of all critical studies will be performed as part of the
critical review and synthesis of evidence for hazard identification for each of the health endpoints
identified in the evidence tables presented in Section 3.
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                   Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1
 2   3.   PRELIMINARY EVIDENCE TABLES AND
 3         EXPOSURE-RESPONSE ARRAYS
 4   3.1.  DATA EXTRACTION FOR EPIDEMIOLOGICAL AND EXPERIMENTAL
 5         STUDIES: PREPARATION OF PRELIMINARY EVIDENCE TABLES
 6         The evidence tables present data from studies related to a specific outcome or endpoint of
 7   toxicity. At a minimum, the evidence tables include the relevant information for comparing key
 8   study characteristics such as study design, exposure metrics, and dose-response information.
 9   Evidence tables will serve as an additional method for presenting and evaluating the suitability of
10   the data to inform hazard identification for DIBP during the analysis of hazard potential and utility
11   of the data for dose-response evaluation. For each critical study selected, key information on the
12   study design, including characteristics that inform study quality, and study results pertinent to
13   evaluating the health effects from subchronic and chronic oral exposure to DIBP are summarized in
14   preliminary evidence tables.
15         Epidemiological studies are presented first where each study per table is listed in reverse
16   chronological order. Animal studies are then presented where each study per health endpoint is
17   presented in alphabetical order by study author, followed by species and strain. Most results are
18   presented as the percent change from the control group; an asterisk (*) indicates a result that has
19   been calculated and reported by study authors to be statistically significant compared to controls
20   (p < 0.05). Unless otherwise noted in a footnote, doses presented in the animal evidence tables
21   were those reported by the study authors.
22         The information in the preliminary evidence tables is also displayed graphically in
23   preliminary exposure-response arrays. In these arrays, a significant effect (indicated by a filled
24   circle) is based on statistical significance by the study authors. The complete list of references
25   considered in preparation of these materials  can be found on the Health and Environmental
26   Research Online (HERO) website at (https://hero.epa.gov/DIBP and
27   http://hero.epa.gov/phthalates-humanstudies).
28
              This document is a draft for review purposes only and does not constitute Agency policy.
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
l    3.2. EPIDEMIOLOGICAL STUDIES

2    3.2.1.  Sexual Differentiation Measures

3           Table 3-1. Evidence pertaining to DIBP and sexual differentiation effects in
4           humans
               Reference and study design
                     Results
     Anogenital distance (AGD)
      Swan (2008) (United States; Minnesota, Missouri,
      California)
      Population:  106 boys from birth cohort study (Study
      for Future Families), 2000-2002, mean age 12.8 mo
      (0-36 mo)
      Outcome: AGD (to posterior genitalia) measured at
      0-36 mo (mean 70.4 mm, 7.1 mm/kg)
      Exposure: Maternal urine sample, 3rd trimester
      MIBP in urine (ng/mL):
                 Median  75th percentile
      Unadjusted    2.5         5.1
      Analysis: Regression analysis using mixed model
      adjusting for age and weight percentile
      Related references: Swan et al. (2005) (exposure
      data and analysis of smaller sample size with less
      robust method of adjustment for variation by size)
Percent change in AGD per interquartile increase in MIBP
concentration (p-value)
MIBP
-3.5 (0.097)
      Cryptorchidism or testicular position
      Swan (2008) (United States; Minnesota, Missouri,
      California)
      Population:  106 boys from birth cohort study (Study
      for Future Families), 2000-2002, mean age 12.8 mo
      (0-36 mo)
      Outcome: Incomplete testicular descent assessed at
      clinical exam (10% prevalence)
      Exposure: Maternal urine sample, 3rd trimester
      MIBP in urine (ng/mL):
                 Median   75th percentile
      Unadjusted    2.5         5.1
      Analysis: Logistic regression, adjusting for age and
      weight percentile
      Related references: Swan et al. (2005) (exposure
      data)
MIBP reported as not associated with testicular position
(quantitative results not reported)
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                Reference and study design
                                                                       Results
      Gender-related play
      Swan et al. (2010) (United States; Minnesota,
      Missouri, California, Iowa)
      Population: 145 children from birth cohort study
      (Study for Future Families), 2000-2002 and
      2002-2005 (Iowa), ages 4-7 yrs; second follow-up
      Outcome:  Gender-specific play based on Pre-School
      Activities Inventory (24 items completed by parent or
      caregiver; subscores of male-oriented items and
      female-oriented items and a composite score
      consisting of male summation minus the female
      summation scores)
      Exposure:  Maternal urine sample, 3rd trimester
      Unadjusted MIBP in urine (ng/mL):
               Median  75th percentile
      Boys       2.4          5.1
      Girls       2.8          5.0
      Analysis:  Regression analysis using Generalized
      Linear Models, considering creatinine, sex and age of
      child, maternal age, parental education, number of
      same and opposite sex siblings, ethnicity, clinic
      location, and parental  attitude as potential covariates
      Related references: Swan et al. (2005) (exposure
      data)
                                                 Regression coefficient (95% Cl) for pre-school activities
                                                 index scores and log-transformed MIBP (adjusted for
                                                 child's age, mother's age, mother's education, parents'
                                                 attitude toward boy's play, and interaction between
                                                 education and attitude; negative value indicates less
                                                 masculine play behavior with higher metabolite level)
                                                                       Boys                Girls
                                                 Masculine       -1.65 (-4.57, 1.28)    1.04 (-1.75, 3.82)
                                                 Composite      -4.53 (-8.12,-0.94)   0.38 (-3.86, 4.63)
1
2
Cl = confidence interval; MIBP = monoisobutyl phthalate
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3.2.2.   Male Reproductive Effects in Humans

        Table 3-2. Evidence pertaining to DIBP and semen parameters or infertility in
        adult men or couples
             Reference3 and study design
                                                                    Results
      Kranvogl et al. (2014) (Slovenia)
      Population:  136 men from couples seeking
      infertility treatment (mean age 36.2 yrs, range
      24-54 yrs), 2012
      Outcome: Semen analysis
      Exposure: Urine sample, collected at same time
      as semen sample
      MIBP in urine:
                       Median  Maximum
      Unadjusted (u.g/L)    21.6     161.8
      Cr-adjusted (u.g/g Cr)  20.8     119.2
      Analysis: Spearman correlation
                                            Spearman correlation coefficient, MIBP and sperm
                                            parameters:
                                            Sperm concentration
                                            Sperm motility
                                            (p > 0.05 for both parameters)
-0.044

-0.075
      Joensen et al. (2012) (Denmark)
      Population:  881 men from general population,
      assessed at military conscript exam*, 2007-2009,
      median age 19.1 yrs (5th-95th percentile:
      18.4-22.0 yrs)
      Outcome: Semen analysis
      Exposure: Urine sample, collected at same time
      as semen sample
      MIBP in urine (ng/mL):
                 Median  95th percentile
      Unadjusted    58         173
      Analysis:  Linear regression, considering age,
      BMI, smoking, alcohol consumption, ethnicity,
      BMI squared, in utero exposure to tobacco
      smoke, previous or current diseases, recent
      fever, recent use of medication, abstinence time,
      and time from ejaculation to analysis as potential
      covariates
      *As reported by Ravnborg et al. (2011)
                                            Results for individual phthalate metabolites (including MIBP)
                                            reported as "few significant associations" with sperm volume,
                                            count, or percentage progressively motile sperm (quantitative
                                            results not reported). Sperm concentration analysis adjusted
                                            for abstinence time (volume, concentration, and count);
                                            sperm motility analysis adjusted for time from ejaculation to
                                            analysis (progressively motile); analysis of percent of
                                            morphologically normal sperm was unadjusted
      Wirth et al. (2008) (United States, Michigan)
      Population:  45 male partners seen in infertility
      clinic, time period not reported; mean age 34 yrs
      Outcome: Semen analysis
      Exposure: Urine sample, collected at same time
      as semen sample (all between 7 and  11 am)
      MIBP in urine (ng/mL) (percentile):
          Median  75th percentile  95th percentile
            5.8        10.0           17.9
      Analysis:  Dichotomized outcomes (above and
      below WHO  reference values), MIBP
      dichotomized at median; age, education (three
      levels), income (three levels), race, BMI (three
      levels), current smoking status, and alcohol use
                                            The combined measure for MIBP and MBP was not associated
                                            with any sperm parameter, nor was MIBP when analyzed
                                            individually (data not shown)
                This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
             Reference3 and study design
                                                                    Results
      (two levels) considered as potential confounders;
      specific gravity also included in all models
      Infertility
      Buck Louis et al. (2014) (United States; Michigan
      and Texas)
      Population:  501 couples discontinuing
      contraception and attempting to achieve
      pregnancy; recruited from 16 counties using
      population sampling; women's mean age
      30.0 yrs, men's mean age 31.8 yrs; 2005-2009
      Outcome: Time to pregnancy as assessed by
      diaries recording intercourse and menstruation,
      home-fertility monitoring to detect ovulation,
      and home pregnancy tests
      Exposure: Urine samples from both partners,
      collected at enrollment (beginning of pregnancy
      attempt)
      Unadjusted MIBP in urine (ng/mL) among
      couples achieving pregnancy:
             Geometric mean (95% Cl)
      Women     5.11(4.58-5.70)
      Men        3.44 (3.09-3.83)
      Analysis:  Fecundability OR calculated using Cox
      models, adjusting for variables shown in results
      column
                                             Fecundability OR (95% Cl) for increase in log-transformed
                                             MIBP scaled by standard deviation (adjusted for female age,
                                             difference in couple's ages, research site, and both partners'
                                             urinary creatinine, BMI, and serum cotinine; in addition,
                                             results for exposure in each partner adjusted for exposure in
                                             the other partner, and models accounted for left truncation or
                                             time off contraception)
                                             Women
                                             Men
0.97 (0.80,1.18)
0.91 (0.76, 1.09)
1
2
3
BMI = body mass index; MBP = monobutyl phthalate; OR = odds ratio; WHO = World Health Organization
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1
2
        Table 3-3. Evidence pertaining to DIBP and reproductive hormones in adult
        men
                   Reference and study design
                                                                         Results
      Joensen et al. (2012) (Denmark)
      Population: 881 men from general population, assessed at
      military conscript exam*, 2007-2009, median age 19.1 yrs
      (5th-95th percentile: 18.4-22.0 yrs)
      Outcome:  Serum steroidal and gonadotropin hormones
      Exposure:  Urine sample, collected at same time as serum
      sample for hormone analysis
      MIBP in urine (ng/mL):
                  Median   95th percentile
      Unadjusted     58        173
      Analysis:  Linear regression considering age, BMI, smoking,
      alcohol consumption, time of blood sampling, assay type,
      ethnicity, BMI squared, in utero exposure to tobacco smoke,
      previous or current diseases, recent fever, and recent use of
      medication as potential covariates
      *As reported by Ravnborg et al. (2011)
                                                       Results for individual phthalate metabolites
                                                       (including MIBP) reported as "few significant
                                                       associations" with free testosterone, estradiol,
                                                       SHBG, LH, inhibin-B, or FSH (quantitative results
                                                       not reported); analyses adjusted for age, BMI,
                                                       smoking, alcohol consumption, and time of blood
                                                       sampling (and assay type for inhibin-B only)
      Mendiola et al. (2011) (United States; Minnesota, Missouri,
      California, Iowa, New York)
      Population: 425 men whose partners enrolled in birth
      cohort study (Study for Future Families), 1999-2005, mean
      age 32 yrs
      Outcome:  Serum steroidal and gonadotropin hormones
      Exposure:  Urine sample, collected at same time as serum
      sample for hormone analysis
      MIBP in urine (ng/mL) (distribution not reported)
      Analysis:  Pearson correlation of log(10)-transformed MIBP
      and hormone measures; linear regression considering age,
      age square, BMI, smoking status, ethnicity, urinary
      creatinine concentration, time of sample collection, time of
      collection squared, season, educational level, center, and
      stressful life events)
                                                      Authors reported "little or no association with
                                                      metabolites of phthalate other than DEHP"
                                                      [including MIBP] with testosterone, estradiol,
                                                      SHBG, LH, inhibin-B, or FSH (quantitative results
                                                      not reported)
3
4
5
6
DEHP = diethylhexyl phthalate; FSH = follicle-stimulating hormone; LH = luteinizing hormone; MOINP = oxo-(mono-
 oxoisononyl) phthalate; SD = standard deviation; SHBG = sex hormone binding globulin
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3.2.3.  Male Pubertal Development in Humans

        Table 3-4. Evidence pertaining to DIBP and the timing of male puberty or sex
        hormones in boys
               Reference and study design
                                                                      Results
      Ferguson et al. (2014c) (Mexico)
      Population: 115 boys ages 8-14 yrs from a birth
      cohort (Early Life Exposure in Mexico to
      Environmental Toxicants, participants enrolled
      during first trimester 1994-2004), follow-up
      initiated in 2010
      Outcome:  Adrenarche or puberty, based on Tanner
      staging by physician (pubic hair stage >2; genitalia
      stage >2 or testicular volume >3 mL); serum
      hormone level
      Exposure:  Maternal urine sample (n = 107) from
      third trimester or child's urine sample (n = 113)
      collected at time of Tanner staging and serum
      collection
      Unadjusted MIBP in urine (ng/mL):
                      Median   95th percentile
      Maternal sample   1.83         6.64
      Child's sample     9.61         36.1
      Analysis: Logistic regression for analysis of puberty
      onset, adjusting for variables shown in results
      column; linear regression for analysis of hormone
      levels, considering age, BMI z-score, socioeconomic
      status, and maternal smoking as potential
      covariates
                                               OR (95% Cl) for adrenarche or puberty per interquartile
                                               increase in In-transformed MIBP (adjusted for child age,
                                               BMI z-score, and urine specific gravity)
                                                                          Exposure basis
                                               Tanner stage or
                                               testicular
                                               volume
                 Maternal urine
                   (prenatal)
                                               Pubic hair
                                               (stage >2)
                0.29 (0.07, 1.30)


Genitalia (stage   0.71 (0.37,1.35)
                                               Testicular        1.60 (0.70, 3.65)
                                               volume (>3 mL)
  Child urine

0.76 (0.32,1.81)


0.76 (0.39, 1.49)


2.17(0.81,5.82)
                                               Percent change (95% Cl) in serum hormone level per
                                               interquartile increase in In-transformed MIBP (adjusted for
                                               urine specific gravity, child age, and BMI z-score)

                                                                          Exposure basis

                                               Serum            Maternal urine
                                               hormone           (prenatal)          Child urine

                                               Testosterone    5.12  (-23.3, 44.0)   -26.2 (-45.6, 0.16)

                                               Free            1.69  (-26.7, 41.1)   -27.9  (-47.8,-0.60)
                                               testosterone

                                               SHBG           5.72  (-5.18, 17.9)    2.20  (-8.41, 14.1)

                                               DHEAS          -2.02 (-15.9, 14.1)   3.02  (-11.4, 19.8)

                                               Estradiol        -1.94 (-11.2, 8.23)  -12.3  (-20.2, -3.54)

                                               Inhibin B        -1.98 (-12.7,10.1)   2.73  (-8.24,15.0)
      Mouritsen et al. (2013b) (Denmark)
      Population:  Boys from population-based cohort
      (COPENHAGEN Puberty Study), 2006-2010; age
      11 yrs (53 boys) or 13 yrs (31 boys)
      Outcome: Adrenarche or puberty, based on Tanner
      staging by physician (pubarche = pubic hair stage >2
      and testicular volume >3 mL); serum hormone level
      Exposure: Urine sample, first morning sample; data
      reported in Mouritsen et al. (2013a, Supplemental
      Material)
                                               Median age (yrs) at development by ZMIBP + MBP level
                                               (evaluation at 11 yrs)
                                               Pubarche
                                               Testicular volume >3 mL
                            Low
                            12.3
                            11.5
       High
   11.0 (p< 0.05)
       11.1
                                               Median hormone concentration by MIBP + MBP level
                                               (evaluation at 11 yrs)
                                                    Testosterone (nmol/L)

                                                    DHEAS (nmol/L)
                                                                           Low

                                                                          <0.23

                                                                           2.02
                                          High

                                          <0.23

                                          1.61
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
MIBP + MBP in urine (ng/mL)a:
Geometric mean Maximum
118 676
(based on larger sample of 84 boys)
Analysis: Two-tailed Mann-Whitney U-test for
comparisons between groups, comparing median
hormone levels and pubertal stage in "high" and
"low" phthalate groups (based on above or below
group mean excretion)











Mieritz et al. (2012) (Denmark)
Population: 38 boys with pubertal gynecomastia
and 190 age-matched controls drawn from 555 boys
from population-based cohort (COPENHAGEN
Puberty Study), 2006-2008; ages 6-19 yrs
Outcome: Anthropometry, pubertal stage (pubic
hair and genital development), presence of
gynecomastia, and serum testosterone
Exposure: Urine sample, first morning sample
MIBP in urine (ng/mL):
Median 95th percentile
Groups 74.88 229.1
(boys without gynecomastia, all ages)
Analysis: Two-tailed Mann-Whitney U-test for
comparisons between groups; linear regression with
age adjustment for association with serum
testosterone; probit analysis with phthalate
concentrations divided in quartiles for analysis of
puberty timing
Results
Adione (nmol/L) 1.28 1.22
Estradiol (pmol/L) <18 <18
FSH (IU/L) 1.28 1.68
LH (IU/L) 0.28 0.27

Median age (yrs) at development by MIBP + MBP level
(evaluation at 13 yrs)
Low High
Pubarche 12.5 12.1
Testicular volume >3 mL 11.6 11.6
Median age (yrs) at development by MIBP + MBP level
(evaluation at 13 yrs)
Low High
Testosterone (nmol/L) 5.1 7.7
DHEAS (u.mol/L) 2.61 3.64
Adione (nmol/L) 2.96 3.85
Estradiol (pmol/L) 19 37
FSH (IU/L) 2.4 2.5
LH (IU/L) 1.8 1.4
MIBP concentration (ng/mL) by group
Group 1 Group 2 Group 3
(n = 38) (n = 189) (n = 517)
Median 68.50 73.96 74.88
95th percentile 178.8 199.5 229.1
Group 1 = boys with palpable gynecomastia
Group 2 = boys without palpable gynecomastia (age-
matched)
Group 3 = boys without palpable gynecomastia (all ages)

No association between MIBP concentration and timing of
puberty or serum testosterone level (quantitative results
not reported)




1
2
3
4
5
6
7
aln this population at this time, MIBP tended to be present at higher concentrations than MBP; EPA includes these
 studies in the DIBP tables, but recognizes the exposure misclassification introduced by the use of the summed
 concentration exposure measure.

DHEAS = dehydroepiandrosterone; EPA = Environmental Protection Agency
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3.2.4.   Female Pubertal Development in Humans

        Table 3-5. Evidence pertaining to DIBP and timing of female puberty or sex
        hormones in girls
               Reference and study design
                                                                      Results
      Precocious puberty and premature thelarche
      Frederiksen et al. (2012) (Denmark)
      Population: 24 girls with precocious puberty (n = 13
      with central precocious puberty, n = 6 with early
      normal puberty, n = 5 with premature thelarche)
      from outpatient clinic, 2008-2009 and 184* age-
      matched controls from population-based cohort
      (COPENHAGEN Puberty Study), recruited from high
      schools 2006-2008; age 7.4-9.9 yrs
      Outcome:  Precocious puberty, early normal
      puberty, or premature thelarche based on Tanner
      staging by physician
      Exposure:  Urine sample (child's), first morning
      sample collected at clinical evaluation
      MIBP and MBP in urine (ng/mL)a, controls (analysis
      based on sum of these two metabolites):
            Median  95th percentile
      MIBP     81         241
      MBP      51         153
      (based on larger sample of 725 controls)
      Analysis: Urine concentrations in cases and controls
      compared with Mann-Whitney U test
      *Study reports number of controls inconsistently;
      text reports 164 controls, while Table 4 reports 184
                                               Median (range) ZMIBP and MBP metabolites in urine
                                               (ng/mL) in cases and controls
                                                    Controls
                                                 147 (22-2,195)
      Precocious
       puberty
     94 (32-383)
(p-value)
(p<0.01)
      Lomenick et al. (2010) (United States, Ohio and
      Kentucky)
      Population: 28 girls with central precocious
      puberty, 28 age- and race-matched controls; all
      recruited from pediatric endocrinology clinic,
      2005-2008; mean age 7 yrs
      Outcome:  Central precocious puberty defined
      based on clinical standards (appearance of physical
      characteristics of puberty before 8 yrs of age, with
      laboratory confirmation of central origin of breast
      development); no cases had received medical
      treatment prior to urine sample collection
      Exposure:  Urine sample (child's), collected at
      clinical evaluation
      MIBP in urine of controls:
                          MeaniSE
      Unadjusted (ng/mL)   22.6 ± 7.6
      Cr-adjusted (ng/g  Cr)   20.2 ± 4.9
      Analysis: MIBP concentrations in cases and controls
      compared with Wilcoxon rank-sum test
                                               Unadjusted
                                               (ng/mL)
                                               Cr-adjusted
                                                    Cr)
             Central
            precocious
 Controls     puberty      (p-value)
22.6 ±7.6    15.4 ±2.9      (0.77)


20.2 ±4.9    16.5 ±2.1      (0.96)
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                Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
          Reference and study design
                      Results
Pubertal development (general population)
Hart et al. (2013) (Australia)
Population: 121 girls from birth cohort study
(Western Australian Pregnancy Cohort), whose
mothers were recruited at 18 wks of gestation,
1989-1991; follow-up at ages 14-16 yrs
Outcome: Age at menarche
Exposure: Maternal serum samples (n = 123)
collected at 18 and 34-36 wks of gestation
(combined aliquot from both time periods)
MIBP in serum (ng/mL):
             Median   90th percentile
Unadjusted    1.77          6.16
Analysis: Correlation between log-transformed
MIBP and age at menarche
Authors reported no association between MIBP and age at
menarche (quantitative results not reported)

Authors reported no correlation between MIBP and serum
SHBG, FSH, total testosterone, free androgen index, anti-
Miillerian hormone, or inhibin B in adolescents
(quantitative results not reported by study authors)
Mouritsen et al. (2013b) (Denmark)
Population: Girls from population-based cohort
(COPENHAGEN Puberty Study), 2006-2010; age
10 yrs (47 girls) and 13 yrs (33 girls)
Outcome:  Adrenarche or puberty, based on Tanner
staging by physician (pubarche = breast stage >2 and
pubic hair stage >2); serum hormone level
Exposure:  Urine sample, first morning sample; data
reported in Mouritsen et al. (2013a, Supplemental
Material)
MIBP + MBP in urine (ng/mL)a:
      Geometric mean    Maximum
            122            904
(based on larger sample of 84 girls)
Analysis: Two-tailed Mann-Whitney U-test for
comparisons between groups, comparing median
hormone levels and pubertal stage in "high" and
"low" phthalate groups (based on above or below
group mean excretion)
Median age (yrs) at development by MIBP + MBP level
(evaluation at 10 yrs)
Pubarche (pubic hair stage
Pubarche (breast stage >2)
 Low
 10.7


 10.6
 High
 11.2


 10.3
Median hormone concentration by MIBP + MBP level
(evaluation at 10 yrs)
Testosterone (nmol/L)

DHEAS (u.mol/L)

Adione (nmol/L)

Estradiol (pmol/L)

FSH (IU/L)

LH (IU/L)
 Low

<0.23

 1.1

 2.03

 20

 1.86

 0.06
 High

<0.23

 0.83

 1.29

 22

 2.25

 0.1
                                               Median age (yrs) at development by MIBP + MBP level
                                               (evaluation at 13 yrs)
                                               Pubarche (pubic hair stage
                                               Pubarche (breast stage >2)
                              Low
                              10.7


                              10.7
              High
              11.2


              10.5
                                               Median hormone concentration by MIBP + MBP level
                                               (evaluation at 13 yrs)
                                               Testosterone (nmol/L)

                                               DHEAS (u.mol/L)

                                               Adione (nmol/L)
                              Low

                              1.1

                              2.23

                              6.40
              High

          0.5 (p< 0.05)

         1.27 (p< 0.05)

              3.91
           This document is a draft for review purposes only and does not constitute Agency policy.
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1
2
3
4
5
Reference and study design
Frederiksen et al. (2012) (Denmark)
Population: 725 healthy girls ages 5.6-19.1 yrs from
COPENHAGEN Puberty Study cohort, recruited from
high schools during 2006-2008
Outcome: Stage of breast or pubic hair
development based on Tanner staging by physician;
Serum steroid and gonadotropin hormones
Exposure: Urine sample (child's), collected at time
of pubertal stage assessment
Unadjusted MIBP and MBP in urine (ng/mL)a, all
725 participants:
Median 95th percentile
MIBP 81 241
MBP 51 153
Analysis: Probit analysis, results verified using Pool-
Adjacent-Violators algorithm
Results
Estradiol (pmol/L) 194 131
FSH(IU/L) 4.9 5.8 (p< 0.05)
LH (IU/L) 3.8 3.8
Mean age (95% Cl) (yrs) at entry into breast stage 2 or
pubic hair stage 2, by quartile of JMIBP + MBP
metabolites:
IMIBP +
MBP Breast stage 2 Pubic hair stage
quartile (n = 394) (n not reported)
l(low) 10.12(9.61,10.62) 10.83(10.54,11.12)
2 9.97 (9.48, 10.46) 10.97 (10.67, 11.28)
3 9.89 (9.40, 10.37) 11.22 (10.93, 11.52)
4 (high) 9.79 (9.30, 10.30) 11.54*(11.21, 11.88)
*Significantly different from quartile 1; p < 0.05
Levels of FSH, LH, estradiol, and testosterone were similar
across JMIBP + MBP metabolite exposure groups when
adjusted for age distribution (quantitative results not
reported)
aln this population at this time, MIBP tended to be present at higher concentrations than MBP; EPA includes these
studies in the DIBP tables, but recognizes the exposure misclassification introduced by the use of the summed
concentration exposure measure.
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-11            DRAFT—DO NOT CITE OR QUOTE

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2
3
                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.2.5.   Female Reproductive Effects in Humans

        Table 3-6. Evidence pertaining to DIBP and reproductive hormones in adult
        women
                  Reference and study design
                                                                        Results
      Maternal hormones during pregnancy
      Sathvanaravana et al. (2014) (United States; Minnesota,
      Missouri, California)
      Population:  180 mothers from birth cohort (Study for
      Future Families), recruited during pregnancy, 1999-2002
      Outcome: Serum hormone levels, samples collected
      during prenatal clinic visit
      Exposure: Maternal urine sample, collected during 2nd or
      3rd trimester
      MIBP in urine (ng/mL):
                  Median   75th percentile
      Unadjusted    2.7         4.85
      Analysis: Linear regression, log-transformed MIBP and log-
      transformed hormone level
                                                     Regression coefficient (95% Cl) for change in
                                                     maternal log-transformed serum hormone level
                                                     with unit increase in log-transformed MIBP,
                                                     stratified by sex of fetus
                                                    Testosterone
                                                    (total)
                                                    Testosterone
                                                    (free)
                                                    Estradiol
Mothers with
 male fetus
  (n = 94)

   -0.03
(-0.18,0.13)

   -0.03
(-0.20,0.14)

   0.003
(-0.12,0.12)
 Mothers with
 female fetus
    (n = 86)

    -0.10
 (-0.28, 0.07)

    -0.11
 (-0.30, 0.08)

     0.03
 (-0.14,0.20)
      Hart et al. (2013) (Australia)
      Population:  123 mothers from birth cohort (Western
      Australian Pregnancy Cohort), whose mothers were
      recruited at 18 wks of gestation between 1989 and 1991
      Outcome: Reproductive and gonadotropin hormone levels
      in maternal serum collected at 18 and 34-36 wks of
      gestation
      Exposure: Maternal serum samples (n = 123) collected at
      18 and 34-36 wks of gestation (combined aliquot from
      both time periods)
      MIBP in serum (ng/mL):
                  Median    90th percentile
      MIBP         1.77           6.16
      Analysis: Correlation  between quartiles of serum MIBP
      and log-transformed hormone levels
                                                     Correlation coefficient between log-transformed
                                                     maternal serum hormone level and quartiles of
                                                     MIBP in maternal serum
                                                    Androstene-
                                                    dione (nmol/L)

                                                    DHEAS (u.mol/L)

                                                    Testosterone
                                                    (pmol/L)

                                                    SHBG (nmol/L)

                                                    Free
                                                    testosterone
                                                    (pmol/L)

                                                    Free
                                                    testosterone
                                                    index
At 18 wks of
  gestation
  (n = 119)

   0.023
   -0.042

   0.003


   0.108

   -0.061



   -0.051
At 34-36 wks of
   gestation
   (n = 114)
    -0.060
    -0.084

    -0.101


    -0.020

    -0.063



    -0.064
                                                         p > 0.10 for all correlations
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                    3-12              DRAFT—DO NOT CITE OR QUOTE

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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1
2
Table 3-7. Evidence pertaining to DIBP and gynecological conditions in
humans
                   Reference and study design
                                                                  Results
      Endometriosis
      Buck Louis et al. (2013) (United States, California and Utah)
      Population: 473 women undergoing laparoscopy or
      laparotomy and 127 population age- and residence-matched
      referents, 2007-2009; ages 18-44 yrs; confirmed cases of
      endometriosis matched to women without endometriosis
      within each cohort:  operative cohort 190 cases,
      238 controls; population cohort 14 cases,  127 controls
      Outcome:  Endometriosis confirmed by surgery (operative
      cohort) or MRI (population cohort)
      Exposure:  Urine sample
      MIBP in urine (ng/mL), unadjusted:
                            Geometric mean
      Operative cohort-controls      6.82
      Population cohort-controls     7.59
      Analysis: Student's t-test or Wilcoxon test for continuous
      data; logistic regression, adjusting for age, BMI, and
      creatinine; sensitivity analyses conducted  restricting cohort
      to endometriosis stages 3 and 4 diagnoses or visually and
      histologically confirmed endometriosis, and referent group
      consisting of women with postoperative diagnosis of normal
      pelvis
                                               OR (95% Cl) for endometriosis per unit increase in
                                               In-MIBP, by cohort (adjusted for age, BMI, and
                                               creatinine)
                                               Operative cohort

                                               Population cohort
1.02 (0.80,1.29)

2.22 (0.98, 5.04)
                                               Adjusted OR (95% Cl) for endometriosis per unit
                                               increase in In-MIBP in operative cohort (sensitivity
                                               analysis)
                                               Endometriosis stage 3 and 4
                                               (n = 339)

                                               Visual/histological confirmed
                                               endometriosis (n = 473)

                                               Comparison with women with
                                               postoperative diagnosis
                                               normal pelvis (n = 320)
   0.96 (0.67, 1.38)


   1.08 (0.77, 1.51)


   1.09 (0.82, 1.46)
                                               Note: Concentrations were log transformed and
                                               rescaled by their SDs for analysis
      Upson et al. (2013) (United States, Washington)
      Population: 92 incident endometriosis cases, 195 controls
      frequency-matched on age, all members of a large health
      care system and enrolled in Women's Risk of Endometriosis
      Study, 1996-2001; ages 18-49 yrs
      Outcome:  Endometriosis confirmed by surgery; for each
      case, reference date assigned by date of first visit for
      symptoms leading to diagnosis; reference dates randomly
      assigned to controls based on case distribution
      Exposure:  Urine sample, collected after enrollment
      (2001-2002)
      MIBP in urine, controls:
                        Median 75th percentile
      Unadjusted (ng/mL)  1.5        3.1
      Analysis: Logistic regression (quartiles of exposure),
      covariates considered based on directed acyclic graph; final
      model adjusted for variables shown in  results column
                                               OR (95% Cl) for endometriosis by quartile MIBP
                                               (adjusted for In-transformed urinary creatinine,
                                               age, and reference yr)
                                               MIBP quartile (ng/mL)

                                               1 (<0.7)

                                               2 (0.7-1.5)

                                               3(1.5-3.1)

                                               4(>3.1)

                                               (trend p-value)
   OR (95% Cl)

  1.0 (referent)

  0.9 (0.4, 2.0)

  0.8 (0.3, 2.2)

  0.8 (0.3, 2.6)

     (0.84)
                                               Adjustment for education, smoking status and
                                               alcohol consumption did not alter the results;
                                               similar results in analyses based on summation of
                                               MIBP and MBP
      Polycystic ovarian syndrome
      Hart et al. (2013) (Australia)
      Population: 121 girls from birth cohort study (Western
      Australian Pregnancy Cohort), whose mothers were
      recruited at 18 wks of gestation between 1989 and 1991;
      follow-up at ages 14-16 yrs
                                               Correlation coefficient (p-value) between log-
                                               transformed MIBP and pubertal development
                                               parameter
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                     3-13             DRAFT—DO NOT CITE OR QUOTE

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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                   Reference and study design
                                                                         Results
      Outcome: Uterine volume, ovarian volume, and antral
      follicle count measured by ultrasound; PCO defined as
      >1 ovary more than 10 cm3 or >12 follicles between 2 and
      9 mm in diameter; PCOS defined either as (1) presence of at
      least two of: polycystic ovarian morphology, clinical or
      biochemical hyperandrogenism, or oligo-anovulation; or
      (2) oligo-anovulatory menstrual cycles with either clinical or
      biochemical hyperandrogenism; all clinical assessments
      conducted on d 2-5 of menstrual cycle
      Exposure: Maternal serum samples (n = 123) collected at
      18 and 34-36 wks of gestation (combined aliquot from both
      time periods)
      MIBP in serum (ng/mL):
              Median  90th percentile
      MIBP     1.77       6.16
      Analysis: Correlation  between log-transformed MIBP and
      uterine volume, ovarian volume, and antral follicle counts;
      MIBP concentrations in PCO or PCOS cases and controls
      compared calculated using t-tests or Mann-Whitney U-tests
                                                      Uterine volume (mL)

                                                      Ovarian volume (cm3)

                                                      Antral follicle count
r < 0.20 (p> 0.17)

r < 0.10 (p> 0.29)

r < 0.12 (p> 0.20)
                                                      Authors reported no association between MIBP
                                                      and polycystic ovarian syndrome using either
                                                      definition (quantitative results not reported).
1
2
3
PCO = polycystic ovarian morphology; PCOS = polycystic ovarian syndrome
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                    3-14             DRAFT—DO NOT CITE OR QUOTE

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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

1    3.2.6.  Pregnancy Outcomes in Humans

2            Table 3-8. Evidence pertaining to DIBP and pregnancy outcomes in humans
               Reference and study design
                       Results
      Fetal growth (birth weight, birth length, head circumference)
      Huang etal. (2014b) (China)
      Population: 207 women delivering at one hospital
      in Chongqing between 2011 and 2012, aged
      18-35 yrs and with no history of tobacco or alcohol
      use; mean age 28 yrs
      Outcome: Standard clinical measures at birth
      Exposure: Cord blood sample
      DIBP in  cord blood (ug/L)
                 Median   75th percentile 95th percentile
      All samples    16.7        26.9          114
      Analysis:  Linear regression, adjusting for variables
      shown in results column
Regression coefficient (95% Cl) for change in clinical
measurement at birth per unit increase in In-transformed
DIBP (ug/L) (adjusted for gestational age):
Birth weight (g)
Birth length
(cm)
Head
circumference
(mm)
                       Girls
                   -27 (-90, 36)
                 -0.06 (-0.45, 0.33)
             Boys
        -87 (-195, 200)
         -0.75 (-1.35,
            -0.15)
                 -3.85 (-9.47, 1.76)   -2.76 (-7.62, 2.11)
      Philippat et al. (2012) (France)
      Population: 72 cases with undescended testis or
      hypospadias, 215 matched controls from two birth
      cohorts (EDEN and PELAGIE), 2002-2006
      Outcome:  Standard clinical  measurements at birth
      Exposure:  Maternal urine sample, collected
      between 6 and 19 (PELAGIE) or between 24 and
      30 (EDEN) wks of gestation
      MIBP in urine (ng/mL):
                     Median    95th percentile
      Measured        45.9        219.0
      Standardized*    64.7        365.3
      Analysis:  Cases and controls combined for this
      analysis; weighted linear regression using tertiles or
      In-transformed urine concentrations, adjusting for
      variables shown  in results column; analysis by
      tertiles for evaluation  of possible non-monotonic
      relationship; analyses  corrected for oversampling of
      malformation cases
      *Standardized for sampling  conditions and
      gestational age at collection
Regression coefficient (95% Cl) for change in birth outcome
by MIBP tertile and per unit change in In-MIBP
(standardized, ng/mL) (adjusted for gestational duration,
maternal pre-pregnancy weight and height, maternal
smoking, maternal education, parity, recruitment center,
urine creatinine, and mode of delivery as potential
covariate; head circumference model also adjusted for
mode of delivery)
MIBP tertile
(Ug/L)
1 (<48.2)


2 (48.2-97.9)


3 (>97.9)


(trend p-value)
In (MIBP)
   Birth
weight (g)
    0
(referent)
    61
(-77, 200)
   -31
(-190, 129)
  (0.48)
   -44
(-110, 23)
  Birth
  length
  (cm)
    0
(referent)
   0.4
(-0.3,1.1)
   0.3
(-0.4, 1.0)
  (0.54)
   0.0
(-0.3, 0.3)
                                            Head
                                        circumference
                                            (cm)
                                             0
                                          (referent)
                                            -0.1
                                          (-0.6, 0.4)
                                            0.2
                                          (-0.5, 0.9)
                                           (0.40)
                                            -0.1
                                          (-0.4,0.1)
      Wolff et al. (2008) (United States, New York City)
      Population: 382 singleton live births without
      medical complications from birth cohort (Mt. Sinai
      Children's Environmental Health study),  1998-2002
      Outcome:  Standard clinical measurements at birth
      Exposure:  Maternal urine sample, third  trimester
      MIBP in urine (ng/mL):
                  Median   75th percentile
      Unadjusted    6.2         12
      Analysis: Linear regression, adjusting for variables
      shown in results column
Regression coefficient (95% Cl) for change in birth outcome
with unit increase in In-MIBP (ng/mL) (adjusted for
race/ethnicity, infant sex, gestational age at delivery, In-
creatinine, prenatal smoking, pre-pregnancy BMI, maternal
education, and marital status)
Birth weight (g)
Birth length (cm)
Head circumference (cm)
       -14 (-57, 28)
     0.04 (-0.19, 0.28)
     0.05 (-0.11, 0.21)
Restricted to observations with creatinine >20 mg/dL
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                     3-15              DRAFT—DO NOT CITE OR QUOTE

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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
          Reference and study design
                       Results
Preterm birth (<37 wks) and gestational age
Ferguson et al. (2014b): Ferguson et al. (2014a)
(United States; Boston)
Population: 130 cases, 352 controls from
pregnancy cohort (study of predictors of pre-
eclampsia, enrolled during first trimester,
2006-2008); controls randomly selected from
among those delivering >37 wks of gestation; mean
age 33 yrs
Outcome:  Preterm birth (<37 wks of gestation;
gestation estimated from first trimester ultrasound);
additional analysis of subgroup with spontaneous
preterm labor or preterm premature rupture of
membranes ("spontaneous preterm," n = 57)
Exposure:  Maternal urine sample, one to three
samples collected at median times of 9.7,17.9, or
26.0 wks of gestation; geometric mean of results
from visits 1-3 used in analyses.
MIBP in urine, SG-adjusted (ng/L):
           Geometric mean  75th percentile
Controls        6.71            10.3
All cases        6.85            10.5
Analysis: Logistic regression (In-transformed
metabolites), considering average specific gravity,
maternal age, race/ethnicity, education level, health
insurance provider, BMI at first study visit, smoking
status, alcohol use, parity, use  of assisted-
reproductive technology, and sex of infant as
potential covariates
Related reference: Ferguson et al. (2014a) (analysis
by individual sample results for the four visits)
OR (95% Cl) for preterm birth per unit increase in In-
transformed MIBP (adjusted for average specific gravity,
maternal age, race/ethnicity, education level, and
insurance provider)
All preterm                        0.98 (0.72,1.34)
Spontaneous preterm               1.52 (0.97, 2.38)
[Results weaker than those seen with DEHP metabolites]


Results by study visit from Ferguson et al. (2014a), all pre-
term births
Visit 1

Visit 2
Visit 3
Visit 4
                         0.92

                         0.88

                         0.75

                         0.66
(0.57, 1.47)

(0.54, 1.41)

(0.50, 1.13)

(0.28, 1.55)
Huang etal. (2014b) (China)
Population: 207 women delivering at one hospital in
Chongqing between 2011 and 2012; aged 18-35 yrs
and with no history of tobacco or alcohol use; mean
age 28 yrs
Outcome: Preterm birth (<37 wks of gestation;
gestational  age estimated from last menstrual
period)
Exposure: Cord blood sample
DIBP in cord blood (ng/L)
           Median  75th percentile 95th percentile
All samples   16.7         26.9           114
Analysis: Logistic and linear regression, adjusting
for variables shown in results column
OR (95% Cl) for preterm delivery comparing In-DIBP above
and below the median (adjusted for maternal age, BMI,
frequency of prenatal exam, and pregnancy history), with
additional stratification by history of intravenous infusions
Total sample (n = 207)
No intravenous infusions (n = 154)
Intravenous infusions (n = 53)
                                   6.01 (3.24, 11.17)
                                   4.78 (1.68, 13.57)
                                   6.07 (2.66, 13.83)
[History of intravenous infusions present in 26% of total
and 55% of preterm birth group]

Regression coefficient (95% Cl) for change in gestational
age (wks) per unit increase in In-transformed DIBP (ng/L)
(adjusted for maternal age, BMI, frequency of prenatal
examination, history of intravenous infusions therapy, and
pregnancy history)
                  -0.75 (-1.03, -0.46)
           This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
               Reference and study design
                       Results
      Meeker et al. (2009) (Mexico)
      Population: 30 cases, 30 controls (term births) from
      pregnancy cohort, 2001-2003.
      Outcome: Preterm  birth (<37 wks of gestation),
      determined using maternal recall of last menstrual
      period
      Exposure: Maternal urine sample, third trimester
      MIBP in urine, among term births
                        Median  75th percentile
      Unadjusted          2.0        4.1
      SG-adjusted (jjg/L)    2.3        5.0
      Cr-adjusted (ng/g Cr)  3.7        6.6
      Analysis:  Logistic regression, considering maternal
      age, pre-pregnancy  BMI, parity, education, marital
      status,  infant's sex, and gestational age at urine
      sample as potential  covariates
OR (95% Cl) for preterm birth by MIBP above compared
with below the median (adjusted for marital status,
maternal education, and infant sex and gestational age at
time of urine sample)
Cr-unadjusted (ng/L)
SG-adjusted (ng/L)

Cr-adjusted (ng/g Cr)
3.6(1.1,12.2)

 2.0 (0.7, 6.0)

 1.5 (0.5, 4.5)
      Wolff et al. (2008) (United States, New York City)
      Population: 382 singleton live births without
      medical complications from birth cohort (Mt. Sinai
      Children's Environmental Health study), 1998-2002
      Outcome:  Standard clinical measurements at birth
      Exposure:  Maternal urine sample, third trimester
      MIBP in urine (ng/mL):
                  Median  75th percentile
      Unadjusted    6.2        12
      Analysis: Linear regression, adjusting for variables
      shown in results column
Regression coefficient (95% Cl) for change in birth outcome
with unit increase in In-MIBP (ng/mL) (adjusted for
race/ethnicity, infant sex, gestational age at delivery, In-
creatinine, prenatal smoking, pre-pregnancy BMI, maternal
education, and marital status)

Gestational age (wks)             0.03 (-0.20, 0.14)
Restricted to observations with creatinine >20 mg/dL
1
2
                 This document is a draft for review purposes only and does not constitute Agency policy,
                                                      3-17              DRAFT—DO NOT CITE OR QUOTE

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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

1    3.2.7.  Immune Effects in Humans

2            Table 3-9. Evidence pertaining to DIBP and allergy/immune effects in humans
           Reference and study design
                           Results
      Ait Bamai et al. (2014)a (Japan)
      Population: Children (n = 122, ages
      <15 yrs) and adults (n = 374, ages >15 yrs)
      living in 148 detached dwellings in which
      at least 25 mg of dust was collected; 2006
      follow-up of 2003 baseline survey
      Outcome: Allergic condition assessed by
      self-administered questionnaire (positive
      response to: in the past 2 yrs have you
      been seen at a hospital for allergic rhinitis,
      allergic conjunctivitis, or atopic
      dermatitis?); parents completed
      questionnaires for children <6 yrs
      Exposure: Dust samples
      DIBP in dust (ng/g dust):
                             Median  75th
                                   percentile
      Floor dust (n = 148)         2.4     5.5
      Multi-surface dust (n = 120)  1.9     3.5
      Analysis: Generalized linear mixed effects
      model, considering gender, age strata
      (<15, >15 yrs), smoking status (personal
      and environmental tobacco smoke), furry
      pets in home, signs of dampness, Der 1
      (not defined by authors), other phthalates
      dust, airborne fungi, formaldehyde, total
      VOC, and building characteristics as
      potential covariates
OR (95% Cl) for allergic condition by tertile of DIBP in floor dust (ng/g
dust)(adjusted for adjusted for gender, age strata, smoking status,
dampness index, furry pets inside the home, Der 1, and sum of other
phthalates)
DIBP
tertile
1 (low)
2


3 (high)


(trend
p-value)


1 (low)
2
3 (high)
(trend
p-value)


1 (low)
2


3 (high)
 Full sample          Children          Adults
          Allergic rhinitis
1.0 (referent)       1.0 (referent)     1.0 (referent)
             1.87 (0.83, 4.22)    3.54 (0.86, 14.5)


             1.05 (0.47, 2.32)    2.30 (0.60, 8.89)


                 (0.91)              (0.23)
                                    0.99 (0.47,
                                       2.05)

                                    0.48 (0.22,
                                       1.02)

                                       (0.06)
                      Allergic conjunctivitis

              1.0 (referent)       1.0 (referent)     1.0 (referent)

             1.07 (0.38, 3.01)    1.97 (0.35, 11.1)  0.59 (0.19, 1.8)

             1.64(0.64,4.18)    3.27(0.68,15.7)  0.82(0.31,2.2)

                 (0.30)              (0.14)           (0.69)


                       Atopic dermatitis

              1.0 (referent)       1.0 (referent)     1.0 (referent)

             5.52 (1.68, 18.1)    11.95 (1.37, 104)    2.55 (0.89,
                                                     7.31)
                                                          4.84 (1.46, 16.0)     15.0 (1.91,118)


                                                              (0.01)              (0.01)


                                             p-value for age interaction >0.05 for all endpoints
(trend
p-value)
                                    1.56 (0.44,
                                       5.53)

                                       (0.49)
                                             No increased aORs (either in the full sample or stratified by age)
                                             were observed in analyses using DIBP measurements in multisurface
                                             dust.
                 This document is a draft for review purposes only and does not constitute Agency policy,
                                                      3-18              DRAFT—DO NOT CITE OR QUOTE

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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
     Reference and study design
                           Results
Callesen et al. (2014b): Callesen et al.
(2014a)a (Denmark)
Population:  81 rhinoconjunctivitis cases,
88 atopic dermatitis cases, 242 healthy
controls group from population-based
survey (Indoor Environment and Children's
Health); ages 3-5 yrs
Outcome: Clinical exam and parent
interview; allergic rhinoconjunctivitis:
recurrence of at least two or more nasal
symptoms (pruritus, runny nose, sneezing
spells >20, nasal stenosis/mouth
breathing) and ocular symptoms (itching,
conjunctival  injection, or watery secretion
in both eyes) when exposed to allergens;
atopic dermatitis: presence of at least 3 of
4 major features and 3 of 23 minor
features;  70% of rhinoconjunctivitis and
50% of atopic dermatitis cases were IgE
positive based on 20 allergen tests
Exposure: DIBP concentrations in dust
samples from bedroom and day care
centers; total DIBP exposure estimated as
a weighted mass fraction
DIBP in dust  among controls (ng/g):
              Median
Home          27.0
Day care        22.6
Area-weighted  27.2
(weighted by assumed hrs in each
environment)
Analysis:  Mann-Whitney U-test
Related study: Callesen etal. (2014a)
(same study  population, with exposure
measured in  urine sample from
participants
MIBP in urine: median 74.2  ng/mL
(controls)
Median DIBP in dust (ng/g), by case-control status assessed by
clinical examination
                                       Cases
Home
Day care
Area-
weighted
Controls
(n = 242)

  27.0

  22.6

  27.2
Rhinoconjunctivitis
     (n = 81)

      30.4

      22.3

      26.8
Atopic dermatitis
    (n = 88)

      33.4

      22.5

      33.1
Similar results when based on case status defined by parent-
questionnaire data (n = 56 rhinoconjunctivitis, n = 83 atopic
dermatitis)

Results from Callesen etal. (2014a):
OR (95% Cl) by quartile of MIBP (urine sample), adjusting for sex,
breastfeeding less than 3 mo, smoking in the home, and single
allergic predisposition
         Rhinoconjunctivitis

       (76 cases, 222 controls)

1          1.0 (referent)

2         1.18(0.54,2.55)

3         0.89 (0.39, 2.02)

4         1.07 (0.52, 2.22)
               Atopic dermatitis

            (76 cases, 216 controls)

                 1.0 (referent)

                1.11 (0.53, 2.34)

                0.88 (0.41, 1.91)

                0.97 (0.48, 1.94)
           This document is a draft for review purposes only and does not constitute Agency policy,
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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
     Reference and study design
                           Results
Hoppin et al. (2013)a (United States,
NHANES)
Population: 2,325 participants in
population-based survey (NHANES),
2005-2006; ages >6 yrs
Outcome: Self-administered
questionnaire current allergy symptoms
(hay fever, allergy, itchy rash, rhinitis) in
past yr; allergic sensitization as measured
by serum IgE (19 allergen specific IgEs,
>0.35kU/L)
Exposure: Urine sample collected same
day as serum sample
MIBP in urine (ng/L):      Percentile
           Median      75th    95th
Children     8.93      16.38   45.97
Adults       5.42      10.53   28.98
Analysis: Logistic regression, adjusting for
variables shown in results column and
sampling weights; separate analyses for
children (ages 6-17 yrs) and adults
(>17yrs)
Prevalence and OR (95% Cl) for allergy symptoms and allergic
sensitization per unit change in log-transformed urinary MIBP level
(adjusted for age, race/ethnicity, gender, BMI, creatinine, and
cotinine)
Children (n = 779)
  Hay fever (n = 23)
  Rhinitis (n = 188)
  IgE sensitization
  (any)
Adults (n = 1,546)
  Hay fever (n = 88)
  Rhinitis (n = 498)
  IgE sensitization
  (any)
 3.6%

27.6%
46.1%
 7.4%
35.4%

44.0%
0.12(0.04,0.39)

0.84 (0.53, 1.33)
0.93 (0.51, 1.70)
0.93 (0.46, 1.87)
0.99 (0.76, 1.29)

1.32 (0.99, 1.76)
Authors reported that adjustment for poverty income ratio did not
alter ORs.
Sunetal. (2009)a (China)
Population:  Cases of rhinitis (n = 240) or
eczema (n = 61) and controls (n = 204 and
119 for rhinitis and  eczema analysis,
respectively), all students of Tianjin
University who had participated in a cross-
sectional study of allergic symptoms and
environmental factors; 2006-2007
Outcome: Self-reported symptoms from
questionnaire: rhinitis = in past 12 mo, had
a problem with sneezing, or a runny, or a
blocked nose when not having a cold or
the flu, or  sneezing, or a runny, or a
blocked nose, or itchy-watery eyes after
contact with furred animals or after
contact with pollen; eczema = in past
12 mo, had an itchy rash; controls
responded no to questions on
asthma/wheeze, rhinitis, and eczema
Exposure: Surface dust sample in dorm
rooms
DIBP in dust (ng/g):
       Median 75th percentile
       20.24       34.77
Analysis:  Logistic regression for OR
considering age, gender, passive smoking,
smoking, pet raising, atopy, and building
age as potential covariates; Mann-
Whitney U-test for comparison between
DIBP concentrations of cases and controls;
OR for rhinitis and eczema comparing DIBP in dust (|Jg/g dust) above
and below the median (adjusted for age, gender, smoking, atopy and
building age) reportedly did not reach statistical significance
(quantitative results not reported)

Median Concentration DIBP in dust (|Jg/g dust)
                                Cases             Controls
Rhinitis                          20.17              28.76*
Eczema                          28.68              22.56

*p = 0.019 by Mann-Whitney test; p = 0.051 by t-test.
           This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
           Reference and study design
                                                                 Results
      t-test for comparisons between log-
      transformed concentrations
      Bornehag et al. (2004) (Sweden)
      Population: 198 cases, 202 controls from
      population-based cohort (Dampness in
      Buildings and Health cohort); n = 10,852;
      2001-2002; ages 3-8 yrs
      Outcome:  Eczema, wheezing, or rhinitis
      (Cases report at least two incidents of
      eczema, or wheezing or rhinitis without a
      cold, in the preceding yr, and at follow-up
      1.5 yrs later)
      Exposure: Surface dust samples from
      children's bedrooms
      DIBP in dust (mg/g):
                  Median
      All homes    0.045
      Analysis:  Mann-Whitney U-test for
      comparing concentrations in all homes;
      t-test for comparing log-transformed
      concentrations in homes with
      concentrations above detection limit.
                                       Concentration in dust (mg/g dust)
                                                      Median, all
                                                        homes
                                                       (n = 346)
                                       Controls          0.048
                                       Cases (all)         0.042
                                       p > 0.4 in both tests
Geometric mean (95% Cl), homes
 with phthalate > detection limit
           (n = 290)

      0.055 (0.046, 0.065)

      0.058 (0.048, 0.070)
1
2
3
4
5
6
Additional results for this study presented in asthma table.

aOR = adjusted odds ratio; IgE = immunoglobin E; NHANES = National Health and Nutrition Examination Survey;
 VOC = volatile organic compound
                This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1
2
Table 3-10. Evidence pertaining to DIBP and asthma/wheezing and
hypersensitivity in humans
             Reference and study design
                                                            Results
      Ait Bamai et al. (2014)a (Japan)
      Population: Children (n = 122, ages <15 yrs)
      and adults (n = 374, ages >15 yrs) living in
      148 detached dwellings in which at least
      25 mg of dust was collected; 2006 follow-up of
      2003 baseline survey.
      Outcome:  Bronchial asthma assessed by self-
      administered questionnaire (positive response
      to: in the past 2 yrs have you been seen  at a
      hospital for bronchial asthma?); parents
      completed questionnaires for inhabitants
      <6yrs
      Exposure:  Dust samples from floor and  other
      surfaces
      DIBP (ng/g dust):
                             Median   75th
                                    percentile
      Floor dust (n = 148)         2.4     5.5
      Multi-surface dust (n = 120)  1.9     3.5
      Analysis: Generalized linear mixed effects
      model, considering gender, age strata (<15,
      >15 yrs), smoking status (personal and
      environmental tobacco smoke), furry pets  in
      home, signs of dampness, Der 1 (not defined
      by authors), other phthalates dust, airborne
      fungi, formaldehyde, total VOC, and building
      characteristics as potential covariates.
                                   OR (95% Cl) for bronchial asthma by tertile of DIBP in floor dust
                                   (adjusted for adjusted for gender, age strata, smoking status,
                                   dampness index, furry pets inside the home, Der 1, and sum of
                                   other phthalate dusts)
                                   1 (low)
                                   2


                                   3 (high)


                                   (trend
                                   p-value)
 Full sample

1.0 (referent)

 2.25 (0.48,
   10.57)

 5.09(1.17,
   22.15)

   (0.03)
   Children          Adults

 1.0 (referent)    1.0 (referent)

4.37(0.36,53.6)  1.16(0.16,8.17)


8.94 (0.86, 93.0)  2.90 (0.52, 16.2)


    (0.067)          (0.22)
                                   p-value for age interaction = 0.51

                                   No significantly increased aORs (either in the full sample or
                                   stratified by age) were observed in analyses of bronchial asthma
                                   using DIBP measurements in multisurface dust.
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                     3-22             DRAFT—DO NOT CITE OR QUOTE

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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
       Reference and study design
                         Results
Callesen et al. (2014b): Callesen et al.
(2014a)a (Denmark)
Population: 72 asthma cases, 242 healthy
controls group from population-based survey
(Indoor Environment and Children's Health);
ages 3-5 yrs
Outcome: Clinical exam and parent interview;
asthma: recurrence of at least two of the
three symptoms: cough, wheeze, and
shortness of breath within the previous 12 mo
(symptoms other than those
triggered by respiratory infections); and
doctor diagnosis of asthma in combination
with ongoing treatment; 47% of asthma cases
were IgE positive based on 20 allergen tests
Exposure: DIBP concentrations in dust
samples from bedroom and day care centers;
total DIBP exposure estimated as a weighted
mass fraction
DIBP in dust among controls (ng/g):
                Median
Home            27.0
Day care          22.6
Area-weighted    27.2
(weighted by assumed hrs in each
environment)
Analysis:  Mann-Whitney U-test
Related study: Callesen etal. (2014a) (same
study population, with exposure measured in
urine sample from participants
MIBP in urine: median 74.2 ng/mL (controls)
Median DIBP in dust (ng/g), by case-control status assessed by
clinical examination
Home
Day care
Area-weighted
Controls (n = 242)

      27.0

      22.6

      27.2
Asthma (n = 72)

     25.8

     21.5

     25.7
Similar results when based on case status defined by parent-
questionnaire data (n = 110 asthma cases)

Results from Callesen etal. (2014a):
OR (95% Cl) by quartile of MIBP (urine sample), adjusting for sex,
breastfeeding <3 mo, smoking in the home, and single allergic
predisposition
Bronchial asthma
(60 cases, 216 controls)
                                      1.0 (referent)

                                    0.49 (0.22, 1.09)

                                    0.91(0.41,1.69)

                                    0.61 (0.27, 1.34)
Bertelsen et al. (2013) (Norway)
Population: 623 children from birth cohort
(Environment and Childhood Asthma study),
born 1992-1993; children with current asthma
over-sampled (follow-up 2001-2004); ages
10 yrs
Outcome: Current asthma (parental report of
history of asthma plus >1 of the following:
dyspnea, chest tightness, and/or wheezing in
previous 12 mo; use of asthma medications in
previous 12 mo; positive exercise challenge
test)
Exposure: First morning urine sample
(child's), collected at study examination
MIBP in urine (ng/L)          Percentile
             Median     75th      95th
Unadjusted    49.2      88.4      231.0
SG-adjusted    50.1      90.5      239.6
Analysis: Logistic regression, adjusting for
variables shown in the results column
OR (95% Cl) for current asthma by quartile of MIBP (ng/L)
(adjusted for urine specific gravity, sex, parental asthma, and
household income)
1: <31.4 (referent)

2: >31.4-49.2

3: >49.2-88.4

4: >88.4
                 1 (referent)

                1.3 (0.74, 2.4)

                1.4(0.73,2.5)

                1.5 (0.80, 2.7)
Increase in odds of current asthma per logio IQR MIBP
(95% Cl) = 1.1 (0.87,1.5)
           This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
             Reference and study design
                                                                    Results
      Hoppin et al. (2013)a (United States, NHANES)
      Population: 2,325 participants in population-
      based survey (NHANES), 2005-2006; ages
      >6yrs
      Outcome:  Self-administered questionnaire
      (asthma, wheeze in past yr)
      Exposure:  Urine sample collected same day as
      serum sample
      Unadjusted MIBP in urine (ng/L):
                                  Percentile
                     Median    75th       95th
      Children         8.93     16.38     45.97
      Adults           5.42     10.53     28.98
      Analysis:  Logistic regression, adjusting for
      variables shown in results column and
      sampling weights; separate analyses for
      children (ages 6-17 yrs) and adults (>17 yrs)
                                           Prevalence and OR (95% Cl) for asthma symptoms per unit
                                           change in log-transformed urinary MIBP level (adjusted for age,
                                           race/ethnicity, gender, BMI, creatinine, and cotinine)
                                           Children (n = 779)
                                           Asthma (n = 65)
                                           Wheeze (n = 80)
                                           Adults (n = 1,546)
                                           Asthma (n = 116)
                                           Wheeze (n = 219)
8.4%
10.7%


7.4%
16.6%
0.92 (0.26, 3.29)
1.08 (0.49, 2.35)


1.39 (0.77, 2.50)
0.92 (0.57, 1.48)
                                           Authors reported that adjustment for poverty income ratio did
                                           not alter ORs.
      Sun et al. (2009)a (China)
      Population: 92 cases asthma/wheezing, cases
      and 346 controls, all students of Tianjin
      University who had participated in a cross-
      sectional study of allergic symptoms and
      environmental factors; 2006-2007
      Outcome: Self-reported symptoms from
      questionnaire; asthma/wheezing = in past
      12 mos, have you had wheezing or whistling
      the in the chest; have you had dry cough at
      night for more than 2 wks, apart from a cough
      associated with a cold or chest infection;
      controls responded no to questions on
      asthma/wheeze, rhinitis, and eczema
      Exposure: Surface dust sample in dorm rooms
      DIBPin dust (|jg/g):
             Median   75th percentile
              20.24       34.77
      Analysis:  Logistic regression for OR,
      considering age, gender, passive smoking,
      smoking, pet raising, atopy,  and building age
      as potential covariates; Mann-Whitney U-test
      (nonparametric) for comparison between
      DIBP concentrations of cases and controls;
      t-test for comparisons between log
      transformed concentrations
                                           OR for asthma comparing DIBP in dust (ng/g dust) above and
                                           below the median (adjusted to age, gender, smoking, atopy, and
                                           building age) reportedly did not reach statistical significance
                                           (quantitative results not reported)
                                           Median concentration DIBP in dust (ng/g dust)
                                                                     Cases
                                           Wheezing                  23.13
                                           (p > 0.46 by Mann Whitney or t-test)
                  Controls
                   22.73
1
2
3
4
Additional results for this study presented in allergy/immune table.

IQR = interquartile range
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                     3-24             DRAFT—DO NOT CITE OR QUOTE

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2
3
                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.2.8.  Neurodevelopmental Effects in Humans

        Table 3-11. Evidence pertaining to DIBP and neurodevelopmental effects in
        humans
               Reference and study design
                                                                      Results
      Attention and executive function in school-aged children
      Kobroslv et al. (2014) (United States; Minnesota,
      Missouri, California, Iowa)
      Population: 153 children (n = 76 girls, n = 77 boys)
      from birth cohort study (Study for Future Families),
      born 2000-2005, ages 6-10 yrs in 2010 follow-up
      Outcome:  Child Behavior Checklist (completed by
      parent)
      Exposure:  Maternal urine sample, 3rd trimester
      (mean 26.6 wks)
      Unadjusted MIBP in urine (ng/mL):
             Geometric mean    (95% Cl)
                  2.3          (2.0,2.8)
      Analysis: Linear regression, considering sex, age,
      mother's education, urinary creatinine, family stress
      measure, and race/ethnicity, as potential covariates.
      Related references: Swan et al. (2005) (exposure
      data)
                                                Regression coefficient (95% Cl) for change in raw score on
                                                child behavior checklist per unit increase in In-transformed
                                                MIBP (adjusted for sex, age, mother's education and
                                                urinary creatinine, and family stress score)
                                                Anxiety/
                                                depression
                                                Withdrawn
                                                Somatic
                                                complaints
                                                Social problems
                                                Thought
                                                problems
                                                Attention
                                                problems
                                                Rule-breaking
                                                behavior *
                                                Aggressive
                                                behavior
                                                Internalizing
                                                behavior
                                                Externalizing
                                                behavior
      Boys              Girls
0.11 (-0.13, 0.34)  -0.03 (-0.29, 0.22)


-0.01 (-0.21, 0.18)  -0.04 (-0.25, 0.17)
-0.03 (-0.23, 0.16)  -0.07 (-0.28, 0.13)


0.18 (-0.02, 0.37)  -0.06 (-0.27, 0.16)
0.15 (-0.05, 0.35)   0.07 (-0.15, 0.29)
                                                                       0.27 (0.04, 0.50)


                                                                       0.20 (0.01, 0.38)

                                                                       0.34 (0.09, 0.59)
                   0.12 (-0.12, 0.36)


                  -0.04 (-0.23, 0.16)

                   0.12 (-0.14, 0.39)
                                                                      0.09 (-0.18, 0.37)   -0.07 (-0.37, 0.22)


                                                                       0.32 (0.06, 0.58)   0.06 (-0.22, 0.34)
                                                     Total problems     0.42 (0.05, 0.80)   0.07 (-0.33, 0.47)
                                                     *Sex interaction p-value = 0.04; all other interaction
                                                     p-values >0.05
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                     3-25              DRAFT—DO NOT CITE OR QUOTE

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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
          Reference and study design
                       Results
Engel et al. (2010) (United States; New York City)
Population: 177 children from original birth cohort
studied by Engel etal. (2009), 54% boys, three
follow-up exams at ages 4.5-5.5, 6-6.5, 7-9 yrs
Outcome:  Behavior assessed by maternal reporting
on BRIEF and BASC-PRS
Exposure:  Maternal urine sample, 25-40 wks
gestation*
                 Median   75th percentile
MIBP (ng/L)*       2.6         12.2
Sum LMW (U.M/L)   1.88        4.59
(sum of MBP, MEP, MIBP, and MMP)
Analysis: Generalized linear regression model,
adjusting for variables shown in results column;
other variables (not specified) were considered
Related references:  Engel et al. (2009) (exposure
data for n = 295 children in the cohort)
*MIBP concentrations not reported in (Engel et al.,
2010); values reported here  are from  an earlier
analysis of this cohort described in Engel et al.
(2009)
Regression coefficient for change in behavioral score
(BASC-PRS) per unit increase in In-phthalate level (nM/L) in
boys (adjusted for race, educational level and marital
status of the primary caretaker, and urinary creatinine)
                        MIBP
 Low molecular
weight phthalate
      sum
Clinical scales (higher score = more problem behaviors)
Aggression              -0.12
Anxiety                -0.25
Attention                0.66
problems
Atypicality               0.53
Conduct problems        0.23
Depression               0.29
Hyperactivity            0.85
Somatization            1.04
     1.24*
     0.78
     1.29*


     0.95
     2.40*
     1.18*
     1.03
     0.36
                                                Withdrawal             -0.01              0.46
                                                Adaptive scales (lower score = more problem behaviors)
                                                Adaptability            -1.32*             -1.08*
                                                Leadership             -1.30             -0.88
                                                Social skills             -0.93             -1.04
                                                Composite scales (higher score =  more problem behaviors)
                                                                        0.33              1.75*
Externalizing
problems
Internalizing
problems
Adaptive skills
Behavioral
Symptoms Index
                                                                        0.46


                                                                       -1.17
                                                                        0.47
      0.99


     -0.98
     1.55*
                                                Significant sex-phthalate interactions (p < 0.05) for
                                                aggression, conduct problems, hyperactivity, externalizing
                                                problems, and behavioral symptoms index, as reported by
                                                study authors.
                                                Regression coefficient for change in behavioral score
                                                (BRIEF scores; higher score = worse executive functioning)
                                                per unit increase in In-phthalate level (nM/L) in boys and
                                                girls (adjusted for race, sex, educational level and marital
                                                status of the primary caretaker, and urinary creatinine)
                                                Emotional control
                        0.09
     1.33*
           This document is a draft for review purposes only and does not constitute Agency policy,
                                                3-26              DRAFT—DO NOT CITE OR QUOTE

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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
          Reference and study design
                       Results
                                                Behavioral               0.30               1.13
                                                regulation index
                                                Initiate                  0.83               0.81
                                                Working memory         1.11               1.03
                                                Plan/organize            0.76               1.02
                                                Metacognition           0.70               1.05
                                                index
                                                Global executive          0.56              1.23*
                                                composite
                                                *p < 0.05

                                                Study authors reported that there were few significant
                                                associations between phthalate concentration and
                                                behavior among girls (quantitative results not reported).
Neurobehavioral outcomes in infants and preschool-aged children
Braun et al. (2014) (United States)
Population:  175 children from birth cohort in Ohio
(HOME cohort, recruited during pregnancy,
2003-2006); follow-up at ages 4-5 yrs
Outcome: Autistic behaviors based on Social
Responsiveness Scale completed by mother; 65 item
scale, higher score = more autistic behaviors
Exposure: Maternal urine samples, 16-26 wks of
gestation
MIBP in urine (u.g/g Cr):
                        Percentile
           Median    75th       95th
Cr-adjusted   5.6       8.6        17
Analysis: Semi-Bayesian hierarchical regression
model
Regression coefficient (95% Cl) for change in total score
per unit increase in log-transformed Cr-adjusted MIBP
(adjusted for maternal demographic and perinatal factors,
depressive symptoms, caregiving environment, and serum
cotinine):
                    0.7 (-1.4, 2.8)
Tellez-Roio et al. (2013) (Mexico)
Population: 135 children from birth cohort (Early
Life Exposure in Mexico to Environmental Toxicants
cohort; mothers recruited during first trimester,
1997-2003)
Outcome: Mental and psychomotor development
based on Bayley Scales of Infant Development-ll
(assessed by trained examiner, videotaped for
quality control assessment) tested at 24, 30, and
36 mo of age
Exposure: Maternal urine sample, 3rd trimester
MIBP in urine (ng/mL):
            Geometric mean (95% Cl)
SG-adjusted     2.30 (1.92, 2.76)
Analysis: Linear regression for longitudinal data,
stratified by sex and adjusted for variables shown in
results column
Related reference: Ettinger et al. (2009)
Regression coefficient (95% Cl) for change in
neurodevelopment score per unit increase in maternal In-
MIBP (adjusted for birthweight, breastfeeding practices,
weight-for-age, child's age, mother's age, mother's
education, and laboratory)
              Total sample
                (n = 135)
 Boys
(n = 64)
                          Girls (n = 71)
MDI
PDI
    0.53         0.32          -0.12
(-0.85, 1.91)  (-1.62, 2.28)   (-1.94, 1.69)

    0.57         0.63          0.37
(-0.67, 1.82)  (-0.68, 1.95)   (-1.67, 2.43)
           This document is a draft for review purposes only and does not constitute Agency policy,
                                                3-27             DRAFT—DO NOT CITE OR QUOTE

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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
          Reference and study design
                       Results
Whvattetal. (2012) (United States, New York City)
Population:  297 children from birth cohort
(Columbia Center for Children's Environmental
Health), born 1999-2006; 3-yr follow-up, mean age
36 mo (range 27-42 mo)
Outcome: Mental, psychomotor and  behavioral
development at 3 yrs based on Bayley Scales of
Infant Development-ll (assessed by trained
examiners) and Child Behavior Checklist (completed
by parent)
Exposure: Maternal urine sample, 3rd trimester
MIBP in urine (ng/mL):
          Geometric mean
Unadjusted     9.3
Analysis: Linear and logistic regression adjusting for
variables shown in results column; Wald test used to
detect sex differences
Regression coefficient (95% Cl) for change in
neurodevelopment score per unit increase in maternal In-
MIBP (adjusted for specific gravity, race/ethnicity,
maternal marital status and prenatal alcohol consumption,
child's gestational age and sex, and quality of care-taking
environment)
                        Boys (n = 140)   Girls (n = 157)
MDI
PDI
                            0.59
                        (-1.40, 2.58)

                           -2.21
                        (-4.61,0.19)
    -1.33
 (-3.20, 0.54)

    -2.33
(-4.59, -0.08)
OR (95% Cl) for risk of mental or psychomotor delay (score
<85) per In-unit increase in maternal In-MIBP (each model
adjusted for one or more of the following: specific gravity,
race/ethnicity, maternal marital status and prenatal
alcohol consumption, child's gestational age and sex, and
quality of care-taking environment)
                                                                        Boys (n = 140)   Girls (n = 157)
                                                MDI
                                                PDI
                            0.87
                         (0.60, 1.28)

                            1.80
                         (1.13, 2.87)
                                           0.98
                                        (0.62, 1.56)

                                           1.98
                                        (1.02, 3.83)
                                                Regression coefficient (95% Cl) for change in
                                                neurobehavior per unit increase in maternal In-MIBP
                                                (adjusted for specific gravity; ethnicity; maternal IQ,
                                                demoralization, hardship, satisfaction during pregnancy
                                                and prenatal exposure to PAH and BPA; and child's sex and
                                                age at testing)
                                                                        Boys (n = 129)   Girls (n = 148)
                                                Emotionally reactive


                                                Anxious/depressed


                                                Somatic complaints


                                                Withdrawn behavior
                            0.42
                        (-0.005, 0.85)

                            0.12
                        (-0.38, 0.61)

                            0.31
                        (-0.18,0.81)

                            0.36
                        (-0.05, 0.77)
                                           0.34
                                       (-0.11,0.78)

                                           0.16
                                       (-0.34, 0.66)

                                           0.24
                                       (-0.22, 0.70)

                                           0.47
                                       (-0.007, 0.94)
                                                                            1.21
                                                                        (-0.16,2.56)
Internalizing behavior


No effect modification by gender was observed (p-values
                                           1.20
                                        (-0.15, 2.55)
                                                OR (95% Cl) for child's score in the borderline or clinical
                                                range (compared to normal) per unit increase in maternal
                                                In-MBP (adjusted for specific gravity, maternal
           This document is a draft for review purposes only and does not constitute Agency policy.
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
               Reference and study design
                                                                    Results
                                                    demoralization and satisfaction during pregnancy, and
                                                    child's sex and age at testing)
                                                    Somatic complaints
                                                    Withdrawn behavior
                                                    Internalizing behavior
                                                                       Borderline

                                                                          1.29
  Clinical

   0.76
                                                                            (0.84, 1.99)     (0.42, 1.36)
                                                                         0.81
                                                                       (0.44, 1.51)

                                                                         1.98
   1.62
(0.97, 2.73)

   1.41
                                                                            (1.24, 3.23)     (0.91, 2.18)
1
2
3
4
5
BASC-PRS = Behavior Assessment System for Children —Parent Rating Scales; BPA = bisphenol A; BRIEF = Behavior
 Rating Inventory of Executive Function; HOME = Health Outcomes and Measures of the Environment; LMW = low
 molecular weight; MDI = mental delay index; MMP = monomethyl phthalate; PAH = polycyclic aromatic
 hydrocarbon; PDI = psychomotor delay index
                This document is a draft for review purposes only and does not constitute Agency policy,
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1
2
                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.2.9.  Thyroid Hormone Effects in Humans

        Table 3-12.  Evidence pertaining to DIBP and thyroid hormones in humans
               Reference and study design
                                                                     Results
      Dirtuetal. (2013) (Belgium)
      Population: 152 overweight or obese adults from
      weight loss cohort (ENDORUP) seen at weight
      management clinic, 43 age- and sex-matched
      controls from hospital staff and other volunteers,
      enrolled 2009-2012; among obese/overweight
      group, 65 received bariatric surgery and 87
      received standard diet and lifestyle counseling;
      follow-up 3, 6, and 12 mo
      Outcome:  Serum thyroid hormone levels (details of
      blood collection were not reported)
      Exposure:  Urine sample (24-hr)
      MIBP in urine (ng/mL):
                      Median
      Controls           65
      Obese (at baseline)  58
                              Percentile
                            75th      90th
                            93       133
                            89       129
                                               Regression coefficient (p-value) for change in hormone level
                                               with unit change in In-MIBP (adjusted for age, weight loss,
                                               and sex, or stratified by sex) (0.0 = no effect)

                                                           Full sample     Men         Women

                                               Overweight/obese group

                                               FreeT4      0.07(0.41)  0.11(0.47)      0.05(0.66)

                                               TSH        -0.01(0.93)  0.09(0.58)      -0.01(0.94)

                                               Referent group

                                               FreeT4      0.24(0.14)  0.49(0.12)      0.16(0.44)

                                               TSH         0.23(0.16)  -0.43(0.19)     0.32(0.10)
      Analysis:  Linear regression, adjusting for variables
      shown in results column
3
4
5
      Meeker and Ferguson (2011) (United States)
      Population: Participants in population-based
      survey (NHANES), 2007-2008; 1,346 ages >20 yrs
      and 329 adolescents ages 12-19 yrs
      Outcome:  Serum thyroid hormone levels
      Exposure:  Urine sample collected same day as
      serum sample
      Cr-adjusted MIBP in urine (u.g/g Cr):
                                 Percentile
                  Median      75th        95th
      Adults        6.67       11.1        24.1
      Adolescents   8.24      13.73        28.78
      Analysis: Linear regression adjusting for variables
      shown in results column.
                                               Regression coefficient (95% Cl) for change in hormone level
                                               with unit increase in In-MIBP (adjusted for age, sex, race,
                                               BMI, In-serum cotinine, In-urinary creatinine, and In-urinary
                                               iodine, and weighted for sampling strategy)
                                               TotalT3(ng/dL)

                                               Ln(FreeTS)
                                               (pg/mL)
                                               TotalT4(u.g/mL)

                                               Ln(FreeT4)
                                               (ng/dL)
                                               Ln (TSH)
                                               (ulU/mL)
                                               Ln (Tg) (ng/mL)
     Adults
      0.77
  (-0.59, 2.12)
    -0.0012
(-0.0074, 0.0051)
     0.020
  (-0.075,0.11)
     0.0010
(-0.0094, 0.011)
     -0.013
 (-0.054, 0.028)
     -0.018
 (-0.081, 0.045)
  Adolescents
     2.30
  (-0.81, 0.52)
    0.0083
(-0.0062, 0.023)
    -0.034
  (-0.25,0.19)
   -0.0001
 (-0.021, 0.021)
     0.003
 (-0.076, 0.081)
    -0.047
 (-0.12,0.074)
T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid stimulating hormone
                This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

1    3.2.10. Obesity and Metabolic Effects in Humans

2            Table 3-13. Evidence pertaining to DIBP and obesity in humans
           Reference and study design
                           Results
      Buser et al. (2014) (United States,
      NHANES)
      Population: Participants in population-
      based survey (NHANES), 2007-2010ages
      >6 yrs [sample size not reported]
      Outcome:  BMI measured at exam; divided
      into obese (BMI z-score >95th percentile in
      children, BMI >30 in adults) and
      overweight (BMI z-score 85th-95th
      percentiles in children, BMI 25-29.9 in
      adults).
      Exposure:  Urine sample, collected at same
      time as exam
      Unadjusted MIBP in  urine (ng/mL)
                Geometric mean (SE)
      Ages 6-19 yrs   10.43 (0.39)
      Ages >20 yrs     6.75(0.23)
      Analysis: Logistic regression, considering
      age, race/ethnicity, sex, urinary creatinine,
      poverty income ratio, calorie intake, and
      serum cotinine as potential covariates in
      analyses of ages 6-19 yrs; or age,
      race/ethnicity, sex, education, diabetes,
      alcohol  consumption, cigarette smoking,
      calorie intake, vigorous  recreational
      activities, urinary creatinine, and serum
      cotinine as potential covariates in analysis
      of ages  >20yrs
OR (95% Cl) in children (6-19 yrs of age) for obesity or overweight
comparing highest quartile urinary MIBP (>20.84 ng/mL) with lowest
quartile (<5.38 ng/mL) (adjusted for age, race/ethnicity, calorie
intake, serum cotinine, urinary creatinine, income level)
All
Boys
Girls
     Obese

 1.82 (0.73, 4.57)

4.26 (1.32, 13.74)

 0.57 (0.18, 1.83)
  Overweight

1.85 (0.78, 4.40)

2.22 (0.78, 6.28)

1.57 (0.58, 4.25)
OR (95% Cl) in adults (>20 yrs of age) for obesity or overweight
comparing highest quartile urinary MIBP (>14.40 ng/mL) with lowest
quartile (<3.49 ng/mL) (adjusted for age, gender, race/ethnicity,
calorie intake, recreational activity, serum cotinine, education level,
smoking status, alcohol intake, diabetes)
All
Men
Women
     Obese

 1.40(0.90,2.16)

 0.98 (0.57, 1.67)

 1.81 (0.94, 3.48)
  Overweight

1.18 (0.79,1.78)

1.06 (0.60, 1.89)

1.25 (0.66, 2.36)
      Hart et al. (2013) (Australia)
      Population: 121 girls from birth cohort
      study (Western Australian Pregnancy
      Cohort), whose mothers were recruited at
      18 wks of gestation between 1989 and
      1991; follow-up at ages 14-16 yrs
      Outcome:  Offspring BMI (height and
      weight measured at clinic visit on d 2-5 of
      menstrual cycle)
      Exposure:  Maternal serum samples
      (n = 123) collected at 18 and 34-36 wks of
      gestation (combined aliquot from both
      time periods)
      MIBP in serum (ng/mL):
                 Median  90th percentile
      Unadjusted   1.77       6.16
      Analysis: Correlation between log-
      transformed MIBP and BMI
Authors reported no association between adolescent BMI (either as
absolute value or as age- and gender-adjusted z-score) and any
phthalate metabolite in maternal serum (r = -0.10-0.04,
p = 0.345-0.931)
                This document is a draft for review purposes only and does not constitute Agency policy,
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                Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
      Reference and study design
                          Results
Trasande et al. (2013a) (United States,
NHANES)
Population: 2,884 participants in
population-based survey (NHANES),
2003-2008; 6-19 yrs old
Outcome: BMI z-score, obesity (BMI
z-score >95th percentile), and overweight
(BMI z-score >85th percentile) (measured)
Exposure: Urine sample, collected at same
time as BMI measurement
ZLMW phthalates in urine (nM):
          Geometric mean
Not obese     0.701
Obese  0.855
ZLMW phthalates = sum of MEP, MBP,
MIBP, andMCPP
Analysis: Logistic regression for
overweight and obese classification; linear
regression of BMI z-score as continuous
variable; adjusted for variables shown in
results column
Full sample results, no association with In-LMW phthalates: OR or
regression coefficient (95% Cl) per one unit increase in £LMW
phthalates (nM) (adjusted for urinary creatinine, sex, poverty-
income ratio, parental education, serum cotinine, age, and
race/ethnicity, caloric intake, and television watching)
Overweight
Obese
BMI z-score
       OR (95% Cl)

       OR (95% Cl)

        P (95% Cl)
              1.01(0.90,1.13)

              1.02(0.90,1.17)

              0.03 (-0.03, 0.09)
Interaction by ethnicity seen, with associations seen between In-
LMW phthalates and each of the obesity measures in blacks, but not
in whites or Hispanics. The patterns seen with ZLMW phthalates
were also seen in analyses for MIBP.  Using same adjustment factors
as above, the associations with In-MIBP are:
               ILMW phthalates
             Hispanic       White
                                  MIBP
                            Black         Black
Over-
weight OR
(95% Cl)

Obese OR
(95% Cl)

BMIz-
score P
(95% Cl)
   0.88
(0.72, 1.08)
   0.97
(0.78, 1.22)
                 1.21
             (1.05, 1.39)
   1.16
(0.99, 1.37)
                                                      0.97         0.94
                                                   (0.83, 1.14)   (0.69, 1.29)
                             1.22         1.17
                         (1.07, 1.39)   (0.97, 1.41)
                                                      -0.04
                                                     (-0.15,
                                                      0.06)
   0.02
(-0.08,0.12)
                            0.09
                         (0.003,0.18)
                            0.08
                           (-0.01,
                            0.17)
Wang et al. (2013) (China)
Population: 259 primary and middle
school students, 8-15 yrs old, stratified
sample from six schools, selected based on
sex and BMI
Outcome: BMI, waist circumference
(measured)
Exposure: First morning urine sample,
collected at same time as BMI
measurement
MIBP in urine (ng/mL):
     Geometric mean (SD)
          38.9(1.1)
Low molecular  weight phthalate
metabolites included MMP, MEP, MBP,
MIBP, and MHBP
Analysis: Linear regression, sampling
weights applied to adjust for sampling
strategy; adjusted for variables shown in
the results column
Regression coefficient (95% Cl) for change in BMI or waist
circumference per unit increase in SG-adjusted InMIBP (adjusted for
age and sex in Model 1; plus sum  of DBP, MMP, and MEP in Model
2)
BMI

Waist
circumference
          Model 1

     0.027 (0.006, 0.048)

     0.022(0.005, 0.038)
                    Model 2

              0.020 (-0.005, 0.045)

              0.019 (-0.001, 0.038)
           This document is a draft for review purposes only and does not constitute Agency policy,
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                Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
      Reference and study design
                          Results
Dirtuetal. (2013) (Belgium)
Population:  152 overweight or obese
adults from weight loss cohort (ENDORUP)
seen at weight management clinic, 43 age-
and sex-matched controls from hospital
staff and other volunteers, enrolled
2009-2012; among obese/overweight
group, 65 received bariatric surgery and
87 received standard diet and lifestyle
counseling; follow-up 3, 6, and 12 mo
Outcome: Waist circumference measured
at each follow-up visit
Exposure: Urine sample (24-hr sample)
MIBP, in urine (ng/mL):
                Percentile
        Median  75th   90th
Controls   65     93    133
Obese     58     89    129
(at baseline)
Analysis: Linear regression, adjusting for
variables shown in results column;
treatment of repeated urinary phthalate
measures was not specified
Regression coefficient (p-value) for change in waist circumference
with unit change in In-MIBP (adjusted for age, weight loss, and sex,
or stratified by sex) (0.0 = no effect)
Overweight/ obese
group

Referent group
Full sample

0.07 (0.40)
   Men

-0.16(0.30)
 Women

0.03 (0.76)
-0.16(0.30)   0.07(0.81)      -0.01(0.98)
Lind et al. (2012a) (Sweden)
Population:  1,016 (507 men, 509 women),
from population-based cohort (Prospective
Investigation of Vasculature in Uppsala
Seniors study), 2001-2003; age 70 yrs at
enrollment
Outcome: BMI, waist circumference
measured at enrollment; DXA (n = 890
participated) and MRI of abdominal region
(n = 287 randomly selected) 2 yrs later
Exposure: Serum sample (fasting),
collected at baseline
MIBP in serum (ng/mL):
         Median  75th percentile
Women   13.4        24.5
Men      13.5        33.3
Analysis: Linear regression, adjusted for
variables shown in results column
Related reference: Olsen etal. (2012)
reports cross-sectional analysis of BMI
from this study population, see Table 14
Regression coefficient (95% Cl) for change in body metric per unit
increase in In-MIBP (ng/mL) (adjusted for serum cholesterol and
triglycerides, education, exercise, and smoking)
       Outcome

BMI (kg/m2)


Waist circumference
(cm)

DXA total fat (kg)

MRI visceral adipose
tissue (cm2)
          Males
        P (95% Cl)

       -0.083 (-0.35,
          0.19)

       -0.025 (-0.80,
          0.75)

      -73 (-754, 608)

       -5.9 (-24, 13)
               Females
              P (95% Cl)

           0.39 (0.002, 0.79)


            1.3 (0.425, 2.3)


           1,079 (283, 1875)

              14 (1.4, 26)
           This document is a draft for review purposes only and does not constitute Agency policy,
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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
      Reference and study design
                           Results
Teitelbaum et al. (2012) (United States,
New York City)
Population: 387 children (80 boys, 307
girls) in child development cohort (Growing
Up Healthy Study), 2004-2008; Hispanic
and black), 6-8 yrs at enrollment
Outcome: BMI and waist circumference
measured 1 yr after enrollment; normal
weight = BMI <85th percentile (n = 2,284);
overweight = BMI >85th percentile (n = 578)
Exposure: Urine sample, collected at
enrollment
Cr-adjusted phthalates in urine (u.g/g Cr),
median:
         MIBP  ILMW phthalates
Boys     22.7        253.2
Girls     22.2        294.0
Low molecular weight phthalate
metabolites included MEP, MBP, MIBP, and
MCPP.
Analysis: Linear regression, considering
sex, age at baseline, sedentary hrs,
metabolic equivalent hrs, caloric intake,
race, ethnicity, season of urine collection,
family income, and parent education as
potential covariates; restricted to children
with creatinine >10 mg/dL
Full sample results, regression coefficient (95% Cl) for change in
body metric per unit change in In-MIBP (ng/g Cr) (adjusted for
creatinine, age, sex, sedentary hrs, metabolic equivalent hrs,
Hispanic ethnicity, caloric intake, season, and parental education
level)
BMI (kg/m2)
Waist circumference (cm)
                                   -0.27 (-0.73, -0.18)

                                   -0.62 (-1.84, -0.61)
Olsenetal. (2012) (Sweden)
Population:  1,016 (507 men, 509 women),
from population-based cohort (Prospective
Investigation of Vasculature in Uppsala
Seniors study), 2001-2003; age 70 yrs at
enrollment
Outcome: BMI measured at study visit
Exposure: Serum sample, collected at time
of examination; results not shown
Analysis: Linear regression, adjusted for
the variables shown in results column
Regression coefficient for change in outcome per unit increase in In-
MIBP (adjusted for sex, smoking, diabetes (except for glucose) and
the other variables in the table; model for Framingham Risk Score
only adjusted for sex)
BMI
                                     0.094 (-0.13, 0.32)
           This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
           Reference and study design
                                                                 Results
      Kasper-Sonnenberg et al. (2012)
      (Germany)
      Population: 104 mothers (and children)
      enrolled in birth cohort study, children
      born between 2000 and 2002, follow-up in
      2007-2009; mean age 39.2 yrs (mothers),
      6.8yrs (children)
      Outcome:  BMI based on questionnaire
      (mothers) and measurements (children)
      Exposure:  Urine sample (first morning),
      collected on same day as exam
      Cr-adjusted MIBP and OH-MIBP in urine
      (ug/gCr):
               Geometric mean (95% Cl)
      Children
                  64.6 (55.2, 75.7)
                  34.2 (28.5, 41.0)
                   101 (87.2, 118)
                                       Spearman correlation coefficient between £DIBP anc| BMI jn
                                       Children                              -0.035 (p> 0.05)
                                       Mothers                              -0.137 (p> 0.05)
  MIBP
  OH-MIBP
  IDIBP
Adults
  MIBP
  OH-MIBP
  IDIBP
                   37.2(31.8,43.5)
                   17.4(15.1,20.0)
                   55.9 (48.4, 64.5)
      Analysis: Spearman's rank correlation
      analysis
1
2
3
4
      Svensson et al. (2011) (Mexico)
      Population: 182 women; healthy controls
      without diabetes from case-control study
      of breast cancer, 2007-2008; mean age
      54 yrs
      Outcome:  BMI, waist circumference, and
      waist:height ratio
      Exposure:  First morning urine sample
      collected at time of clinical evaluation
      Cr-adjusted MIBP in urine (u.g/g Cr):
               Geometric mean (SD)
      No diabetes     9.1(2.3)
      Analysis: Spearman correlation coefficient
      Related references:  Lopez-Carrillo et al.
      (2010)
                                       Spearman correlation coefficient between anthropometric measure
                                       and In-MIBP in urine (u.g/g Cr)
                                       BMI (kg/m2)
                                       Waist circumference (cm)
                                       Waist/height ratio
                                       (p > 0.05 for all parameters)
0.0457

0.0151

-0.0156
DXA = dual energy x-ray absorptiometry; MCPP = mono-(3-carboxypropyl) phthalate; MHBP = mono-
 (S-hydroxybutyl)phthalate; MRI= magnetic resonance imaging; SE = standard error
                This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1
2
Table 3-14. Evidence pertaining to DIBP and diabetes/insulin resistance in
humans
           Reference and study design
                                                           Results
      Diabetes diagnosis
      James-Todd et al. (2012) (United States,
      NHANES)
      Population: 215 cases, 1,235 controls from
      population-based survey (NHANES),
      2001-2008; women age 20-79 yrs
      Outcome:  Positive response to, "Other
      than during pregnancy, have you ever been
      told by a doctor or health professional that
      you have diabetes or sugar diabetes?"
      Exposure:  Urine sample, collected  at time
      of survey
      MIBP in urine (units not reported):
                Geometric mean
      Unadjusted      3.7
      (based on larger sample of 2,350 women)
      Analysis: Logistic regression, adjusting for
      variables shown in the results column
                                OR (95% Cl) for diabetes by quartile of MIBP (adjusted for urinary
                                creatinine, age, race/ethnicity, education, poverty status, fasting
                                time, total caloric intake, total fat intake, smoking status, and
                                physical activity; little change with additional adjustment for BMI
                                and waist circumference)
                                MIBP quartile
                                1 (low)
                                2
                                3
                                4 (high)
 1.0 (referent)
1.04 (0.66-1.67)
1.69 (0.93-3.06)
1.95 (0.99-3.85)
      Lind et al. (2012b) (Sweden)
      Population: 1,003 (501 men, 502 women),
      from population-based cohort (Prospective
      Investigation of Vasculature in Uppsala
      Seniors study), 2001-2003; age 70 yrs at
      enrollment
      Outcome:  Diabetes (n = 88; history of
      diabetes or fasting glucose >7.0 mmol/L,
      mean duration 8.9 yrs);
      Exposure: Serum sample (fasting),
      collected at time of clinical assessment
      MIBP in serum (ng/mL):
               Median 75th percentile
      Women    13.4       24.5
      Men      13.5       33.3
      Analysis: Logistic regression for diabetes
      classification, adjusting for variables shown
      in results column
                                OR (95% Cl) per unit increase in serum In-MIBP (adjusted for sex,
                                serum cholesterol and triglycerides, BMI, smoking, exercise, and
                                education)
                                                       1.30(1.10,1.55)

                                OR (95% Cl) by quintile of In-MIBP (adjusted for sex, serum
                                cholesterol and triglycerides, BMI, smoking, exercise, and education)
                                MIBP quintile
                                1 (low)
                                2
                                3

                                4
                                5 (high)
                                (trend p)
 1.0 (referent)
1.19(0.59,2.38)
0.84 (0.41, 1.76)

 1.37 (0.7, 2.66)
2.00 (1.03, 3.99)
    (0.038)
                This document is a draft for review purposes only and does not constitute Agency policy,
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                 Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
      Reference and study design
                           Results
Svensson et al. (2011) (Mexico)
Population: 221 women with diabetes,
182 healthy without diabetes from case-
control study of breast cancer, 2007-2008;
mean age 54 yrs
Outcome: Self-reported diabetes
Exposure: First morning urine samples
MIBP in urine (u.g/g creatinine):
         Geometric mean (SD)
No diabetes    9.1(2.3)
Diabetes       7.9(2.1)
Analysis: Logistic regression, adjusted for
variables shown in the results column (age
and waist-height ratio not found to be
potential confounders)
OR (95% Cl) per unit increase in In-MIBP (adjusted for creatinine and
education)
                       1.01 (0.65,1.55)
Markers of insulin resistance
Huang etal.(2014a) (United States,
NHANES)
Population:  3,083 participants in
population-based survey (NHANES),
2001-2008; ages 12-<80 yrs; self-reported
non-diabetic, non-pregnant participants
Outcome: Fasting blood glucose; fasting
insulin; HOMA-IR
Exposure: Urine sample at time of clinical
exam
Cr-adjusted MIBP in urine (ng/g Cr):
          Median   75th percentile
Men        3.8         6.6
Women     4.9         8.9
Analysis: Logistic regression, adjusting for
variables shown in the results column
Median change (95% Cl) in biomarkers for diabetes by quartile of
MIBP (adjusted for age, gender, race/ethnicity, fasting time, urinary
creatinine, total caloric intake, triglycerides, education, and poverty
and smoking status)
MIBP
quartile
1 (low)
2
3
4 (high)
(pfor
trend)
 Fasting glucose
 1.0 (referent)
1.87 (0.83, 2.92)
2.77 (1.75, 3.80)
3.69 (2.60, 4.78)
   (<0.0001)
 Fasting insulin       HOMA-IR
 1.0 (referent)      1.0 (referent)
1.45 (0.85, 2.04)   0.38 (0.23, 0.52)
1.23 (0.57, 1.89)   0.35 (0.19, 0.51)
1.73 (0.92, 2.54)   0.53 (0.33, 0.72)
    (0.0028)          (0.0002)
Trasande et al. (2013c) (United States,
NHANES)
Population: 766 participants in the
2003-2008 NHANES, 12-19 yrs old
Outcome:  HOMA, calculated as fasting
glucose (mmol/L) multiplied by fasting
insulin (nU/mL divided by 22.5.
Exposure:  Urine sample, collected at same
time as insulin resistance measurements.
ZLMW phthalates in urine (nM):
           Median   75th percentile
Unadjusted  0.83         1.89
ZLMW phthalates = sum of MEP,  MBP, and
MIBP
Urinary concentration of MIBP alone not
reported.
Analysis: HOMA-IR assessed as continuous
or categorical variable; categorical analysis
OR (95% Cl) for insulin resistance and In-urinary metabolite
concentration (nM), adjusted for urinary creatinine, BMI category,
continuous age, race/ethnicity, caregiver education, poverty-income
ratio, gender, serum cotinine, and caloric intake
Ln-MIBP
Ln-ZLMW
                           1.57(1.18,2.09)
                           0.92 (0.71, 1,19)
Regression coefficient (95% Cl) for increase in In-HOMA-IR per unit
increase in In-urinary metabolite concentration (nM), adjusted for
urinary creatinine, BMI category, continuous age, race/ethnicity,
caregiver education, poverty-income ratio, gender, serum cotinine,
and caloric intake.
Ln-MIBP
Ln-ZLMW
                           0.15(0.04,0.26)
                          -0.07 (-0.18, 0.04)
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
used cut point of 4.39, reflecting >2 SD
above the mean HOMA-IR for normal
weight adolescents with normal fasting
glucose in NHANES 1999-2002. Linear and
logistic regression analyses, adjusting for
variables shown in results column. HOMA-
IR and urinary phthalate measures natural-
log transformed for analysis.
James-Todd et al. (2012) (United States,
NHANES)
Population: 2,092 women without history
of diabetes with various measures of
insulin resistance from population-based
survey (NHANES), 2001-2008; women age
20-79 yrs
Outcome: Among women without history
of diabetes, FBG (n = 985), HOMA-IR
(n = 971), glycosolated hemoglobin Ale
(n = 2,092)
Exposure: Urine sample, collected at time
of survey
MIBP in urine (units not reported):
Geometric mean
Unadjusted 3.7
Analysis: Logistic regression, adjusting for
variables shown in the results column








Results

Among women
without diabetes, difference (from first quartile) in
median value (95% Cl) of glucose and insulin parameters by quartile
of MIBP (Model
education level,
total fat intake,
1 adjusted for urine creatinine, age, race/ethnicity,
poverty status, fasting time, total caloric intake,
smoking status, and physical activity; Model 2 also
adjusted for BMI and waist circumference)
MIBP Quartile
FBG (mg/dL)
1 (low)

2
3
4 (high)
Ln (HOMA)

1 (low)
2
3
4 (high)
Ale (%)
1 (low)
2
3
4 (high)
Model 1 Model 2

(referent) (referent)

3.08 (1.22, 4.93) 3.03 (1.05, 5.00)
3.50(1.45,5.54) 3.17(1.17,5.17)
5.86 (3.55, 8.17) 6.04 (3.81, 8.28)


(referent) (referent)
0.13 (-0.02, 0.28) 0.13 (0.01, 0.25)
0.08 (-0.08, 0.25) 0.10 (-0.01, 0.21)
0.22(0.06,0.38) 0.18(0.06,0.31)

(referent) (referent)
0.03 (-0.01, 0.08) 0.03 (-0.01, 0.08)
0.03 (-0.02, 0.09) 0.04 (0.00, 0.09)
0.01 (-0.05, 0.07) 0.01 (-0.04, 0.07)
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
           Reference and study design
                                                                  Results
      Lind et al. (2012b) (Sweden)
      Population: 1,003 (501 men, 502 women),
      from population-based cohort (Prospective
      Investigation of Vasculature in Uppsala
      Seniors study), 2001-2003; age 70 yrs at
      enrollment
      Outcome:  Ratio of fasting proinsulin to
      insulin; HOMA
      Exposure: Serum sample (fasting),
      collected at time of clinical assessment
      MIBP in serum (ng/mL):
              Median 75th percentile
      Women   13.4        24.5
      Men      13.5        33.3
      Analysis:  Linear regression for continuous
      outcomes (proinsulin/insulin and HOMA-
      IR); adjusting for variables shown in results
      column
      Related reference: Olsen etal. (2012)
      presents blood glucose data for this study
      population; the regression coefficient per
      unit increase in serum In-MIBP was 0.024
      (0.01, 0.04) (see Table 14)
                                        Regression coefficient (95% Cl) for insulin measures per unit
                                        increase in serum In-MIBP (adjusted for sex, serum cholesterol and
                                        triglycerides, BMI, smoking, exercise, and education)
                                        Proinsulin/insulin
                                        HOMA
   0.06 (0.03, 0.089)
  0.014 (-0.015, 0.043)
                                        The magnitude of the association between proinsulin/insulin and
                                        MIBP was similar to that for MEHP, but in the opposite direction of
                                        MEP and MMP (-0.05 and -0.005, respectively).  The magnitude of
                                        the association between HOMA-IR and MIBP was lesser than that
                                        for MEP and MMP. The magnitude of the association between
                                        prevalent diabetes and  MIBP was greater than that for MEHP, and
                                        less than that for MEP and MMP in the highest quintile.
      Olsen etal. (2012) (Sweden)
      Population: 1,016 (507 men, 509 women),
      from population-based cohort (Prospective
      Investigation of Vasculature in Uppsala
      Seniors study), 2001-2003; age 70 yrs at
      enrollment
      Outcome:  Fasting serum sample for
      glucose
      Exposure:  Serum sample, collected at time
      of examination; results not shown
      Analysis:  Linear regression, adjusted for
      the variables shown in results column.
                                        Regression coefficient for change in outcome per unit increase in In-
                                        MIBP (adjusted for sex, smoking, diabetes (except for glucose), and
                                        the other variables in the table; model for Framingham Risk Score
                                        only adjusted for sex)
                                        Fasting serum glucose
0.024 (0.01, 0.04; p = 0.0001
1
2
FBG = fasting blood glucose; HOMA-IR = homeostatic model assessment of insulin resistance
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1
2
Table 3-15. Evidence pertaining to DIBP and cardiovascular disease risk
factors in humans
            Reference and study design
                                                            Results
      Shiue (2014) (United States, NHANES)
      Population: 2,489 participants in
      population-based survey (NHANES),
      2011-2012; ages >20 yrs
      Outcome:  High blood pressure (systolic
      blood pressure >140 mmHg and diastolic
      blood pressure >90 mmHg)
      Exposure:  Urine sample collected at time
      of clinical exam
      MIBP in urine (units not given)
                  Mean ±SD
      Normal BP  13.13 ±22.17
      High BP     15.71 ±25.15
      Analysis: Survey-weighted logistic
      regression, adjusting for variables shown in
      results column; t-test for comparison
      between concentrations
                                 OR (95% Cl) for high blood pressure with increased log-transformed
                                 MIBP (adjusted for urinary creatinine, age, sex, ethnicity, BMI, and
                                 sampling weights)
                                                        1.14 (0.92,1.41)
                                 Mean ± SD MIBP in urine (units not given) in participants with
                                 normal and high BP
                                 Normal BP (n = 2,180)
                                 High BP (n = 309)
                  13.13 ±22.17

                  15.71 ±25.15
      Trasande et al. (2013b) (United States,
      NHANES)
      Population: 2,447 children in population-
      based survey (NHANES), 2003-2008; ages
      8-19 yrs old
      Outcome: Systolic BP and diastolic BP
      z-score (based on height-, sex-, and age-
      normalized values); prehypertension (BP
      >90th percentile for age/height/sex); fasting
      serum triglycerides (n = 906;  high =
      >100 mg/dL); nonfasting high density
      cholesterol (HDL; n = 2,555;
      low = <40 mg/dL)
      Exposure: Urine sample, collected at time
      of BMI measurement
      ILMW phthalates in urine (nM):
                    Geometric mean
      BP <90th percentile    0.817
      BP >90th percentile    1.002
      ILMW phthalate = sum of MEP, MBP, and
      MIBP
      Analysis: Logistic regression  for pre-
      hypertension (BP >90th percentile)
      classification; linear regression for systolic
      BP and diastolic BP z-score and triglycerides
      and HDL as continuous variable; all models
      adjusted for variables shown in results
      column
                                 Changes in z-score (95% Cl) per unit increase in In-phthalates
                                 (adjusted for sex, caloric intake, television watching,
                                 poverty:income, parental education, serum cotinine, urinary
                                 creatinine, BMI, race/ethnicity, and age)
                                 Systolic BP
                                 Diastolic BP
                                 Triglycerides
                                 HDL
ILMW phthalates

0.03 (-0.02, 0.07)

0.02 (-0.04, 0.07)

-0.22 (-4.40, 0.07)

0.13 (-0.60, 0.85)
      MIBP

0.03 (-0.02, 0.08)

-0.02 (-0.09, 0.04)

   not reported

   not reported
                                 OR (95% Cl) for BP >90th percentile per unit increase in In-
                                 phthalates
                                 BP >90th percentile
                                 High triglycerides
                                 Low HDL
ILMW phthalates

 1.19 (0.96, 1.47)

 0.85 (0.71, 1.01)

 1.00(0.87,1.15)
      MIBP

 1.00 (0.74, 1.35)

   not reported

   not reported
                                 Interactions with covariates examined in supplemental analyses;
                                 stratified analyses showed no statistically significant associations
                                 between ILMW phthalates and systolic BP for gender, age,
                                 race/ethnicity, cotinine level, or BMI
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Olsen et al. (2012) (Sweden)
Population: 1,016 (507 men, 509 women),
from population-based cohort (Prospective
Investigation of Vasculature in Uppsala
Seniors study), 2001-2003; age 70 yrs at
enrollment
Outcome: Blood pressure measured at
study visit; fasting serum sample for LDL
and HDL cholesterol, and triglycerides;
Framingham risk score
Exposure: Serum sample, collected at time
of examination; results not shown
Analysis: Linear regression, adjusted for
the variables shown in results column
Lind and Lind (2011) (Sweden)
Population: 1,016 (507 men, 509 women),
from population-based cohort (Prospective
Investigation of Vasculature in Uppsala
Seniors study), 2001-2003; age 70 yrs at
enrollment
Outcome: Carotid artery intima media
thickness (IMT); grey scale media of the
intima media complex (IM-GSM); plaque in
carotid artery
Exposure: Serum sample (fasting),
collected at time of clinical assessment
MIBP in serum (ng/mL):
Median 75th percentile
13.5 29.3
Analysis: Linear regression for continuous
outcomes (IMT, IM-GSM) and ordinal
logistic regression for number of carotid
arteries with plaques (0, 1, 2), adjusted for
variables shown in results column













Results
Regression coefficient for change in outcome per unit increase in In-
MIBP (adjusted for sex, smoking, diabetes and the other variables in
the table; model for Framingham Risk Score only adjusted for sex)
/D rc ci \
(P [SE])
LDL 0.044 (-0.01, 0.09)

HDL 0.017 (-0.01, 0.09)
Triglycerides -0.009 (-0.03, 0.01)

Systolic BP -0.05 (-1.28, 1.18)
Diastolic BP 0.35 (-0.20, 0.90)
Framingham risk score 0.13 (-0.05, 0.31)
Median IMT by quintile of MIBP (adjusted for sex, BMI, fasting
blood glucose, systolic BP, diastolic BP, HDL and LDL cholesterol,
triglycerides, smoking, antihypertensive treatment, statin use)
MIBP
quintile IMT IM-GSM
Median IM-
Median IMT (p-value) GSM (p-value)
1 (low) 0.87 (referent) 80 Referent
2 0.89 (0.91) 72 (0.0001)

3 0.86 (0.13) 68 (0.0001)
4 0.89 (0.91) 69 (0.0001)

5 (high) 0.85 (0.074) 102 (0.0001)
Regression coefficient (P [p-value]) per unit increase in serum MIBP
(adjusted for sex, BMI, fasting blood glucose, systolic BP, diastolic
BP, HDL and LDL cholesterol, triglycerides, smoking,
antihypertensive treatment, statin use)
IMT -0.0045(0.14)
IM-GSM 5.5 (O.OOOlp
OR for presence of plaques and median value of plaque GSM by
quintile of MIBP (adjusted for sex, BMI, fasting blood glucose,
systolic BP, diastolic BP, HDL and LDL cholesterol, triglycerides,
smoking, antihypertensive treatment, statin use)
MIBP
quintile Plaque prevalence Plaque GSM
OR (p-value) Median (p-value)
1 (low) 1.0 (referent) 65 (referent)
2 0.70 (0.059) 69 (0.37)
3 0.74 (0.17) 59 (0.11)
4 1.00 (0.78) 62 (0.074)
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
           Reference and study design
                                                                Results
                                            5 (high)         0.64       (0.011)        99        (0.0001)

                                            OR or regression coefficient per unit increase in serum MIBP
                                            Plaque
                                            prevalence

                                            Plaque GSM
                                                         OR (95% Cl)
                                                           (p-value)
0.88 (0.79, 0.98)
  8.0 (0.0001)
                                            The regression models did not show evidence of interaction by
                                            gender, except for IMT (interaction term p-value = 0.030).
1
2
3
4
BP = blood pressure; HDL= high-density lipoprotein; IM-GSM = grey scale media of the intima media complex;
 IMT = intima media thickness; LDL = low-density lipoprotein
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                                                   3-42             DRAFT—DO NOT CITE OR QUOTE

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         Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

Table 3-16. Evidence pertaining to DIBP and cancer in humans
 Reference and study design
                                                                   Results
                                    Geometric mean (95% Cl) DBP in urine (u.g/g Cr), all subjects
                                    and by menopausal status
Lopez-Carrillo et al. (2010) (Mexico)
Population: 233 incident cases, 221 population
controls matched by age and residency, >18 yrs
of age, >1 yr in study area, 2007-2008; mean
age 53 yrs; participation rates: 94.8% of cases
and 99.5% of controls
Outcome:  Histologically-confirmed breast
cancer
Exposure:  Urine sample (for cases, urine
collected on average 2 mo after diagnosis, but
before treatment)
MIBP in urine, controls:
                 Geometric mean
Cr-adjusted (u.g/g Cr)    8.85
Analysis: Logistic regression, adjusting for
variables shown in results column
                                    All
                                    Pre-menopause
                                    Post-menopause
    Controls

8.85 (7.95, 9.84)

9.99 (8.42, 11.85)

8.32 (7.27, 9.52)
     Cases

7.81(6.93,8.81)

8.31 (6.85, 10.09)

7.53 (6.45, 8.78)
                                    OR (95% Cl) for breast cancer, by tertile of MIBP (adjusted for
                                    current age, age at menarche, parity, menopausal status, and
                                    other phthalate metabolites)
                                    MIBP tertile
                                    (Hg/g Cr)
                                    1 (0.23-7.44)

                                    2 (7.45-12.07)

                                    3 (12.08-86.22)

                                    (trend p)
                                                               1.0 (referent)

                                                              0.59 (0.35, 0.98)

                                                              0.73 (0.43, 1.24)

                                                                  (0.365)
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                  Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
1   3.3.  Experimental Studies

2   3.3.1.  Developmental Effects

3          Table 3-17. Evidence pertaining to developmental effects in animals following
4          oral exposure to DIBP
Reference and study design
Results3
Fetal survival
See Table 3-19
Fetal growth
BASF (2007)
Rat (Wistar); 22-23 dams/group
0, 88, 363, 942 mg/kg-day
Diet
CDs 6-20 (GD 20 c-section)
Borch et al. (2006)
Rat (Wistar); 11-12 dams/group
0, 600 mg/kg-day
Gavage
GDs 7-19 (GD 19 c-section) or
G Ds 7-20/2 1(GD 20/21
c-section); 5-6 dams/group per
time point
Saillenfait et al. (2006)
Rat (Sprague-Dawley);
20-22 dams/group
0, 250, 500, 750, 1,000 mg/kg-day
Gavage
GDs 6-20
(GD 21 c-section)
Fetal body weight (percent
Doses
MBW
FBW
Fetal body weight (percent
Doses (M)
BW(GD19)
(data presented in graphb)
B W (GD 20/21)
(data presented in graphb)
Doses (F)
BW(GD19)
(data presented in graphb)
B W (GD 20/21)
(data presented in graphb)
change compared to control)
0 88
0% -3%
0% -3%
change compared to control)
0
0%
0%
0
0%
0%

363 942
-3% -5%**
-3% -6%**

600
-27%*
-12%
600
-28%*
12%
Fetal body weight (mean percent change compared to control)
Doses
M and F (all fetuses) BW
MBW
FBW
0 250 500
0% 0% -7%**
0% 0% -6%*
0% -1% -8%**
750 1,000
-17%** -24%**
-17%** -25%**
-18%** -26%**
              This document is a draft for review purposes only and does not constitute Agency policy,
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Results3
Postnatal survival
Saillenfait et al. (2008)
Rat (Sprague-Dawley);
11-14 dams/group
0, 125, 250, 500, 625 mg/kg-day
GDs 12-21 (dams allowed to
deliver)
Pup survival (percent change compared to control [litter means])
Doses 0 125 250 500
percentage pup survival 0% -1% -1% -1%
PNDs 1-4
percentage pup survival 0% 2% 5% 3%
PNDs 4-21
625
-7%
5%
Postnatal and adult growth
Eastman Kodak (1954)
Rat (no strain designation);
5 male and 5 females/group
0, 0.1, 1, 5% DIBP (0, 97, 1,000,
7,800 mg/kg-day for males; 0,
110, 1,100, 6,400 mg/kg-day for
females)0
Diet
Weaning to 8 weeks
post-weaning
Body weight gain (percent change compared to control)
Doses (M) 0 97 1,000
BW gain (weaning to 0% 3% -2%
4 weeks post-weaning)
Doses (F) 0 110 1,100
BW gain (weaning to 0% -9% 1%
4 weeks post-weaning)
7,800
-61%
6,400
-34%
Body weight (percent change compared to control)
Doses (M) 0 97 1,000
4 weeks post-weaning 0% 2% -1%
BW
Doses (F) 0 110 1,100
4 weeks post-weaning 0% -5% 1%
BW
7,800
-41%
6,400
-19%
Body weight gain (percent change compared to control)
Doses (M) 0 97 1,000
BW gain (weaning to 0% 3% -3%
8 weeks post-weaning)
Doses (F) 0 110 1,100
BW gain (weaning to 0% -11% 0%
8 weeks post-weaning)
7,800
-58%
6,400
-34%
Body weight (percent change compared to control)
Doses (M) 0 97 1,000
8 weeks post-weaning 0% 2% -3%
BW
Doses (F) 0 110 1,100
8 weeks post-weaning 0% -7% 0%
BW
Note: Statistical analysis not reported in study.
7,800
-44%
6,400
-22%
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Hazleton Laboratories (1992,
1987): NIOSH (1983)
Mouse (CD-I); 50 control
females; 10 females/treated
group (6.5-9 weeks old)
0, 1,000, 1,795, 3,225, 5,790,
10,400 mg/kg-day
Gavage
8 days
Oishi and Hiraga (1980d)
MIBP
Rat (Wistar) (JCL);
10 males/group
0, 2% in diet (0,
l,100mg/kg-day)d
Diet
1 week
Oishi and Hiraga (1980a)
Mouse (JCLICR); 10 males/group
0, 2%(0,2,100mg/kg-day)d
Diet
1 week
Saillenfait et al. (2008)
Rat (Sprague-Dawley);
11-14 dams/group
0, 125, 250, 500, 625 mg/kg-day
Gavage
Results3
Body weight change (g)
Doses (F) 0
BW change 0
(days 1-8 of study)
1,000 1,795 3,225
010
5,790 10,400
1 1
Body weight change (g)
Doses (F) 0
BW change (days 1
1-12 of study)
1,000 1,795 3,225
110
5,790 10,400
1 1
Body weight change (g)
Doses (F) 0
1,000 1,795 3,225
BW change (days 1111
1-16 of study)
Note: Statistical analysis not reported in study.
Body weight and weight gain
Doses (M)
5,790 10,400
1 1
(percent change compared to control)
0
B Wat 6 weeks 0%
B W gain (5-6 weeks* ) 0%
Note: Statistical analysis was not performed on BW gain.
Body weight and weight gain
Doses (M)
B W at 6 weeks
BW gain (5-6 weeks6)
1,100
-10%*
-31%
(percent change compared to control)
0
0%
0%
2,100
-13%*
-54%
Note: Statistical analysis was not performed on BW gain.
Body weight (percent change compared to control [litter means])
Doses 0
M postnatal (PND 1) 0%
BW
M postnatal (PND 21) 0%
BW
F postnatal (PND 21) 0%
BW
125 250
-1% -2%
-1% -3%
-3% -5%
500 625
-2% -10%**
-6% -10%*
-3% -10%
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
GDs 12-21 (dams allowed to
deliver)
University of Rochester (1953)
Rat (Albino; no other strain
designation); 5 males/group
0, 0.01, 0.1, 1, 2, 5% (0, 15, 140,
1,400, 3,000, 8,900 mg/kg-day)f
Diet
Weaning to 1 month
post-weaning
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and
5 females/group
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for
females)h
Diet
Weaning to 4 months
post-weaning
Results3
M BW at day of PPS 0% -8%* -5%* 7%
M adult (PNDs 77-84) 0% -6% -4% -7%*
BW
2%
-9%**
Body weight gain (percent change compared to control)
Doses (M) 0 15 140 1,400 3,000
BWgaine(weaningto 0% -22% -19% -22% -27%
1 month post-weaning)
Note: Statistical analysis was not performed on BW gain.
8,900
-51%
Body weight (percent change compared to control)
Doses (M) 0 15 140 1,400 3,000
1 month post-weaning 0% -16% -14% -16% -20%
BW
Note: Statistical analysis not reported in study.
8,900
-38%
Body weight gain (percent change compared to control)
Doses (M) 0 15 140 1,400 3,000
BW gain (weaning to 0% -20% -18% -21% -27%
PND 49 [-after PPS^])
Note: Statistical analysis was not performed on BW gain.
8,900
-49%
Body weight (percent change compared to control)
Doses (M) 0 15 140 1,400 3,000
PND 49 Rafter PPSg) 0% -14% -13% -15% -19%
BW
Note: Statistical analysis not reported in study.
8,900
-35%
Body weight gain (percent change compared to control)
Doses (M) 0 65 710
BW gain (weaning to 0% 5% -6%
1 month
post-weaning)8
Doses (F) 0 82 770
BW gain (weaning to 0% -7% 3%
1 month
post-weaning)8
Note: Statistical analysis was not performed on BW gain.
5,800
-60%
4,700
-27%
Body weight (percent change compared to control)
Doses (M) 0 65 710
BW at 1 month 0% 4% -4%
post-weaning
5,800
-42%
This document is a draft for review purposes only and does not constitute Agency policy,
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

Results3
Doses (F) 0 82 770
BW at 1 month 0% -4% 2%
post-weaning
Note: Statistical analysis not reported in study.
4,700
-16%
Body weight gain (percent change compared to control)
Doses (M) 0 65 710
BW gain (weaning to 0% 3% -2%
PND 49 [-after PPS^]f
Doses (F) 0* 82 770
BW gain (weaning to 0% -8% 3%
PND 49 [-after V(y]e
Note: Statistical analysis was not performed on BW gain.
5,800
-61%
4,700
-33%
Body weight (percent change compared to control)
Doses (M) 0 65 710
BW at PND 49 (-after 0% 2% -1%
PPSQ)
Doses (F) 0 82 770
BW at PND 49 (-after 0% -4% 1%
VCft)
Note: Statistical analysis not reported in study.
5,800
-41%
4,700
-18%
Body weight gain (percent change compared to control)
Doses (M) 0 65 710
BW gain (weaning to 0% 5% -11%
4 months
post-weaning)
Doses (F) Oy 82 770
BW gain (weaning to 0% -1% 10%
4 months
post-weaning)
Note: Statistical analysis was not performed on BW gain.
5,800
-53%
4,700
-19%
Body weight (percent change compared to control)
Doses (M) 0 65 710
BW at 4 months 0% 4% -9%
post-weaning
Doses (F) 0 82 770
BW at 4 months 0% -1% 7%
post-weaning
Note: Statistical analysis not reported in study.
5,800
-43%
4,700
-13%
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Results3
Fetal morphological development
Saillenfait et al. (2006)
Rat (Sprague-Dawley) rats;
20-22 dams/group
0, 250, 500, 750, 1,000 mg/kg-day
Gavage
CDs 6-20 (GD 21 c-section)
Malformations
External malformations (incidence; number of affected fetuses [litters])
Doses 0 250 500
total fetuses (litters) 281 276 237
examined for external (22) (21) (21)
malformations
anasarca 00 0
exophthalmos 00 0
(unilateral) and
absence of eyelids
(bilateral)
exencephaly 00 0
meningoencephalocele 00 0
microstomia 00 0
ectopia cordis 00 0
omphalocele 00 0
750 1,000
212 111
(21) (18)
0 1(1)
1(1) 0
2(2) 0
3 (3) 3 (2)
0 1(1)
0 1(1)
0 1(1)
Combined total with external malformations (incidence [percent])
Doses 0 250 500
total number (%) 000
fetuses with external
malformations
total number (%) litters 000
with external
malformations
mean % fetuses with 0% 0% 0%
external
m a If arm ations/litter
750 1,000
5 (2%)* 6 (5%)**
4 (19%) 4 (22%)
2% 4%
Visceral malformations (incidence; number of affected fetuses [litters])
Doses 0 250 500
total fetuses (litters) 141 138 119
examined for visceral (22) (21) (21)
malformations
anophthalmia, uni- or 0 0 0
bilateral
aorta and/or 000
pulmonary artery
transposed
diaphragmatic hernia 0 2(1) 2 (2)
kidney and ureter, 000
absent, uni- or bilateral
750 1,000
106 56
(21) (18)
6 (4) 4 (3)
6 (5) 3 (3)
2 (2) 1 (1)
KD 3(3)
This document is a draft for review purposes only and does not constitute Agency policy,
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design














Results3
kidney, small, uni- or
bilateral
0
0
Combined total with visceral malformations
Doses
total number (%)
fetuses with visceral
malformations
total number (%) litters
with visceral
malformations
mean % fetuses with
visceral
malformations/litter
Skeletal malformations
Doses
total number of fetuses
(litters) examined for
skeletal malformations
mandible, small
stern ebrae, fused
sternebrae, fused and
scrambled
sternebrae, total
cleft sternum
sternebrae,
checkerboard
ribs, fused
cervical arches, fused
thoracic or lumbar
vertebral arches, fused
thoracic or lumbar
vertebral centra, fused
thoracic or lumbar
centrum, hemicentric
thoracic or lumbar
vertebral centra,
misaligned
0
0
0
0%
250
2 (1%)
1
(5%)
1%
0
(incidence
500
2 (2%)
KD
[percent])
750
13
(12%)**
2 8(38%)**
(10%)
2%
13%*
KD

1,000
10 (18%)**
8(44%)**
16%*
(incidence; number of affected fetuses [litters])
0
140
(22)
0
0
0
0
0
0
0
0
0
0
0
0
250
138
(21)
0
0
0
0
0
0
0
0
0
0
0
0
500
118
(21)
0
0
0
0
KD
0
0
0
1(1)
1(1)
1(1)
2(2)
750
106
(21)
0
7(6)
5(3)
12 (7)*
KD
2(2)
0
3(3)
2(2)
0
4(3)
3(2)
1,000
55
(18)
KD
14(9)**
12(7)
26 (13)**
2(2)
0
2(2)
3(3)
2(2)
4(3)
3(3)
5(4)
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-50            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design












Results3
Combined total with skeletal malformations (incidence [percent])
Doses
total number (%)
fetuses with skeletal
malformations
total number (%) litters
with skeletal
malformations
mean % fetuses with
skeletal
malformations/litter
0
0
0
0%
250 500
0 4 (3%)
0 4
(19%)
0% 3%
750
18
(17%)**
11
(52%)**
18%**
1,000
34 (62%)**
15 (83%)**
67%**
Variations
External variations (incidence; number of affected fetuses [litters])
Doses
total fetuses (litters)
examined for external
variations
clubfoot
tail, curly
tail tip, haemorrhage
0
281
(22)
0
0
0
250 500
276 237
(21) (21)
2(1) 0
0 0
1(1) 0
750
212
(21)
0
KD
0
1,000
111
(18)
0
0
0
Visceral variations (incidence; number of affected fetuses [litters])
Doses
total fetuses (litters)
examined for visceral
variations
dilated cerebral
ventricle, slight
dilated renal pelvis
ureter (all)
hydroureter
distended ureter
ovaries, displaced
testis, ectopic
degree of trans-
abdominal testicular
migration (mean)
0
141
(22)
0
1(1)
3(3)
0
3(3)
0
0
2.6
250 500
138 119
(21) (21)
0 0
0 0
0 2(2)
0 0
0 2(2)
0 0
0 3(2)
3.8 13.6**
750
106
(21)
KD
2(2)
10(8)
4(4)
6(5)
5(4)
30(16)**
42.2**
1,000
56
(18)
0
5(4)
12 (8)*
6(5)
6(4)
2(2)
30(16)**
58.1**
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-51            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design





























BASF (2007V
Rat (Wistar); 25 dams/group
0, 88, 363, 942 mg/kg-day
Diet
CDs 6-20 (GD 20 c-section)






Results3
Skeletal variations (incidence; number of affected fetuses [litters])
Doses
total fetuses (litters)
examined for skeletal
variations
parietals or
supraoccipital,
incomplete ossification
hyoid, absent or
incomplete ossification
stern ebrae, fused,
1st and 2nd only
stern ebrae, bipartite
stern ebrae, incomplete
ossification
ribs, cervical,
rudimentary
ribs, 14th, any
supernumerary
ribs, 14th, long
supernumerary
ribs, short or reduced
ossification (unilateral)
thoracic or lumbar
vertebral centra,
incomplete ossification
vertebrae, 27 presacral
Note: A single fetus may
variations.
0 250 500 750
140 138 118 106
(22) (21) (21) (21)

0 0 0 3(2)


0 0 0 1(1)

1(1) 0 8(4) 29(11)**

0 1(1) 2(2) 7(5)
0 1(1) 5(5) 9(6)

0 0 2(2) 12(9)*

23(11) 32(14) 42(18) 72(20)**

1(1) 1(1) 2(2) 15(9)*

0 0 0 1(1)

3(2) 8(6) 7(7) 18(14)**


000 0
1,000
55
(18)

1(1)


8(7)

5(4)

4(4)
6(5)

9(6)

52 (18)**

9(9)*

KD

16 (8)*


2(2)
be represented more than once in the individual


Malformations
External malformations (incidence; number of affected fetuses [litters])
Doses
total fetuses (litters)
examined for external
malformations and
variations
malformed head
anophthalnia
0 88 363
208 (23) 197 (22) 182 (22)


0 1(1) 0
0 1(1) 0
942
211(23)


0
0
Combined total with external malformations (incidence [percent])
Doses
fetuses
0 88 363
0 1 (1%) 0
942
0
This document is a draft for review purposes only and does not constitute Agency policy.
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

Results3
litters 0 1 (5%)
0 0
Combined total with soft tissue malformations
No fetuses affected at any dose
Skeletal malformations (incidence; number of affected fetuses [litters])
Doses 0 88
total fetuses (litters) 109 (23) 101 (22)
examined for skeletal
malformations/
variations
severely malformed 0 1(1)
skull bones
shortened scapula 0 0
(cartilage present)
malpositioned and 1(1) 1(1)
bipartite sternebral
(unchanged cartilage)
branched rib 1(1) 0
misshapen humerus 0 2 (2)
shortened humerus 0 0
363 942
97 (22) 110 (23)
0 0
1(1) 0
0 0
0 0
0 2(2)
2(1) 0
Combined total with skeletal malformations (incidence [percent])
fetuses 1 (1%) 4 (4%)
litters 1 (4%) 4 (18%)
2 (2%) 2 (2%)
1 (5%) 2 (9%)
Variations
Combined total with external variations
No fetuses affected at any dose
Soft tissue variations (incidence; number of affected fetuses [litters])
Doses 0 88
total fetuses (litters) 99 (23) 96 (22)
examined for external
soft tissue
malformations and
variations
dilated renal pelvis 10 (7) 7 (5)
dilated ureter 2 (2) 2 (1)
363 942
85 (22) 101 (23)
9 (8) 7 (5)
1 (1) 1 (1)
Combined total with soft tissue variations (incidence (percent))
Doses 0 88
fetuses 10 (10%) 7 (7%)
litters 7 (30%) 5 (23%)
363 942
9 (11%) 7 (7%)
8 (36%) 5 (22%)
This document is a draft for review purposes only and does not constitute Agency policy,
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design










































Results3
Skeletal variations (incidence; number of affected fetuses [litters])
Doses
supraoccipital hole(s)
incomplete ossification
of basisphenoid
incomplete ossification
of interparietal
(unchanged cartilage)
incomplete ossification
of parietal (unchanged
cartilage)
incomplete ossification
of supraoccipital
(unchanged cartilage)
incomplete ossification
of skull (unchanged
cartilage)
incomplete ossification
ofhyoid (cartilage
present)
incomplete ossification
of cervical arch
(cartilage present)
incomplete ossification
of thoracic centrum
(unchanged cartilage)
dumbbell ossification of
thoracic centrum
(unchanged cartilage)
dumbbell ossification of
thoracic centrum
(dumbbell-shaped
cartilage of centrum)
bipartite ossification of
thoracic centrum
(dumbbell-shaped
cartilage of centrum)
supernumerary thoracic
vertebra
unossified thoracic
centrum (dumbbell-
shaped cartilage of
centrum)
0
31(15)
7(3)

24 (15)


16 (10)


9(7)


5(4)


KD


KD


0


4(3)


14(9)



2(2)



KD

KD



88
23 (13)
2(2)

11(7)


13(9)


14 (11)


2(1)


0


0


3(3)


3(3)


13(9)



2(2)



2(1)

0



363
15 (12)
6(5)

22 (12)


22 (13)


14 (12)


7(4)


KD


0


3(3)


2(2)


13 (13)



0



KD

0



942
38 (19)
8(4)

13(9)


13(7)


13(6)


2(2)


KD


0


0


9(7)


17 (14)



KD



3(2)

0



This document is a draft for review purposes only and does not constitute Agency policy,
                                3-54            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design













































Results3
dumbbell ossification of
lumbar centrum
(dumbbell-shaped
cartilage of centrum)
incomplete ossification
of lumbar arch
(cartilage present)
misshapen sacral
vertebra
fused sacral centrum
and arch (unchanged
cartilage)
incomplete ossification
of sacral arch (cartilage
present)
unossified sternebra
(unchanged cartilage)
incomplete ossification
of sternebra
(unchanged cartilage)
misshapen sternebral
(unchanged cartilage)
unilateral ossification of
sternebra (unchanged
cartilage)
extra sternebral
ossification site
(unchanged cartilage)
bipartite ossification of
sternebral (unchanged
cartilage)
supernumerary rib
(14th) (cartilage
present)
supernumerary rib
(14th) (cartilage not
present)
cervical rib (cartilage
present)
cervical rib (cartilage
not present)
wavy rib
incomplete ossification
ofpubis (cartilage
present)
KD



0


KD

3(2)


5(4)


4(4)

42 (17)


32 (19)

0


0


KD


6(5)


50 (15)


0

5(5)

6(5)
0


0



0


1(1)

7(3)


2(2)


11(7)

44 (17)


26 (17)

0


0


0


KD


33 (15)


KD

5(4)

2(2)
KD


0



KD


KD

5(4)


0


KD

44 (19)


20 (12)

0


KD


0


6(6)


40 (17)


0

5(3)

10(4)
0


0



0


4(4)

5(3)


0


6(4)

75(22*)


28 (16)

4(4)


0


2(2)


6(5)


62(22*)


0

4(4)

9(9)
0


This document is a draft for review purposes only and does not constitute Agency policy,
                                3-55            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

Results3
Combined total with skeletal variations (incidence (percent))
Doses 0 88 363
fetuses 101 87(86%) 85(88%)
(93%)
litters 23 22(100%) 22(100%)
(100%)
942
107 (97%)
23 (100%)
Unclassified observations
Unclassified external observations (incidence; number of affected fetuses
[litters])
Doses 0 88 363
total fetuses (litters) 208(23) 197(22) 182(22)
examined for
unclassified
observations
discolored amniotic 00 0
fluid
942
211(23)
KD
Combined total with external unclassified observations (incidence [percent])
Doses 0 88 363
fetuses 000
litters 000
942
1 (1%)
1 (4%)
Combined total with unclassified soft tissue observations
No fetuses affected at any dose
Skeletal unclassified cartilage observations (incidence; number of affected
fetuses [litters])
Doses 0 88 363
total fetuses (litters) 109(23) 101(22) 97(22)
examined for skeletal
unclassified
observations
notched cartilage 2(2) 0 0
between basiphenoid
and basioccipital
fused cervical arch 1(1) 0 0
cartilage
dumbbell-shaped 0 1(1) 0
cartilage of cervical
centrum
hole in processus 0 1(1) 0
coracoideus
bipartite processus 36(14) 30(15) 30(15)
xiphoideus
942
110 (23)
KD
0
0
0
40 (14)
This document is a draft for review purposes only and does not constitute Agency policy,
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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

Borch et al. (2006)
Rat (Wistar); 11-12 dams/group
0, 600 mg/kg-day
Gavage
GDs 7-19 (GD 19 c-section) or
CDs 7-20/21 (GD 20/21
c-section); 5-6 dams/group per
time point
Results3
notched manubrium
fused rib cartilage
branched rib cartilage
5 (5) 7 (5)
0 0
0 0
3 (3) 1 (1)
1(1) 0
1(1) 0
Combined total with skeletal unclassified cartilage observations (incidence
[percent])
Doses
fetuses
litters
AGO change in females
Doses
AGO at GD 19 (data
shown in graphb)
AGO at GD 20/21) (data
shown in graphb)
0 88
39 (36%) 35 (35%)
16 (70%) 17 (77%)
363 942
32 (33%) 41 (37%)
16 (73%) 15 (65%)
(percent change compared to control)
0
0%
0%
600
16%
26%*
AGD/cubic root of BW change in females (percent change compared to
control)
Doses
at GD 19 (data shown
in graphb)
at GD 20/21 (data
shown in graphb)
0
0%
0%
600
27%**
27%*
 1
 2    Response is % control (indicated by %) or in cases when % control was not possible to present (e.g., if control
 3     value was 0), response levels are presented. Equation used to calculate percent change compared to control:
 4                                        treated value - control value x 100
 5                                                 control value
 6    bGrablt Software used to estimate % control from graph.
 7    °Dose conversions were performed using this information: For the Eastman Kodak (1954) study, average BWs were
 8     183,186,180, and 115 g for males, and 132,126,133, and 110 g for females at 0, 0.1,1.0, and 5.0%, respectively.
 9     Reference values for food consumption of 0.018 and 0.014 kg/day for male and female rats of an unspecified
10     species (U.S. EPA, 1988) were used.
11    dDose conversions were performed using this information: In Oishi and Hiraga (1980d), average BWs for rats and
12     mice in these studies were 145 and 25 g, respectively, and the default food consumption rates of 0.008 kg/day for
13     male Wistar rats and 0.0025 kg/day for male B6C3Fi mice (U.S. EPA, 1988) were applied. In Oishi and Hiraga
14     (1980a), average BWs over the week-long studies were 132 and 24 g for rats and mice, respectively, and the
15     default food consumption rates of 0.008 kg/day for male Wistar rats and 0.0025 kg/day for male B6C3Fi mice
16     were applied (U.S. EPA, 1988).
17    eChange in body weight was calculated by EPA.
18    fDose conversions were performed using this information: For University of Rochester (1953), average BWs were
19     139,124,127,127,121, and 101 g at 0, 0.01, 0.1,1.0, 2.0, and 5.0%, respectively). Reference values for food
20     consumption of 0.018 and 0.014 kg/day for male and female rats of an unspecified strain (U.S. EPA,  1988) were
21     used.
                  This document is a draft for review purposes only and does not constitute Agency policy,
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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    gPND 49 was selected among the periodic weight measurement ages to present in this table because it
 2     corresponds to the age when VO and PPS, the developmental markers of puberty, would be expected to have
 3     completed in the male and female rat.
 4    hDose conversions were performed by EPA using this information: For University of Rochester (1954), average BWs
 5     were 269, 277, 252, and 155 g for males, and 178,170,182, and 148 g for females at 0, 0.1,1.0, and 5.0%,
 6     respectively. Reference values for food consumption of 0.018 and 0.014 kg/day for male and female rats of an
 7     unspecified species (U.S. EPA, 1988) were used.
 8    'Male reproductive organs were not evaluated in the BASF study.
 9
10    * = Statistically significant difference at p < 0.05 from control value, as reported by study authors; ** = Statistically
11     significant difference at p < 0.01 from control value, as reported by study authors; *** = Statistically significant
12     difference at p < 0.001 from control  value, as reported by study authors; BW = body weight; GD = gestation day;
13     PND = postnatal day; PPS = preputial separation; VO = vaginal opening
                 This document is a draft for review purposes only and does not constitute Agency policy.
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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
       O
       (5
       OJ

       Q.
       s
       Q
                              fetal body weight
                             BASF, 2007; rat, male
                              fetal body weight
                            BASF, 2007; rat, female
                         fetal body weight; GD 19
                        Borch et al., 2006; rat, male
                        fetal body weight; GD 19
                       Borch et al., 2006; rat, female
                        fetal body weight; GD 20/21
                        Borch et al., 2006; rat, male
fetal body weight; GD 20/21
Borch et al., 2006; rat, female
                           fetal body weight
                     Saillenfait et al., 2006; rat, male
                          fetal body weight
                    Saillenfait et al., 2006; rat, female
           ro          pup survival; PND 1-4
           '>  Saillenfait et al., 2008; rat, male & female
           i:
           i/l
           £         pup survival; PND 4-21
           S.  Saillenfait et al., 2008; rat, male & female
• significantly changed
O not significantly changed

G.




•
•
0
O




                                                                 10                 100

                                                              Doses (mg/kg-day)
                                                                                                       1000
2      Figure 3-1.  Exposure-response array of effects on developmental growth and
3      survival following developmental oral exposure to DIBP.
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                                                      3-59              DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                    Eastman Kodak, 1954; 4 wk; rat, male

                   Eastman Kodak, 1954; 4 wk; rat, femah

                    Eastman Kodak, 1954; 8 wk; rat, mal<

                   Eastman Kodak, 1954; 8 wk; rat, femahr

                    NIOSH, 1983; day 1-8; mouse, female

                    NIOSH, 1983; day 1-12; mouse, female

                    NIOSH, 1983; day 1-16; mouse, female

                       Oishi and Hiraga, 1980d; 6 wk; rat

                      Oishi and Hiraga, 1980d; 5-6 wk; rat

                      Oishi and Hiraga, 1980a; 6 wk; mice

                    Oishi and Hiraga, 1980a; 5-6 wk; mice

             Saillenfait et al., 2008; PND 1, PND 21; rat, male

                     Saillenfait et al., 2008; pps; rat, male

                 Saillenfait et al., 2008; PND 21; rat, female

                Saillenfait et al., 2008; PND 77-84; rat, male

              University of Rochester, 1953; 1 mo; rat, male

             University of Rochester, 1953; PND 49; rat, male

              University of Rochester, 1954; 1 mo; rat, male

             University of Rochester, 1954; 1 mo; rat, female

             University of Rochester, 1954; PND 49; rat, male

           University of Rochester, 1954; PND 49; rat, female

              University of Rochester, 1954; 4 mo; rat, male

             University of Rochester, 1954; 4 mo; rat, female
                                                              10
3
C
• significantly changed
O not significantly changed
D statistical analysis not
reported
^
^
t
3
3
3
3
3
3
3













[
[
[

0/-\ i^Y^i

















•
3
•
0








                                                                         100
                                                                  Doses (mg/kg-day)
                                                                                     1000
                                                                                                 10000
                                                                                                             100000
2        Figure 3-2.  Exposure-response array of effects on postnatal and adult body weight
3        following developmental oral exposure to DIBP.
                  This document is a draft for review purposes only and does not constitute Agency policy,
                                                           3-60               DRAFT—DO NOT CITE OR QUOTE

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                          Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                             total malformations
                    Saillenfait et al., 2006; rat, male & female
                              total variations
                    Saillenfait et al., 2006; rat, male & female
                                total malformations
                            BASF, 2007; rat, male & female
                                   total variations
                            BASF, 2007; rat, male & female
                             total unclassfied observations
                            BASF, 2007; rat, male & female
                             total malformations
                    Saillenfait et al., 2006; rat, male & female
                            variation: ureter (all)
                   Saillenfait et al., 20061; rat, male & female
                varaiation trans abdominal testicular migration
                      Saillenfait et al., 20061; rat, male
                                variation: testis, etopic
                            Saillenfait et al., 20061; rat, male
                             total malformations
                    Sailenfait et al., 2006; rat, male & female
                         variation: sternebrae, fused
                   Saillenfait et al., 20061; rat, male & female
                      variation: ribs, cervical, rudimentary
                   Saillenfait et al., 20061; rat, male & female
                   variation: ribs,  14th, any supernumentary
                   Saiilenfait et al., 20061; rat, male & female
                   variation: ribs, 14th, long supernumentary
                   Saiilenfait et al., 20061; rat, male & female
                         variation: thoracic/ lumbar
                   Saillenfait et al., 20061; rat, male & female
                                total malformations
                            BASF, 2007; rat, male & female
                                   total variations
                            BASF, 2007; rat, male & female
                     total unclassified cartilage observations
                        BASF, 2007; rat, male & female
                                total malformations
                            BASF, 2007; rat, male & female
                                   total variations
                            BASF, 2007; rat, male & female
                            total unclassified observations
                            BASF, 2007; rat, male & female
                           AGO change; GD 19
                Borch et al., 2006 (female); rat, male & female
                         AGO change; GD 20/21
                Borch et al., 2006 (female); rat, male & female
                                                       1
         1 Individual variation incidences shown here were included because statistical
         significance was reported. Incidence for other individual malformations and
         variations are shown in Table 3-1 (Saillenfait et al., 2006).
                                                                             _L
                                            • significantly changed
                                            O not significantly changed































X-N X">









O
•
                                                                             10
                                                        Doses (mg/kg-day)
                                                                                                   100
                                                                                                                         1000
2
3
Figure 3-3. Exposure-response array of effects on fetal morphological
developmental following developmental oral exposure to DIBP.
                    This document is a draft for review purposes only and does not constitute Agency policy,
                                                               3-61                DRAFT—DO NOT CITE OR QUOTE

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2
3
               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.3.2.  Male Reproductive Effects

       Table 3-18. Evidence pertaining to male reproductive effects in animals
       following oral exposure to DIBP
Reference and study design
Results3
Morphological development (assessed in fetal or postnatal development or adults)
Borch et al. (2006)
Rat (Wistar); 11-12 dams/group
0, 600 mg/kg-day

Gavage

CDs 7-19 (GD 19 section) or CDs
7-20/21 (GD 20/21 c-section); 5-6
dams/group per time point





AGO change in fetus (percent

Doses
At GD 19 (data shown
in graphb)
At GD 20/21 (data
shown in graphb)
change compared to control)

0 600
0% -15%**

0% -11%**

AGD/cubic root BW change in fetus (percent change compared to
control)

Doses (M)
GD 19 (data shown in
graph")
GD 20/21 (data
shown in graphb)


0 600
0% -5%

0% -9%**

Histologic lesions in fetal testis
Borch et al. (2006)

Rat (Wistar); 11-12 dams/group
0, 600 mg/kg-day
Gavage

CDs 7-19 (GD 19 section) or CDs
7-20/21 (GD 20/21 c-section); 5-6
dams/group per time point;
1-3 males/litter










Testicular histological changes (incidence; percentage incidence in
fetuses)
Doses
Fetuses GD 19
clustering of small
Leydig cells




Sertoli cell
vacuolization

central localization of
gonocytes

multinucleated
gonocytes

Fetuses GD 20/21

0 600

2/13 9/9***

15% 100%***



0/13 1/9

0% 11%
0/13 2/9

0% 22%
1/13 0/9

8% 0%

              This document is a draft for review purposes only and does not constitute Agency policy,
                                              3-62            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

Results3
clustering of small
Ley dig cells
Sertoli cell
vacuolization
central localization of
gonocytes
multinucleated
gonocytes
0/10 13/15***
0% 87%***
0/10 14/16***
0% 88%***
0/10 14/16***
0% 88%***
1/10 10/16*
10% 63%*
Fetal testicular testosterone production
Borch et al. (2006)
Rat (Wistar); 11-12 dams/group
0, 600 mg/kg-day
Gavage
GDs 7-19 (GD 19 c-section) or
CDs 7-20/21 (GD 20/21 c-section);
5-6 dams/group per time point
Hannasetal. (2011)
Rat (Harlan Sprague-Dawley);
3 dams/group; 3 males/dam
0, 100, 300, 600, 900 mg/kg-day
Gavage
GDs 14-18
Testicular testosterone (T)
control)
Doses
T content (GD 19 M)
(data shown in
graphb)
T content (GD 20/21
M) (data shown in
graph")
Testicular testosterone (T)
to control)
Doses
testicular T production
ex vivo (GD 19 M)
(data shown in
graph")
testicular T production
ex vivo (GD 20/21 M)
(data shown in
graph")
content (percentage change compared to
0 600
0% -70%
0% -90%***
production (percentage change compared
0 600
0% -21%
0% -96%***
Fetal testicular testosterone (T) production (percentage change
compared to control)
Doses 0
Tproduction 0%
100 300 600 900
10% -56%** -80%** -87%**
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-63            DRAFT—DO NOT CITE OR QUOTE

-------
     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Hannas et al. (2012)
Rat (Sprague-Dawley); 3 dams/group;
3 males/dam
0, 500 mg/kg-day
Gavage
CDs 14-18
Howdeshell et al. (2008)
Rat (Sprague-Dawley); 5-8 dams/group;
3 males/dam
0, 100, 300, 600, 900 mg/kg-day
Gavage
GDs 8-18; c-section on GD 18
Results3
Fetal testicular testosterone (T) production (percentage change
compared to control)
Doses 0 500
T production (data 0% -73%**
shown in graphb)
Fetal testicular testosterone (T) production (percentage change
compared to control)
Doses 0 100 300 600 900
T production (litter 0% -5% -40%** -59%** -63%**
mean)
Morphological development assessed in postnatal development and adults
Saillenfait et al. (2008)
Rat (Sprague-Dawley);
11-14 dams/group
0, 125, 250, 500, 625 mg/kg-day
Gavage
GDs 12-21 (dams allowed to deliver)
Postnatal effects (percent change in litter mean compared to control)
Doses 0 125 250 500 625
AGD(PNDl) 0% -4% -11%* -21%** -22%**
ageatPPS 0% -4%* -1% 10%** 6%*
Postnatal effects (incidence; percentage incidence)
Doses 0 125 250 500 625
retained 0/76 0/78 8/96 47/79 56/76
nipples or
areolas at
PNDs 12-14
0% 0% 8% 59% 74%
Note: No statistical analysis was reported by the authors for this
endpoint.
Male adult effects at necropsy (PNDs 77-84 or 112-119; percentage
incidence)
Doses 0 125 250 500 625
retained 0/46 0/40 4/55 24/44 29/38
nipples or
areolas
0% 0% 7% 55% 76%
hypospadias 0/46 0/40 0/55 5/44 22/39
0% 0% 0% 11% 56%
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-64            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design







Results3
exposed os 0/46 0/40 0/55
penis
0% 0% 0%
cleft prepuce 0/46 0/40 0/55
0% 0% 0%
nonscrotal 0/46 0/40 0/55
testis
0% 0% 0%
Note: No statistical analysis was reported by the
endpoint.
4/44
9%
0/44
0%
11/44
25%
11/39
28%
10/39
26%
30/39
77%
authors for this
Histopathologic lesions in adult testis and epididymis
Saillenfait et al. (2008)
Rat (Sprague-Dawley);
11-14 dams/group
0, 125, 250, 500, 625 mg/kg-day
Gavage
GDs 12-21 (dams allowed to deliver)





Adult effects0 (PNDs 77-84; incidence)
Doses 0 125 250
number of males 24(12) 20(10) 28(14)
(litters)
examined
500
22(11)
625
20 (10)
Epididymides (number of males with effect)
Doses 0 125 250
oligospermia 013
azoospermia 013
granulomatous 000
inflammation
500
2
10
4
625
1
18
3
Testes (number of males with effect)
Doses 0 125 250
tubular 227
degeneration-
atrophy/
hypoplasia
tubular necrosis 001
interstitial cell 000
hyperplasia
Note: No statistical analysis was reported by the
endpoints.
500
16
3
1
625
20
5
9
authors for these
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-65            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Results3
Testes weight
Zhu et al. (2010)
Rat (Sprague-Dawley)
Mouse (C57BI/6N) (number of animals
not specified)
0, 100, 300, 500, 800, 1,000 mg/kg-day
Gavage
7 days
Foster et al. (1982); Foster et al. (1981)
MIBP
Rat (Sprague-Dawley); 6 males/group
0, 800 mg/kg-day
Gavage
6 days
Oishi and Hiraga (1980c)
Rat (JCLWistar); 10 males/treated group;
20 control males
0, 2% (0, 1,200 mg/kg-day)d
Diet
1 week
Oishi and Hiraga (1980d)
MIBP
Rat (JCLWistar); 10 males/group
0, 2% in diet (0, 1,100 mg/kg-day)d
Diet
1 week
Oishi and Hiraga (1980a)
Mouse (JCLICR); 10 males/group
0, 2%(0,2,100mg/kg-day)d
Diet
1 week
Testes weight at
Doses
7 days (percent change compared to control)
0 100 300 500 800 1,000
Rat, absolute 0% -3% -10% -22%*** -32%*** -44%***
weight (data
shown in
graph")
Mouse, 0% 0% 10% 6% 4% -22%**
absolute
weight(data
shown in
graph")
Note: Relative weight not reported by study authors.
Testes weight (percent change compared to control)
Doses
absolute weight
relative weight
0 800
0% -28%***
0% -27%***
Testes weight (percent change compared to control)
Doses
absolute weight
relative weight
0 1,200
0% -37%*
0% -33%*
Testes weight (percent change compared to control)
Doses
absolute weight
relative weight
0 1,100
0% -47%*
0% -40%*
Testes weight (percent change compared to control)
Doses
relative weight
0 2,100
0% 29%*
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-66            DRAFT—DO NOT CITE OR QUOTE

-------
     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Oishi and Hiraga (1980b)
MIBP
Mouse (JCLICR); 10 males/group
0, 2% (0, 2,000 mg/kg-day)d
Diet
1 week
Saillenfait et al. (2008)
Sprague-Dawley rats; 11-14 dams/group
0, 125, 250, 500, 625 mg/kg-day
Gavage
GDs 12-21 (dams allowed to deliver)
Assessed PNDs 77-84 (adults) after in
utero exposure



University of Rochester (1954)
Rat (Albino; no other strain designation);
5 males/group
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day)e
Diet
Weaning to 4 months post-weaning
Results3
Testes weight (percent change compared to control)
Doses
relative weight


0
0%


2,000
45%*


Male reproductive organ weights (percent change compared to
control)
Doses 0
right testis 0%
weight
right 0%
epididymal
weight
left testis 0%
weight
left 0%
epididymal
weight
seminal 0%
vesicles
prostate 0%
Testes weight at 4 months
Doses
absolute weight
relative weight
125 250
1% 0%
-2% -6%
-2% -1%
-4% -8%
1% -6%

-10% -11%*
500 625
-22% -52%**
-22%** -49%**
-13% -59%**
-16%** -49%**
-18%** -33%**

-16%** -30%**
(percent change compared to control)
Oh 65
0% 2%
0% 1%
710 5,800
-1% -70%
12% -45%
Note: Statistical analysis not reported in study.



Seminal vesicle weight
Foster et al. (1982); Foster et al. (1981)
MIBP
Rat (Sprague-Dawley); 6 males/group
0, 800 mg/kg-day
Gavage
6 days
Seminal vesicle weight (percent change compared to control)
Doses

absolute weight
relative weight

0

0%
0%

800

-18%
-11%

This document is a draft for review purposes only and does not constitute Agency policy,
                                3-67            DRAFT—DO NOT CITE OR QUOTE

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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Results3
Prostate weight
Foster et al. (1982); Foster et al. (1981)
MIBP
Rat (Sprague-Dawley); 6 males/group
0, 800 mg/kg-day
Gavage
6 days
Prostate weight (percent change compared to control)
Doses
absolute weight
relative weight
0 800
0% -13%
0% 4%
Testosterone concentration in adults
Oishi and Hiraga (1980c)
Rat (JCLWistar); 10 treated males;
20 control males
0, 2% (0, 1,200 mg/kg-day)d
Diet
1 week
Oishi and Hiraga (1980d)

MIBP
Rat (JCLWistar); 10 males/group
0, 2% (0, 1,100 mg/kg-day)d
Diet
1 week
Oishi and Hiraga (1980a)
Mouse (JCLICR); 10 males/group
0, 2%(0,2,100mg/kg-day)d
Diet
1 week
Oishi and Hiraga (1980b)
MIBP
Mouse (JCLICR); 10 males/group
0, 2% (0, 2,000 mg/kg-day)d
Testosterone (T) concentration (percent change compared to control)
Doses
serum T concentration
(data shown in graphb)
testicular T concentration
(data shown in graphb)
0 1,200
0% 19%
0% 158%*
Dihydrotestosterone (DHT) concentration (percent change compared
to control)
Doses
serum DHT concentration
(data shown in graphb)
0 1,200
0% 40%
Testosterone (T) concentration (percent change compared to control)
Doses
serum T concentration
(data shown in graphb)
testicular T concentration
(data shown in graphb)
Testicular testosterone (T)
to control)
Doses
T concentration
Testicular testosterone (T)
to control)
Doses
T concentration
0 1,100
0% 61%*
0% 161%*
concentration (percent change compared
0 2,100
0% 7%
concentration (percent change compared
0 2,000
0% -83%*
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-68            DRAFT—DO NOT CITE OR QUOTE

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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
            Reference and study design
                                                                 Results3
       Diet

       1 week
       Testes histology in adults
       Foster et al. (1982): Foster et al. (1981)
       MIBP
       Rat (Sprague-Dawley); 6 males/group
       0, 800 mg/kg-day

       Gavage

       6 days
                                      Number of animals with atrophy of seminiferous tubules
                                      Doses
 0
800
                                      0% atrophic tubules
6/6
0/6
                                      <50% atrophic tubules                0/6                3/6
                                      >50% atrophic tubules                0/6                3/6
                                      The study authors noted marked atrophy of the majority of the
                                      seminiferous tubules with decreased spermatocytes and decreased
                                      spermatogonia (data not shown).
       Oishi and Hiraga (1980c)
       Rat (JCLWistar); 10 treated males;
       20 control males
       0, 2% (0,1,212 mg/kg-day)d
       Diet
       1 week
                                      The testes showed decreased spermatocytes and decreased
                                      spermatogonia compared to control (quantitative results not
                                      provided).
 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
aPercent change compared to control = treated value - control value x 100
                                          control value
bGrablt Software used to estimate % change from graph.
cPNDs 112-119 males were also evaluated for these endpoints (see Saillenfait et al., 2008, Table 4).
dDose conversions were performed  by EPA using this information: Oishi and Hiraga (1980a, c) average BWs over
 the week-long studies were 132 and 24 g for rats and mice, respectively, and the default food consumption rates
 of 0.008 kg/day for male weanling Wistar rats and 0.0025 kg/day for male weanling B6C3Fi mice were applied
 (U.S. EPA, 1988). Oishi and Hiraga  (1980b, d) average BWs for rats and mice in these studies were 145 and 25 g,
 respectively, and the default food consumption rates of 0.008 kg/day for male weanling Wistar rats and
 0.0025 kg/day for male weanling B6C3Fi mice (U.S. EPA, 1988) were applied. Note that Table 1-6 of U.S. EPA
 (1988) listed the default food consumption rate for male weanling Wistar rats as 0.080 kg/day. However, it was
 later determined using an equation in Table 1-3 of the document that this value was actually supposed to be
 0.008 kg/day.
eDose conversions were performed  by EPA using this information: University of Rochester (1954) average BWs
 (measured at least once weekly) of the rats were 269, 277, 252, and 155 g for males, and 178,170,182, and 148 g
 for females at 0, 0.1,1.0, and 5.0%, respectively; and the default food consumption rates of 0.018 kg/day for male
 rats and 0.014 kg/day for female rats (U.S. EPA, 1988) for an unspecified strain in a sub-chronic study were
 applied.

* = Statistically significant difference at p < 0.05 from control value, as reported by study authors; ** = Statistically
 significant difference at p < 0.01 from control value, as reported by study authors; *** = Statistically significant
 difference at p < 0.001 from control value, as reported by study authors.
                  This document is a draft for review purposes only and does not constitute Agency policy,
                                                      3-69             DRAFT—DO NOT CITE OR QUOTE

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                        Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
       o
       Q.
                leydig cell clusters; GD19
                  Borch et al., 2006; rat

             sertoli cell vacilization; GD19
                  Borch et al., 2006

          multinucleated gonocytes; GD19
              Borch et al., 2006; rat

             leydig cell clusters; GD20/21
                Borch et al., 2006; rat

          sertoli cell vacilization; GD20/21
               Borch et al., 2006; rat

       multinucleated gonocytes; GD 20/21
             Borch et al., 2006; rat

                    nipple retention
                Saillenfait et al.,2008; rat

                     hypospadis
                Saillenfait et al., 2008; rat

                     cleft prepuce
                Saillenfait et al.,2008; rat

                   nonscrotal testis
                Saillenfait et al., 2008; rat

                   exposed os penis
                Saillenfait et al.,2008; rat

             aligospermia & azoospermia
              Saillenfait et al., 2008; rat

             granulomatous inflammation
              Saillenfait et al., 2008; rat

          tubular degeneration & necrosis
             Saillenfait et al.,2008; rat

                 interstitial hyperplasia
                Saillenfait et al., 2008; rat

                     AGO; PND 1
                Saillenfait et al.,2008; rat

                   AGO change; GD 19
                  Borch et al., 2006; rat

                  AGO change; GD 20/21
                  Borch et al., 2006; rat
• significantly changed
O not significantly changed
1
0
Doses (mg/kg-day)
                                                                                                          -e-e
                                                                                                          -e-e
                                                                                                          -e-e
                                                                                                          -e-e
                                                                                                          -e-e
                                                                                                          -e-e
                                                                                                          -e-e
                                                                                           100
                                                                                                                1000
2
3
Figure 3-4. Exposure-response array of effects on male reproductive
development following developmental oral exposure to DIBP.
                  This document is a draft for review purposes only and does not constitute Agency policy,
                                                           3-70               DRAFT—DO NOT CITE OR QUOTE

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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
        o
        tt
              TesticularT content; GD 19
                Borch etal., 2006; rat
            TesticularT content; GD 20/21
               Borch et al., 2006; rat
      T production; GD 19
      Borch etal., 2006; rat
                 T production; GD 20/21
                  Borch etal., 2006; rat
                  T production; GD 18
                 Hannas etal., 2011; rat
                  T production; GD 18
                 Hannas etal., 2012; rat
                 T production; GD 18
              Howdeshell et al., 2008; rat
                                            • significantly changed
                                            O not significantly changed
                                                        10
                                                                            100
                                                                                                 1000
                                                           Doses (mg/kg-day)
2
3
Figure 3-5.  Exposure-response array of effects on fetal testosterone (T)
following developmental oral exposure to DIBP.
                This document is a draft for review purposes only and does not constitute Agency policy,
                                                    3-71              DRAFT—DO NOT CITE OR QUOTE

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                         Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                        Foster et al., 1981; Foster et al., 1982; rat


                                   Oishi and Hiraga, 1980c; rat


                                  Oishi and Hiraga, 1980d; rat


                    "Si         Oishi and Hiraga, 1980b; mouse
                    'oj

                    £        University of Rochester, 1954; rat


                                         Zhu et el., 2010; rat


                                      Zhu et el., 2010; mouse

                                          testis weight
                                    Saillenfait et al., 2008; rat

                    •gj                 epididymal weight
                    |              Saillenfait et al., 2008; rat

                    ^              Saillenfait et al., 2008; rat
                    _c
                    op

                        Foster et al., 1981; Foster et al., 1982; rat

                    _c
                    •5              Saillenfait etal., 2008; rat
                    g
                    OJ
                    ra
                    g  Foster et al., 1981; Foster et al., 1982; rat
                    Q_

                                   testicular T concentration
                                   Oishi and Hiraga, 1980c; rat
                    c
                    O
                    '•£             serum DHT concentration
                    •£             Oishi and Hiraga, 1980c; rat
                    OJ
                    u
                    2       serum & testicular T concentration
                    £          Oishi and Hiraga, 1980d; rat
                    o
                    y            testicular T concentration
                    &         Oishi and Hiraga, 1980a; mouse
                    £
                                  testicular T concentration
                               Oishi and Hiraga, 1980b; mouse

                    ao            atrophic tubules
                    J  Foster et al., 1981; Foster et al., 1982; rat
                    ra
                    o"             decreased spermatocytes
                    ~             Oishi and Hiraga, 1980c; rat


,
• significantly changed
O not significantly changed
D statistical analysis not reported













c
\.











B(~\ (~\ f~\l
e/~\ 1^^


o
o


o


9


I

I




o
o

o
                                                                      10
                                                                                   100
                                                                                                1000
                                                                                                            10000
                                                                    Doses (mg/kg-day)
2
3
Figure 3-6. Exposure-response array of male reproductive effects following
oral exposure to DIBP.
    This document is a draft for review purposes only and does not constitute Agency policy.
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2
3
               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.3.3.  Female Reproductive Effects

       Table 3-19. Evidence pertaining to female reproductive effects in animals
       following oral exposure to DIBP
Reference and study design
Results
Maternal weight"
Howdeshell et al. (2008)
Rat (Sprague-Dawley);
5-8 dams/group
0, 100, 300, 600,
900 mg/kg-day
Gavage
CDs 8-18 (GD 18 c-section)
Borch et al. (2006)
Rat (Wistar);
11-12 dams/group
0, 600 mg/kg-day
Gavage
GDs 7-19 (GD 19 c-section) or
G Ds 7-20/2 1(GD 20/21
c-section); 5-6 dams/group per
time-point
BASF (2007)°
Rat (Wistar);
22-23 dams/group
0, 88, 363, 942 mg/kg-day
Diet
GDs 6-20 (GD 20 c-section)
Saillenfait et al. (2008)
Rat (Sprague-Dawley);
11-14 dams/group
0, 125, 250, 500,
625 mg/kg-day
Gavage
GDs 12-21 (dams allowed to
deliver)
Maternal body weight (percent change compared to control)
Doses 0 100 300 600
maternal BW gain GDs 8-18 0% 9% 4% -35%
900
-42%*
Maternal body weight
Doses 0 600
— Wo significant effect on maternal
weight gain during pregnancy
(quantitative data not reported by
study authors)
Maternal body weight (percent change compared to control)
Doses 0 88 363
BW change GDs 6-20 0% -3% -6%
gravid uterine weight 0% -3% -8%
corrected BW gain GDs 6-20? 0% -2% -2%
942
-11%*
-3%
-25%*
Maternal body weight (percent change compared to control)
Doses 0 125 250 500
BW gain GDs 12-21 0% 4% 6% 6%
625
-3%
              This document is a draft for review purposes only and does not constitute Agency policy,
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Saillenfait et al. (2006)
Rat (Sprague-Dawley);
20-22 dams/group
0, 250, 500, 750,
1,000 mg/kg-day
Gavage
CDs 6-20 (GD 21 c-section)
Results
Maternal body weight (percent
Doses
BW gain GDs 6-21
gravid uterine weight
corrected BW gain GDs 6-21e
change compared to control13)
0 250 500 750
0% -1% -14% -14%
0% -2% -19%* -28%**
0% 0% 0% 19%

1,000
-39%**
-61%**
19%
Maternal food consumption
Doses
food consumption every
4-6 days, GDs 0-21
0 250 500 750
Wo statistically significant
change from control
1,000

Maternal toxicity
BASF (2007)c
Rat (Wistar); 25 females/group
0, 88, 363, 942 mg/kg-day
Diet
CDs 6-20 (GD 20 c-section)
Abnormalities in dams examined at necropsy
Doses
0 88 363
abnormalities (incidence) 0/25 2/25 0/25
Observed: hemorrhagic thymus,
diaphragmatic hernia, and
dilated renal pelvis
abnormalities (percent 0% 8% 0%
incidence)
Note: Statistical analysis was not performed on these data
942
2/25
8%
Fertility/fetal survival
BASF (2007)c
Rat (Wistar); 25 dams/group
0, 88, 363, 942 mg/kg-day
Diet
CDs 6-20 (GD 20 c-section)
Note: BW and food
consumption measured every
1-3 days through GD 20
Fertility (percent change compared to control13)
Doses
percentage pregnant
0 88 363
23/25 22/25 22/25
942
23/25
Fetal survival (incidence)
Doses
dams with all resorptions
0 88 363
0/23 0/22 0/22
942
0/23
Fetal survival (percent change compared to control)
Doses
percentage preimplantation
loss/litter
percentage postimplantation
loss/litter
percentage resorptions/litter
number of live fetuses/litter
number of live male
fetuses/litter
0 88 363
0% -19% 25%
0% 36% 105%
0% 36% 105%
0% 0% -8%
0% 5% 2%
942
-30%
16%
16%
2%
-2%
This document is a draft for review purposes only and does not constitute Agency policy,
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Borch et al. (2006)
Rat (Wistar); 16 dams/group
0, 600 mg/kg-day
Gavage
GDs 7-19 (GD 19 c-section) or
G Ds 7-20/2 1(GD 20/21 c-
section); 5-6 dams/group per
time-point)
Howdeshell et al. (2008)
Rat (Sprague-Dawley);
5-8 dams/group
0, 100, 300, 600,
900 mg/kg-day
Gavage
GDs 8-18 (GD 18 c-section)
Saillenfait et al. (2006)
Rat (Sprague-Dawley);
23-24 dams/group
0, 250, 500, 750,
1,000 mg/kg-day
Gavage
GDs 6-20 (GD 21 c-section)
Results
Fertility (incidence)
Doses 0 600
number pregnant/dams mated 11/16 12/16
Fetal survival
Doses 0 600
— Wo significant effect on litter size,
fetal viability, or number of
resorptions (quantitative data not
reported by study authors)
Fetal survival (n litters evaluated for endpoint)
Doses 0 100 300 600 900
dams with whole litter loss/total 0/5 0/8 0/5 0/5 1/5
dams
number of implantations/litter 13.7(3) 14.8(4) 16.0(3) 12.7(3) 13.3(5)
number of live fetuses/litter 13.3(3) 13.5(4) 15.3(3) 9.3(3) 5.0* (3)
total resorptions/litter 0.2(5) 1.0(8) 0.4(5) 2.0(5) 7.8* (5)
percentage fetal mortality per 1.3% 4.6% 2.7% 17.2% 59.0%*
litter (3) (4) (3) (5) (5)
Fertility
Doses 0 250 500 750 1,000
number pregnant/mated dams 22/24 22/24 22/23 21/23 20/24
(percent) (91.7%) (91.7%) (95.7%) (91.3%) (83.3%)
Fetal survival (percent incidence)
Doses 0 250 500 750 1,000
percentage postimplantation 6.7% 11.0% 13.9% 28.2%** 59.6%**
loss/litter
percentage dead fetuses per 0% 0% 0.3% 0.7% 0.3%
litter
percentage resorptions/litter 6.7% 11.0% 13.6% 27.6%** 59.3%**
Fetal survival (percent change compared to control)
Doses 0 250 500 750 1,000
percentage live litters 0% -5% -5% 0% -10%
number of live fetuses/litter 0% 2% -12% -21%* -52%**
percentage male fetuses/litter 0% -4% -5% 0% 17%
This document is a draft for review purposes only and does not constitute Agency policy,
                                3-75            DRAFT—DO NOT CITE OR QUOTE

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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Saillenfait et al. (2008)
Rat (Sprague-Dawley);
11-14 dams/group
0, 125, 250, 500,
625 mg/kg-day
Gavage
GDs 12-21 (dams allowed to
deliver)
Results
Fetal survival (percent change compared to control13)
Doses
gestation length
percentage postimplantation
loss per litter
percentage pups born alive per
litter
live pups/litter at PND 1
0 125
0% 1%
0% 41%
0% -1%
0% 5%
250
0%
-35%
-4%
0%
500
0%
-31%
-1%
8%
625
1%
-13%
-8%
1%
 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
aSome studies measured BW at multiple timepoints/lifestages and not all of these data are presented here.  For
 the sake of comparability of data across the available studies, BW data measures presented are similar across
 studies and/or measures of BW change over the dosing period or greatest time period.
bPercent change compared to control = treated value - control value x 100
                                         control value

CBASF (2007): Dams in the 952 mg/kg-day group showed significantly decreased food consumption on days 10-13
 and 15-17 (<10% decreased compared to control); however, overall food consumption  did not differ between
 groups.
dCorrected weight gain = carcass weight (GD 20 body weight - gravid uterine weight) - GD 6 body weight.
Corrected weight gain = BW gain GDs 6-21 - gravid uterine weight.

* = Statistically significant difference at p < 0.05 from  control value, as reported by study authors; ** = Statistically
 significant difference at p < 0.01 from control  value, as reported by study authors; *** = Statistically significant
 difference at p < 0.001 from control value, as  reported by study authors.
                  This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                                                           I
      CD
      3=
                         BASF, 2007; rat
                  gravid uterine weight
                 Saillenfait et al., 2006; rat
                     BW AGO 6-21
                 Saillenfait et al., 2006; rat
        BW AGO 12-21
    Saillenfait et al., 2008; rat
                    gravid uterine weight
                       BASF, 2007; rat
                         BW AGO 6-20
                         BASF, 2007; rat
                    body weight; GD7-21
                    Borch etal., 2006; rat
                  body weight; GD8-18
                Howdeshell et al. 2008; rat
                            • significantly changed

                            O not significantly changed

                            Dstatistical analysis not
                              reported	
                                                                  &
                                                                  &
                                                                                 O-
                                                                                 -e—e-
                                                                                   o
                                                                                  -e—•
                                                          10
                                                                               100
                                                                                                   1000
                                                            Doses (mg/kg-day)
2
3
Figure 3-7.  Exposure-response array of female reproductive effects, maternal
weight and toxicity, following oral exposure to DIBP.
                This document is a draft for review purposes only and does not constitute Agency policy,
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                         Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
£   I
•p   */)
                    pre/postimplantation loss
                        BASF, 2007; rat
                             % resorptions
                            BASF, 2007; rat
               o         # of resorptions
               Q.       Borchetal., 2006; rat
               o
               J]      # of implantations
               ^   Howdeshell et al., 2008; rat
               £       total resorptions
               '•>=   Howdeshell et el., 2008; rat
               _§     % postimplantation loss
               |-    Saillenfait et al., 2006; rat
                        % resorptions
                    Saillenfait etal., 2006; rat
                     % postimplantation loss
                    Saillenfait et al., 2008; rat
                          # of fetuses/litter
                           BASF, 2007; rat
                          fetal viability
                       Borchetal., 2006; rat
                            litter size
               £       Borch et al., 2006; rat
               ;§       # of fetuses/litter
               -   Howdeshell et al. 2008; rat
               TO
               2         % live litters
                    Saillenfait etal., 2006; rat
                       # of fetuses/litter
                    Saillenfait et al., 2006; rat
                       # of fetuses/litter
                    Saillenfait et al., 2008; rat

               >-   Howdeshell et el., 2008; rat
               ~3
               £       whole litter loss
               2   Howdeshell et el., 2008; rat
               2
               ^    Saillenfait etal., 2006; rat

                            BASF, 2007; rat

                    Saillenfait et al., 2006; rat
                                         • significantly changed

                                         O not significantly changed
                                                                                                       -•-HP
                                                                                                   -e—•—«
                                                                                                   -e-e
                                                                                                     o

                                                                                                     o
                                                                                                       -•-HI
                                                                                                   -e-e
                                                                                                       -e—o
                                                                 10                      100

                                                                    Doses (mg/kg-day)
                                                                                                                1000
2
3
        Figure 3-8.  Exposure-response array of female reproductive effects, fertility
        and fetal survival, following oral exposure to DIBP.
                  This document is a draft for review purposes only and does not constitute Agency policy,
                                                            3-78               DRAFT—DO NOT CITE OR QUOTE

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2
3
               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.3.4.  Liver Effects

       Table 3-20. Evidence pertaining to hepatic effects in animals following oral
       exposure to DIBP
Reference and study design
Results
Liver weight
Foster et al. (1982); Foster et al. (1981)
MIBP
Rat (Sprague-Dawley); 6 males/group
0, 800 mg/kg-day
Gavage
4 days
Oishi and Hiraga (1980c)
Rat (JCLWistar); 10 treated males;
20 control males
0, 2% (0, 1,200 mg/kg-day)b
Diet
1 week
Oishi and Hiraga (1980a)
Mouse (JCLICR); 10 males/group
0, 2%(0,2,100mg/kg-day)b
Diet
1 week
Oishi and Hiraga (1980b)
MIBP
Mouse (JCLICR); 10 males/group
0, 2% (0, 2,000 mg/kg-day)b
Diet
1 week
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82, 770,
4,700 mg/kg-day for females)0
Diet
Weaning to 4 months post-weaning
Liver weight (percent change compared to control")
Doses 0
relative weight 0
800
30%***
Liver weight (percent change compared to control0)
Doses 0
absolute weight 0%
relative weight 0%
1,200
27%*
35%*
Liver (with gallbladder) weight (percent change compared to control")
Doses 0
relative weight 0%
2,100
45%*
Liver weight (percent change compared to control0)
Doses 0
relative weight 0%
2,000
30%*
Liver weight (percent change compared to control")
Doses (M) 0 65 710
absolute weight 0% 6% 11%
relative weight 0% 2% 22%
Doses (F) Of 82 770
absolute weight 0% 0% 16%
5,800
5%
84%
4,700
41%
              This document is a draft for review purposes only and does not constitute Agency policy,
                                              3-79            DRAFT—DO NOT CITE OR QUOTE

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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

University of Rochester (1953)
Rat (Albino; no other strain
designation); 5 males/dose
0, 0.01, 0.1, 1, 2, 5% (0, 15, 140, 1,400,
3,000, 8,900 mg/kg-day)d
Diet
Weaning to 1 month post-weaning
Results
relative weight 0% 0% 8%
62%
Note: Statistical analysis not reported in study.
Liver weight (percent change compared to control")
Doses 0 15 140 1,400 3,000
absolute weight 0% -17% -12% 5% 15%
relative weight 0% -1% 5% 26% 43%
Note: Statistical analysis not reported in study.
8,900
13%
79%
Liver histopathology
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82, 770,
4,700 mg/kg-day for females)0
Diet
Weaning to 4 months post-weaning
University of Rochester (1953)
Rat (Albino; no other strain
designation); 5 males/dose
0, 0.01, 0.1, 1, 2, 5% (0, 15, 140, 1,400,
3,000, 8,900 mg/kg-day)d
Diet
Weaning to 1 month post-weaning
Liver histopathology
Wo treatment-related differences from control were observed at any
dose group.
Note: 4-5 animals per dose group were assessed. Statistical analysis
not reported in study.
Liver histopathology
Histopathological findings only noted in the control group. No
treatment-related differences were observed.
Notes: Number of animals assessed is unclear. Findings limited
"filled with coarse granular cytoplasm." Statistical analysis not
reported in study.
to
 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
aPercent change compared to control = treated value - control value x 100
                                         control value
bDose conversions were performed using this information: Oishi and Hiraga (1980a, c) average BWs over the week-
 long studies were 132 and 24 g for rats and mice, respectively, and the default food consumption rates of
 0.008 kg/day for male weanling Wistar rats and 0.0025 kg/day for male weanling B6C3Fi mice were applied (U.S.
 EPA, 1988).  Oishi and Hiraga (1980b) average BWs for rats and mice in these studies were 145 and 25 g,
 respectively, and the default food consumption rates of 0.008 kg/day for male weanling Wistar rats and
 0.0025kg/day for male weanling B6C3Fi mice (U.S. EPA, 1988) were applied. Note that Table 1-6 of U.S. EPA
 (1988) listed the default food consumption rate for male weanling Wistar rats as 0.080 kg/day.  However, it was
 later determined using an equation in Table 1-3 of the document that this value was actually supposed to be
 0.008 kg/day.
°Dose conversions were performed using this information: University of Rochester (1954) average BWs (measured
 at least once weekly) were 269, 277, 252, and 155 g for male rats, and 178,170,182, and 148 g for female rats at
                 This document is a draft for review purposes only and does not constitute Agency policy,
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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1     0, 0.1,1.0, and 5.0%, respectively; and the default food consumption of 0.018 for male and 0.014 kg/day for
 2     female rats (U.S. EPA, 1988) of an unspecified strain in a subchronic study were applied.
 3    dDose conversions were performed using this information: University of Rochester (1953) average BWs (measured
 4     weekly) of the rats were 139,124,127, 127,121, and  101 g at 0, 0.01, 0.1,1.0, 2.0, and 5.0%, respectively; and
 5     the default food consumption of 0.018 for male rats and 0.014 kg/day for female rats (U.S. EPA, 1988) of an
 6     unspecified strain in a subchronic study were applied.
 7
 8    * = Statistically significant difference at p < 0.05 from control value, as reported by study authors; ** = Statistically
 9     significant difference at p < 0.01 from control value, as reported by study authors; *** = Statistically significant
10     difference at p < 0.001 from control value, as reported by study authors.
11
                  This document is a draft for review purposes only and does not constitute Agency policy.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                                                10
                                                              100
                                                                            1000
                                                                                         10000
University of Rochester, 1954; rat, male
University of Rochester, 1954; rat, female
University of Rochester, 1953; rat, male
00 OJ
1 5
u
-§) Oishi & Hiraga, 1980b; mouse, male
'(U
£
Oishi & Hiraga, 1980a; mouse, male
Oishi & Hiraga, 1980c; rat, male
0)
00
3
5 Foster et al, 1982; rat, male
0

• sign fican
Onot signif
D statist ca




ly changed
cantly changed
analysis not reported











































•



1
•



i
»












                                                      Doses (mg/kg-day)
2
3
Figure 3-9. Exposure-response array of liver effects following oral exposure to
DIBP.
               This document is a draft for review purposes only and does not constitute Agency policy,
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2
3
               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.3.5.  Kidney Effects

       Table 3-21. Evidence pertaining to renal effects in animals following oral
       exposure to DIBP
Reference and study design
Results
Kidney weight
Foster et al. (1982); Foster et al.
(1981)
MIBP
Rat (Sprague-Dawley);
6 males/group
0, 800 mg/kg-day
Gavage
4 days
Oishi and Hiraga (1980c)
Rat (JCLWistar); 10 treated males;
20 control males
0, 2% (0, 1,200 mg/kg-day)b
Diet
1 week
Oishi and Hiraga (1980b)
MIBP
Mouse (JCLICR); 10 males/group
0, 2% (0, 2,000 mg/kg-day)b
Diet
1 week
Oishi and Hiraga (1980a)
Mouse (JCLICR); 10 males/group
Diet
0, 2%(0,2,100mg/kg-day)b
1 week
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for females)0
Diet
Kidney weight (percent change compared to control")
Doses 0
relative weight 0
800
396%***
Kidney weight (percent change compared to control")
Doses 0
absolute weight 0%
relative weight 0%
1,200
-5%
2%
Kidney weight (percent change compared to control0)
Doses 0
relative weight 0%
2,000
-5%
Kidney weight (percent change compared to control")
Doses 0
relative weight 0%
2,100
-10%*
Kidney weight (percent change compared to control")
Doses (M) 0 65 710
absolute weight 0% 10% 9%
relative weight 0% 7% 20%
5,800
-31%
22%
              This document is a draft for review purposes only and does not constitute Agency policy,
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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Weaning to 4 months post-weaning
University of Rochester (1953)
Rat (Albino; no other strain
designation); 5 males/dose
0, 0.01, 0.1, 1, 2, 5% (0, 15, 142,
1,417, 2,975, 8,911 mg/kg-day)d
Diet
Weaning to 1 month post-weaning
Results
Doses (F) 0 82 770 4,
700
absolute weight 0% 4% 11% -2%
relative weight 0% 5% 4% 13%
Note: Statistical analysis not reported in study.
Kidney weight (percent change compared to control")
Doses 0 15 140 1,400 3,000
absolute weight 0% -14% -11% -11% -11%
relative weight 0% 3% 7% 7% 12%
Note: Statistical analysis not reported in study.
8,900
-23%
23%
Kidney histopathology
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for females)0
Diet
Weaning to 4 months post-weaning
University of Rochester (1953)
Rat (Albino; no other strain
designation); 5 males/dose
0, 0.01, 0.1, 1, 2, 5% (0, 15, 142,
1,417, 2,975, 8,911 mg/kg-day)d
Diet
Weaning to 1 month post-weaning
Kidney histopathology
Wo treatment-related differences in males or females were observed
compared with control.
Note: 4-5 animals per dose group were assessed. Findings in males
limited to pyelitis, granuloma, and pyelonephritis; Findings in females
limited to pyelitis and pyelonephritis.
Kidney histopathology
Wo treatment-related differences were observed.
Note: Number of animals assessed is unclear. Findings limited to
eosinophils and inflammatory cells. Statistical analysis not reported
study.
in
 1
 2
 3
 4
 5
 6
 7
 8
 9
10
aPercent change compared to control = treated value - control value x 100
                                          control value
bDose conversions were performed using this information: Oishi and Hiraga (1980a, c) average BWs over the week-
 long studies were 132 and 24 g for rats and mice, respectively, and the default food consumption rates of
 0.008 kg/day for male weanling Wistar rats and 0.0025 kg/day for male weanling B6C3Fi mice were applied (U.S.
 EPA, 1988).  Oishi and Hiraga (1980b) average BWs for rats and mice in these studies were 145 and 25 g,
 respectively, and the default food consumption rates of 0.008 kg/day for male weanling Wistar rats and 0.0025
 kg/day for male weanling B6C3Fi mice (U.S. EPA, 1988) were applied. Note that Table 1-6 of U.S. EPA (1988)
 listed the default food consumption rate for male weanling Wistar rats as 0.080 kg/day.  However, it was later
                 This document is a draft for review purposes only and does not constitute Agency policy,
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                       Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1     determined using an equation in Table 1-3 of the document that this value was actually supposed to be 0.008
 2     kg/day.
 3    °Dose conversions were performed using this information:University of Rochester (1954) average BWs (measured
 4     at least once weekly) were 269, 277, 252, and 155 g for male rats, and 178,170,182, and 148 g for female rats at
 5     0, 0.1,1.0, and 5.0%, respectively; and default food consumption of 0.018 kg/day for male rats and 0.014 kg/day
 6     for female rats (U.S. EPA, 1988) of an unspecified strain in a subchronic study were applied.
 7    dDose conversions were performed using this information: University of Rochester (1953) average BWs (measured
 8     weekly) were 139,124,127,127,121, and 101 g at 0,0.01, 0.1,1.0, 2.0, and 5.0%, respectively; and default food
 9     consumption of 0.018 for male rats and 0.014 kg/day for female rats (U.S. EPA, 1988) of an unspecified strain in a
10     subchronic study were applied.
11
12    * = Statistically significant difference at p < 0.05 from control value, as reported by study authors.
13
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
                   Foster et al., 1982; rat, male
               Oishi and Hiraga, 1980c; rat, male
            Oishi and Hiraga, 1980a; mouse, male
            Oishi and Hiraga, 1980b; mouse, male
           University of Rochester, 1953; rat, male
           University of Rochester, 1954; rat, male
          University of Rochester, 1954; rat, female

• significantly changed
O not significantly changed •
D statistical analysis not reported



B_





O
•
O



                                                       10             100

                                                           Doses (mg/kg-day)
                                                                                    1000
                                                                                                   10000
2
3
Figure 3-10. Exposure-response array of kidney effects following oral
exposure to DIBP.
                This document is a draft for review purposes only and does not constitute Agency policy,
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2
3
               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.3.6.  Hematopoietic Effects

       Table 3-22. Evidence pertaining to hematopoietic effects in animals following
       oral exposure to DIBP
Reference and study design
Results
Hematology
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82, 770,
4,700 mg/kg-day for females)b
Diet
Weaning to 4 months post-weaning



University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82, 770,
4,700 mg/kg-day for females)b
Diet
Weaning to 4 months post-weaning








Hematology at 4 months (percent change compared to control")
Doses (M)
RBCs
WBCs
Hgb
Doses (F)
RBCs
WBCs
Hgb
Note: Statistical
0
0%
0%
0%
0
0%
0%
0%
analysis not
65
-1%
-37%
-4%
82
6%
-19%
0%
reported in study.
Differential counts at 4 months (percent of each
Doses (M)
neutrophils
eosinophils
basophils
lymphocytes
monocytes
myeloids
blast forms
plasma cells
Doses (F)
neutrophils
eosinophils
basophils
lymphocytes
monocytes
0
20%
1%
0%
79%
1%
0%
0%
0%
0
15%
2%
0%
83%
0%
65
18%
5%
0%
76%
0%
0%
0%
0%
82
14%
4%
0%
82%
0%
710
-5%
-15%
-5%
770
1%
-8%
3%

type of WBC)
710
19%
5%
1%
75%
0%
0%
0%
0%
770
22%
4%
0%
73%
0%
5,800
-16%
38%
-9%
4,700
13%
29%
-6%


5,800
17%
2%
1%
80%
0%
0%
0%
0%
4,700
13%
3%
1%
84%
0%
              This document is a draft for review purposes only and does not constitute Agency policy,
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

Results
myeloids 0% 0%
blast forms 0% 0%
Note: Statistical analysis not reported in study.
0%
0%
0%
0%
Spleen weight
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for females)b
Diet
Weaning to 4 months post-weaning
Spleen weight (percent change compared to control")
Doses (M) 0 65
absolute weight 2% 9%
relative weight 0% 5%
Doses (F) 0 82
absolute weight 0% 8%
relative weight 0% 11%
Note: Statistical analysis not reported in study.
710
7%
17%
770
93%
80%
5,800
-13%
52%
4,700
-6%
9%
1
2
3
4
5
6
7
8
9
aPercent change compared to control = treated value - control value x 100
                                        control value
bDose conversions were performed by EPA using this information: University of Rochester (1954) average BWs
 (measured at least once weekly) were 269, 277, 252, and 155 g for male rats, and 178,170,182, and 148 g for
 female rats at 0, 0.1,1.0, and 5.0%, respectively; and the default food consumption of 0.018 kg/day for male
 rats and 0.014 kg/day for female rats (U.S. EPA, 1988) of an unspecified strain in a subchronic study were applied.

Hgb = hemoglobin; RBC = red blood cell; WBC = white blood cell
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                         Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
              =    University of Rochester, 1954; rat, male
              £1   University of Rochester, 1954; rat, female
                   University of Rochester, 1954; rat, male
           >.  oj   University of Rochester, 1954; rat, female
           on  ^
           o  -n
      £   E
      U   OJ
                          differential counts*
                   University of Rochester, 1954; rat male
                         differential counts*
                  University of Rochester, 1954; rat, female
              c    University of Rochester, 1954; rat, male
                  University of Rochester, 1954; rat, female
                   University of Rochester, 1954; rat, male
                  University of Rochester, 1954; rat, female
        * differential counts include neutrophil, eosinophil,
        basophil, lymphocyte, monocyte, myelocyte, blast forms
        and plasma cell measurements
D statistical analysi
reported

not n

































                                                  10              100

                                                     Doses (mg/kg-day)
                                                                                                 1000
                                                                                                                 10000
2
3
Figure 3-11.  Exposure-response array of hematopoeitic effects following oral
exposure to DIBP.
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2
3
               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

3.3.7.  Other Effects

       Table 3-23. Evidence pertaining to other toxicity effects in animals following
       oral exposure to DIBP
Reference and study design
Results
Neurotoxicity effects
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for females)b
Diet
Weaning to 4 months post-weaning
Brain weight (percent change compared to control")
Doses (M) 0 65 710
absolute weight 0% 1% 0%
relative weight 0% -2% 11%
Doses (F) 0 82 770
absolute weight 0% 3% 1%
relative weight 0% 4% -6%
Note: Statistical analysis not reported in study.
5,800
-3%
72%
4,700
2%
17%
Cardiac effects
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for females)b
Diet
Weaning to 4 months post-weaning
Heart weight (percent change compared to control)
Doses (M) 0 65 710
absolute weight 0% 2% -6%
relative weight 0% -3% 3%
Doses (F) 0 82 770
absolute weight 0% 10% -4%
relative weight 0% 10% -11%
Note: Statistical analysis not reported in study.
5,800
-28%
24%
4,700
11%
28%
Lung effects
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for females)b
Diet
Lung weight (percent change compared to control")
Doses (M) 0 65 710
absolute weight 0% 10% 4%
relative weight 0% 5% 16%
5,800
-30%
23%
              This document is a draft for review purposes only and does not constitute Agency policy,
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     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design
Weaning to 4 months post-weaning


Results
Doses (F)
absolute weight
relative weight
Note: Statistical
0
0%
0%
analysis not



reported
82
-20%
-19%
in study.
770
-6%
-12%





4,700
-27%
-16%

Stomach effects
University of Rochester (1954)
Rat (Albino; no other strain
designation); 5 males and 5
females/dose
0,0.1, 1,5% (0,65, 710,
5,800 mg/kg-day for males; 0, 82,
770, 4,700 mg/kg-day for females)b
Diet
Weaning to 4 months post-weaning


Stomach weight (percent change compared to
Doses (M)
absolute weight
relative weight
Doses (F)
absolute weight
relative weight
Note: Statistical
0
0%
0%
0
0%
0%
analysis not






reported
65
10%
7%
82
8%
9%
in study.
control")
710
6%
18%
770
7%
0%










5,800
1%
80%
4,700
18%
35%

Clinical signs
Hazleton Laboratories (1992, 1987);
NIOSH(1983)
Mouse (CD-I); 50 control females,
10 females/treated group
0, 1,000, 1,795, 3,225, 5,790,
10,400 mg/kg-day
Gavage
8 days










Clinical signs of toxicity (incidence /total animals)
Doses
languid
prostrate
ataxia
hunched
tremors
head tilt
thin
wheezing
dyspnea
urine stains
alopecia
rough hair coat
sores
piloerection
opaque eyes
0
1/50
0/50
0/50
0/50
0/50
0/50
1/50
0/50
0/50
0/50
0/50
0/50
0/50
0/50
N/A
1,000
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1,795
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
3,225
2/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
5,790
5/10
4/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1/10
10,400
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
10/10
0/10
10/10
0/10
0/10
0/10
This document is a draft for review purposes only and does not constitute Agency policy,
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
Reference and study design

Results
discoloration N/A 0/10 0/10 0/10 1/10 0/10
(yellow hair)
Note: Statistical analysis not reported in study for clinical signs data.
1
2
3
4
5
6
7
8
aPercent change compared to control = treated value - control value x 100
                                        control value
bDose conversions were performed using this information: University of Rochester (1954) average BWs (measured
 at least once weekly) were 269, 277, 252, and 155 g for male rats, and 178,170,182, and 148 g for female rats at
 0, 0.1,1.0, and 5.0%, respectively; and default food consumption of 0.018 kg/day for male rats and 0.014 kg/day
 for female rats (U.S. EPA, 1988) of an unspecified strain in a subchronic study were applied.
                This document is a draft for review purposes only and does not constitute Agency policy,
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                   Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
University of Rochester, 1954; rat, male


D no statistical analysis
reported
University of Rochester, 1954; rat, female
University of Rochester, 1954; rat, male
University of Rochester, 1954; rat, female
University of Rochester, 1954; rat, male
University of Rochester, 1954; rat, female
University of Rochester, 1954; rat, male
University of Rochester, 1954; rat, female
H, 1983; Hazleton Laboratories, 1987;
Laboratories, 1992; mouse, male & female
1 1







0 1C
Doses (mg/kg-d








i
)0 10
ay)









00 IOC
                                                                                       100000
2
3
Figure 3-12. Exposure-response array of effects on other toxicities following
oral exposure to DIBP.
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                     Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 l    3.4. PRELIMINARY MECHANISTIC INFORMATION FOR DIBP
 2          The systematic literature search for DIBP also identified studies evaluating mechanisms of
 3    action considered potentially relevant to effects observed following exposure to DIBP.  Studies were
 4    included if they evaluated mechanistic events following exposure to DIBP or the metabolite, MIBP,
 5    or contained information relevant to the mechanistic understanding of DIBP toxicity. Reviews or
 6    analyses that do not contain original data are not included here, but may be considered in later
 7    stages of assessment development
 8          The diverse array of mechanistic studies presented here includes investigations of the
 9    cellular, biochemical, and molecular mechanisms underlying toxicological outcomes. For this
10    preliminary evaluation, information reported in each study was extracted into a database (in the
11    form of an Excel spreadsheet) that will facilitate future evaluation of mechanistic information. This
12    information is being made available to provide an opportunity for stakeholder input, including the
13    identification of relevant studies not captured here.
14          The information extracted from each study and included in the database, corresponds to the
15    column headings in the spreadsheet, and is as follows: link to the HERO record (contained within a
16    URL that links to the study abstract in the HERO database), HERO ID, author(s), year, molecular
17    formulation, in vitro/in vivo, species, cell type, endpoint(s) (i.e., mechanistic outcomes), assay, and
18    mechanistic category.  The database supports sorting capabilities, e.g., data can be organized by
19    assay.  The database is available through HERO at
20    [http://hero.epa.gov/index.cfm?action=reference.details&reference_id=2508641]. To access the
21    database, click on the link at the top of the web page and select "download" and then "ok" to view
22    the spreadsheet in Excel. This spreadsheet may also be saved to your desktop by downloading and
23    selecting "save." The resulting inventory of DIBP mechanistic studies consists of 32 mechanistic
24    outcomes from 13  identified in vivo studies, as well as 28 mechanistic outcomes from 23 in vitro
25    assays. Table 3-24 presents a summary of the mechanistic outcomes recording in the database
26    from each study identified.
27          The mechanistic categories developed here are not mutually exclusive and are designed to
28    facilitate the analysis of similar studies and experimental observations in a systematic manner.
29    This process will allow the identification of mechanistic events that contribute to mode(s) of action
30    (MOAs) and/or adverse outcome pathways (AOPs) following DIBP exposure. The mechanistic
31    categories assigned to each mechanistic outcome reported by an individual study are as follows:
32    (1) mutation, including investigations of gene and chromosomal mutation; (2) DNA damage,
33    including indicator assays of genetic damage; (3) DNA repair; (4) oxidative stress; (5) cell death and
34    division (this captures a broad range of assays, but it is useful to consider them together as
35    observations resulting from cell cycle alterations; (6) pathology, which includes morphological
36    evaluations pertaining to the dysfunction of organs, tissues, and cells; (7) epigenetic effects, which
37    are observations of heritable changes in gene function that cannot be explained by changes in the
38    DNA sequence; (8) receptor-mediated and cell signaling effects; (9) immune system effects;
39    (10) cellular differentiation and transformation; (11) cellular energetics; and (12) "other," to

                This document is a draft for review purposes only and does not constitute Agency policy.
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                      Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate
 1
 2
 3
 4
capture those mechanistic outcomes not easily assigned to a defined category. Mechanistic
outcomes in the "other" category include sex steroid hormone (e.g., testosterone) production and
gene expression.
 5
 6
       Table 3-24.  Summary of mechanistic outcomes evaluated following DIBP or
       MIBP administration
Mechanistic
category
Mutation3
DNA damage
Total #
outcomes/
# studies
5/5
6/4

Total
0
0
In vivo (# outcomes/
# studies)
Human
0
0
Rat
0
0
Mouse
0
0
In vitro (# outcomes/ft studies)
Total
5/5
6/4
Human
0
6/4
Primate
0
0
Rat
0
0
Mouse
0
0
DNA repair
Oxidative stressb
Cell death and
division
Pathology
1/1
7/5
2/2
1/1
3/2
2/2
0
0
0
0
2/2
2/2
0
1/1
0
0
4/3
N/A
0
3/2
0
0
0
0
0
1/1
N/A
Epigenetics
Receptor-
mediated and
cell signaling0
Immune system
Cellular
differentiation
and
transformation
Cellular
energetics
Otherd
Total
14/9
5/3
1/1
1/1
18/11
60/35
8/4
1/1
1/1
0
16/10

1/1
0
1/1
0
0
7/3
1/1
0
0
14/8
0
0
0
0
2/2
32/13
6/5
4/2
0
1/1
2/2
0
1/1
0
0
0
1/1
0
0
0
0
0
2/1
0
1/1
2/2
0
1/1
0
0
0
28/23
 7
 8
 9
10
11
12
13
14
15
16
17
18
aDatabase included five outcomes in five studies utilizing Salmonella typhimuhum.
bDatabase included one outcome from one study utilizing Caenorhabditis elegans.
°Database included two outcomes from one study utilizing cultured hamster cells, two outcomes from two studies
 utilizing yeast, and one cell-free system.
dDatabase primarily composed of hormone (testosterone, estradiol) content or production in tissues from rats and
 mice.

Notes: The number in rows may not sum to "total" amounts as several studies evaluated multiple species or
 employed both in vivo and in vitro models. The mechanistic categories in italics and in gray shading had no DIBP-
 specific information available.
                 This document is a draft for review purposes only and does not constitute Agency policy,
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 1
 2
 3
 4
 5
 6
 7
 8
 9
10
               Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

       Information summarized in Table 3-24 and Figure 3-13 and detailed in the mechanistic
database can be used to ascertain the breadth and scope of available mechanistic studies. At this
preliminary stage, study results are not presented.  Additionally, the inclusion of a study in the
spreadsheet does not reflect conclusions reached as to mechanistic study quality or relevance.
After the epidemiological and experimental studies on each health effect have been synthesized,
mechanistic studies will be reviewed and findings synthesized to evaluate potential MOAs and/or
AOPs, which can be used to inform hazard identification and dose-response assessment, specifically
addressing questions of human relevance, susceptibility, and dose-response relationships.
11
12
13

14
                                       Mechanistic Outcomes
                                  (60 outcomes from 35 reports)
                                                                        I n vivo
I In vitro
                         Mutation

                      DNA damage

                    Oxidative stress

                Cell death and division

                        Pathology
            Receptor-mediated and cell
                  signaling
                    Immune system
            Cellular differentiation and
                 transformation
                  Cellular energetics

                           Other
                                                              10
                                                      Number of endpoints
                                                                             15
                                                                                           20
       Figure 3-13. Summary of in vivo and in vitro mechanistic data for DIBP and
       MIBP by mechanistic category.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate


 1
 2
4.    REFERENCES
 3   Adibl JJ: Hauser. R: Williams. PL: Whyatt RM: Calafat AM: Nelson. H: Herrick. R: Swan.
 4          SH. (2009). Maternal urinary metabolites of Di-(2-Ethylhexyl) phthalate in relation to the
 5          timing of labor in a US multicenter pregnancy cohort study. Am J Epidemiol 169: 1015-
 6          1024. http://dx.doi.org/10.1093/aje/kwp001
 7   Adibl JJ: Whyatt RM: Williams. PL: Calafat AM: Camann. D: Herrick. R: Nelson. H: Bhat
 8          HK; Perera, FP; Silva, MJ: Hauser, R. (2008). Characterization of phthalate exposure
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11   Ait Bamai, Y; Shibata, E; Saito, I; Araki, A; Kanazawa, A; Morimoto, K; Nakayama, K; Tanaka,
12          M: Takigawa. T: Yoshimura. T: Chikara. H: Saijo. Y: Kishi. R. (2014). Exposure to
13          house dust phthalates in relation to asthma and allergies in both children and adults. Sci
14          Total Environ 485-486: 153-163. http://dx.doi.Org/10.1016/j.scitotenv.2014.03.059
15   Aschengrau, A; Coogan,  P; Ouinn, M; Cashins, L. (1998). Occupational exposure to estrogenic
16          chemicals and the occurrence of breast cancer: An exploratory analysis. Am J Ind Med
17          34:6-14. http://dx.doi.org/10.10027(8101)1097-0274(199807)34:1<6::AID-
18          AJIM2>3.0.CO:2-X
19   Baird, DP: Wilcox, AJ. (1985). Cigarette smoking associated with delayed conception. JAMA
20          253: 2979-2983. http://dx.doi.org/10.1001/iama.1985.03350440057031
21   Baird, DP: Wilcox, AJ: Weinberg, CR. (1986). Use of time to pregnancy to study environmental
22          exposures. Am J Epidemiol 124: 470-480.
23   BASF. (1961). AG: Abteilung Toxikologie, unpublished study (IX/418) [Cited in European
24          Commission, 2004, 2000].
25   BASF. (2003). Diisobutyl Phthalate: Prenatal developmental toxicity study in Wistar rats:
26          Administration in the diet (Volume 1 of 3). (Project number 32R0233/02018).
27          [Germany].
28   BASF. (2007). Diisobutylphthalate - prenatal developmental toxicity study in Wistar rats
29          administration in the diet  (2007 Update) [TSCA Submission] (pp. 68-155). (Document
30          Control Number:  86070000046). Submitted to the U.S. Environmental Protection Agency
31          under TSCA Section 8d.
32          http://vosemite.epa.gov/oppts/epatscat8.nsf/bv+Service/82FC6103COE2F95585257B510
33          0479E79/$File/86070000046.pdf
34   Behall KM: Scholfield. DJ: Hallfrisch. JG: Kelsav, JL: Reiser. S. (1984). Seasonal variation in
35          plasma glucose and hormone levels in adult men and women. Am J Clin Nutr 40: 1352-
36          1356.
37   Bertelsen. RJ: Carlsen. KC: Calafat AM: Hoppin. JA: Haland. G: Mowinckel P: Carlsen. KH:
38          L0vik, M. (2013). Urinary biomarkers for phthalates associated with asthma in
39          Norwegian children. Environ Health Perspect 121: 251-256.
40          http://dx.doi.org/10.1289/ehp.1205256
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1    Blair, A; Stewart, P; Lubin, JH; Forastiere, F. (2007). Methodological issues regarding
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36          Perspect 116: 1092-1097. http://dx.doi.org/10.1289/ehp.11007
37    Wolff. MS:  Teitelbaum. SL: Pinnev. SM: Windham. G: Liao. L:  Biro. F: Kushl LH: Erdmann.
38          C: Hiatt RA; Rvbak, ME; Calafat, AM. (2010). Investigation of relationships between
39          urinary biomarkers of phytoestrogens, phthalates, and phenols and pubertal stages in
40          girls. Environ Health Perspect 118: 1039-1046.  http://dx.doi.org/10.1289/ehp.0901690
               This document is a draft for review purposes only and does not constitute Agency policy.
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                    Preliminary Materials for the IRIS Toxicological Review ofDiisobutyl Phthalate

 1   Wormuth, M; Scheringer, M; Vollenweider, M; Hungerbuhler, K. (2006). What are the sources
 2          of exposure to eight frequently used phthalic acid esters in Europeans? Risk Anal 26:
 3          803-824. http://dx.doi.0rg/10.llll/i.1539-6924.2006.00770.
 4   Zhu, XB; Tay, TW: Andriana, BB; Alam, MS: Choi, EK; Tsunekawa, N; Kanai, Y; Kurohmaru,
 5          M. (2010). Effects of di-iso-butyl phthalate on testes of prepubertal rats and mice.
 6          Okajimas Folia Anat Jpn 86:  129-136.
 7   Zota, AR; Calafat AM; Woodruff, TJ. (2014). Temporal trends in phthalate exposures: findings
 8          from the national health and nutrition examination survey, 2001-2010. Environ Health
 9          Perspect 122: 235-241. http://dx.doi.org/10.1289/ehp.1306681
10
11
               This document is a draft for review purposes only and does not constitute Agency policy.
                                               4-15            DRAFT—DO NOT CITE OR QUOTE

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