EPA/635/R-14/242
«• ^_^J^% Preliminary Materials
www.epa.gov/iris
Preliminary Materials for the Integrated Risk Information System (IRIS)
Toxicological Review of Diisononyl Phthalate (DINP)
(CASRNs 28553-12-0, 68515-48-0, 71549-78-5, and 14103-61-8)
August 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 ofDiisononyl 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 ofDiisononyl Phthalate
CONTENTS
PREFACE viii
1. INTRODUCTION 1-1
1.1.DINP 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 1-3
1.2.SCOPE OF THE ASSESSMENT 1-4
2. METHODS FOR IDENTIFYING AND SELECTING STUDIES 2-5
2.1.DRAFT LITERATURE SEARCH AND SCREENING STRATEGY 2-5
2.2.SELECTION OF CRITICAL STUDIES IN EARLY STAGES OF DRAFT DEVELOPMENT 2-15
2.2.1. General Approach 2-15
2.2.2. Exclusion of Studies 2-16
2.3.STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE FUTURE EVALUATION AND
SYNTHESIS OF THE CRITICAL EPIDEMIOLOGICAL STUDIES FOR DINP 2-17
2.4.STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE FUTURE EVALUATION AND
SYNTHESIS OF THE CRITICAL EXPERIMENTAL STUDIES FOR DINP 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. Pregnancy Related Outcomes 3-3
3.2.3. Male Reproductive Effects in Humans 3-5
3.2.4. Male Pubertal Development in Humans 3-7
3.2.5. Female Reproductive Effects in Humans 3-8
3.2.6. Female Pubertal Development in Humans 3-10
3.2.7. Thyroid Effects in Humans 3-12
3.2.8. Immune Effects in Humans 3-13
3.2.9. Immune Effects in Humans 3-14
3.2.10.Obesity Effects in Humans 3-16
3.3.ANIMAL STUDIES 3-17
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 ofDiisononyl Phthalate
3.3.1. Liver Effects 3-17
3.3.2. Kidney Effects 3-34
3.3.3. Male Reproductive Effects 3-44
3.3.4. Female Reproductive Effects 3-59
3.3.5. Developmental Effects 3-70
3.3.6. Hematopoietic Effects 3-75
3.4. PRELIMINARY MECHANISTIC INFORMATION FOR DINP 3-79
4. REFERENCES 4-1
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 ofDiisononyl Phthalate
TABLES
Table 2-1. Database search strategy for DINP 2-6
Table 2-2. Summary of additional search strategies for DINP 2-7
Table 2-3. Inclusion criteria used to identify animal studies of health-related endpoints,
supporting data, or secondary literature 2-11
Table 2-4. Summary of search terms: targeted epidemiology search 2-12
Table 2-5. Inclusion criteria used to identify epidemiology studies of health-related endpoints 2-13
Table 2-6. Primary source epidemiological studies examining health effects of DINP 2-14
Table 2-7. Summary of additional search strategies for epidemiology studies of phthalate
exposure in relation to health-related endpoints 2-14
Table 2-8. DINP metabolites and their synonyms 2-19
Table 2-9. General and outcome-specific considerations for DINP study evaluation 2-27
Table 2-10. Questions and relevant experimental information for the evaluation of
experimental animal studies 2-30
Table 3-1. Evidence pertaining to DINP metabolite(s) and measures of sexual differentiation in
humans 3-2
Table 3-2. Evidence pertaining to DINP metabolite(s) and pregnancy outcomes in humans 3-3
Table 3-3. Evidence pertaining to DINP metabolite(s) and male reproductive effects in humans 3-5
Table 3-4. Evidence pertaining to DINP metabolite(s) and the timing of male puberty in humans 3-7
Table 3-5. Evidence pertaining to DINP metabolite(s) and gynecological conditions or
reproductive and steroidal hormones in humans 3-8
Table 3-6. Evidence pertaining to DINP metabolite(s) and the timing of female puberty in
humans 3-10
Table 3-7. Evidence pertaining to DINP metabolite(s) and thyroid effects in humans 3-12
Table 3-8. Evidence pertaining to DINP metabolite(s) and immune effects in humans 3-13
Table 3-9. Evidence pertaining to DINP metabolite(s) and immune effects in humans 3-14
Table 3-10. Evidence pertaining to DINP metabolite(s) and obesity in humans 3-16
Table 3-11. Evidence pertaining to liver effects in animals following oral exposure to DINP 3-17
Table 3-12. Evidence pertaining to kidney effects in animals following oral exposure to DINP 3-34
Table 3-13. Evidence pertaining to male reproductive effects in animals following oral exposure
to DINP 3-44
Table 3-14. Evidence pertaining to female reproductive effects in animals following oral
exposure to DINP 3-59
Table 3-15. Evidence pertaining to developmental effects in animals following oral exposure to
DINP 3-70
Table 3-16. Evidence pertaining to hematopoietic effects in animals following oral exposure to
DINP 3-75
Table 3-17. Summary of mechanistic outcomes evaluated following DINP administration 3-80
FIGURES
Figure 1-1. Chemical structure of DINP (HSDB, 2009) 1-1
Figure 2-1. Literature search approach for DINP 2-10
Figure 3-1. Exposure-response array of liver weight effects following oral exposure to DINP 3-31
Figure 3-2. Exposure-response array of liver serum chemistry enzyme levels following oral
exposure to DINP 3-32
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 ofDiisononyl Phthalate
Figure 3-3. Exposure-response array of liver histopathological effects following oral exposure to
DINP 3-33
Figure 3-4. Exposure-response array of kidney weight effects following oral exposure to DINP 3-42
Figure 3-5. Exposure-response array of kidney histopathological effects following oral exposure
to DINP 3-43
Figure 3-6. Exposure-response array of male reproductive puberty effects following oral
exposure to DINP 3-55
Figure 3-7. Exposure-response array of male reproductive testosterone effects following oral
exposure to DINP 3-56
Figure 3-8. Exposure-response array of male reproductive histopathological effects following
oral exposure to DINP 3-57
Figure 3-9. Exposure-response array of male reproductive organ weight effects following oral
exposure to DINP 3-58
Figure 3-10. Exposure-response array of female reproductive fertility measures following oral
exposure to DINP 3-67
Figure 3-11. Exposure-response array of other female reproductive effects following oral
exposure to DINP 3-68
Figure 3-12. Exposure-response array of maternal weight gain effects following oral exposure to
DINP 3-69
Figure 3-13. Exposure-response array of developmental effects following oral exposure to DINP 3-74
Figure 3-14. Exposure-response array of hematopoietic effects following oral exposure to DINP 3-78
Figure 3-15. Summary of in vivo and in vitro mechanistic data by mechanistic category 3-81
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 ofDiisononyl Phthalate
ABBREVIATIONS
AGO anogenital distance MEHHP
ALP alkaline phosphatase
ALT alanine aminotransferase MEOHP
AOP adverse outcome pathway MHINP
AST aspartate aminotransferase MIBP
BBP butyl benzyl phthalate MINP
BMI body mass index MNCL
BUN blood urea nitrogen MOA
BW body weight MOINP
CalEPA California Environmental Protection NCEA
Agency
CASRN Chemical Abstracts Service Registry NHANES
Number
CHAP Chronic Hazard Advisory Panel NRC
CI confidence interval NTP
CPSC Consumer Product Safety Commission OR
CPSIA Consumer Product Safety Improvement ORD
Act PCOS
DBF dibutyl phthalate PND
DEP di-ethyl phthalate PNW
DEHP di(2-ethylhexyl)phthalate PVC
DHEAS Dehydroepiandrosterone RBC
DIBP diisobutyl phthalate SD
DIDP di-isodecyl phthalate SHBG
DINP diisononyl phthalate T3
DNA deoxyribonucleic acid T4
DPP dipentyl phthalate TSCA
ED estrous day TSH
EPA Environmental Protection Agency WHO
FIFRA Federal Insecticide, Fungicide, and
Rodenticide Act
FSH follicle stimulating hormone
GD gestational day
Hct hematocrit
HERO Health and Environmental Research
Online
Hgb hemoglobin
IgE immunoglobulin E
ICC intra-class correlation coefficient
IRIS Integrated Risk Information System
LH luteinizing hormone
LOD level of detection
LOQ level of quantification
MBzP mono-benzyl phthalate
MEP monoethyl phthalate
MBP monobutyl phthalate
MCIOP mono-carboxyisooctyl phthalate
MCNP monocarboxyisononyl phthalate
MCOP mono-carboxyoctyl phthalate
MCPP mono(3-carboxypropyl) phthalate
MECCP mono-2-ethyl-carboxypentyl
MEHP mono-(2-ethylhexyl) phthalate
mono-2-ethyl-5-hydroxyhexyl
phthalate
mono-2-ethyl-oxohexyl phthalate
mono-hydroxyisononyl phthalate
monoisobutyl phthalate
monoisononyl phthalate
mononuclear cell leukemia
mode of action
oxo-(mono-oxoisononyl) phthalate
National Center for Environmental
Assessment
National Health and Nutrition
Examination Survey
National Research Council
National Toxicology Program
odds ratio
Office of Research and Development
polycystic ovarian syndrome
postnatal day
postnatal week
polyvinyl chloride
red blood cell
standard deviation
sex-hormone binding globulin
triiodothyronine
thyroxine
Toxic Substances Control Act
thyroid stimulating hormone
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 ofDiisononyl Phthalate
1
2
PREFACE
3 This draft document presents preliminary materials for an assessment of diisononyl
4 phthalate (DINP) 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 DINP. 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 DINP, occurrence of
14 DINP 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 at 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 DINP 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. In May 2014, the NRC released their report reviewing the IRIS assessment
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 ofDiisononyl Phthalate
1 development process. As part of this review, the NRC reviewed current methods for evidence-
2 based reviews and made several recommendations with respect to integrating scientific evidence
3 for chemical hazard and dose-response assessments. In their report, the NRC states that EPA
4 should continue to improve its evidence-integration process incrementally and enhance the
5 transparency of its process. The committee did not offer a preference but suggests that EPA
6 consider which approach best fits its plans for the IRIS process. The NRC recommendations will
7 inform the IRIS Program's efforts in this area going forward. This effort is included in Phase 3 of
8 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 ofDiisononyl Phthalate
2
3 1. INTRODUCTION
4 This introduction contains a planning and scoping summary for the Integrated Risk
5 Information System (IRIS) assessment of diisononyl phthalate (DINP). The planning and scoping
6 summary includes information on the properties, sources, and uses of DINP, occurrence and fate of
7 DINP in the environment, potential for human exposure, and the rationale for the development of
8 this assessment
9 1.1. DINP IN THE ENVIRONMENT
10 1.1.1. Production and Use
11 DINP (Chemical Abstract Service Registry Numbers (CASRNs) 68515-48-0, 28553-12-0,
12 71549-78-5,14103-61-8), is not a pure compound, but rather a mixture of isomers with an average
13 side chain length of nine carbons (Figure 1-1).
14
15 Figure 1-1. Chemical structure of DINP fHSDB. 20091.
16
17 Between 100 and 500 million pounds of DINP was imported or manufactured in US in 2006
18 (US.EPA 2014). It is used in the production of plastics to increase flexibility and is commonly
19 present in products such as toys, vinyl swimming pools, vinyl containing furniture and clothes,
20 flooring, gloves, drinking straws, garden hoses, sealants used in food packaging, and cosmetics
21 (CDC. 2014: (HSDB. 2009). Most DINP is used in PVC products, with less than 10% used in non-
22 PVC products such as different types of rubber, inks, pigments, paints, lacquers, adhesives, and
23 sealants fGal/EPA. 20131 The use of di-2-ethylhexyl phthalate (DEHP) has largely been replaced by
24 DINP, though not in medical products. In 2008, the Consumer Product Safety Improvement Act
25 (CPSIA) placed an interim ban on DINP in children's toys and certain child care articles at
26 concentrations greater than 0.1 percent The Chronic Hazard Advisory Panel (CHAP) recommended
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 ofDiisononyl Phthalate
1 that the interim ban on DINP be made permanent in children's toys and child care products at level
2 greater than 0.1% [CHAP. 20141.
3 DINP has been sold in varying commercial formulations, such as DINP-1, DINP-2, and
4 DINP-3, which are produced with different C8-C10 alcohol feedstocks (Gill etal.. 20011. Production
5 of the DINP-3 formulation was discontinued in 1995 (ECPI. 2010: Gill etal.. 20011. The exact
6 composition of the commercial DINP formulations is not well defined. Gas chromatographic
7 analysis of these mixtures is difficult due to the large number of isomers present at low
8 concentrations and the co-elution of isomers present at higher concentrations (Gill etal., 2001].
9 Based on the available estimates of alkyl chain content, the compositions of DINP-1 and DINP-2 can
10 be expected to be similar, while DINP-3 contained larger proportions of methyl ethyl hexanols than
11 the other formulations (BASF. 2013: Evonik Industries. 2009: ECTRC. 2003: ExxonMobil. 2001]. The
12 correspondence between DINP formulations and CASRNs is as follows:
13 • DINP-1: CASRN 68515-48-0.
14 • DINP-2 and DINP-3: CASRN 28553-12-0
15 • Santicizer 900 and DINP-A: CASRN 71549-78-5;
16 • Bis(3,5,5-trimethylhexyl] phthalate: CASRN 14103-61-8
17 As noted above, DINP-2 and DINP-3 were assigned the same CASRN, and, thus, the specific
18 formulation used in some studies was not readily distinguishable. Throughout this document, the
19 general term, DINP, will be used to describe the test materials used and evidence tables will provide
20 the specific formulation in the reference design column if this information is available.
21 1.1.2. Environmental Fate
22 As noted by Wormuth etal. (2006]. the major portion of phthalates that are found in the
23 environment comes from their slow releases from plastics and other phthalate containing articles.
24 The presence of phthalates in food is due to their use in packaging materials and food preparation.
25 Certain waste streams, sludges, and contaminated sites may contain higher levels of phthalates.
26 Based on its vapor pressure, DINP, if released to air, is expected to exist in both the vapor
27 and particulate phases. Vapor-phase DINP will be photolytically degraded with a half-life of less
28 than a day. Particulate-phase diisononyl phthalate will be removed from the atmosphere by wet or
29 dry deposition. Once in soil, DINP will be tightly sorbed given a high organic carbon partition
30 coefficient, Koc. DINP's binding to soil limits its volatilization. Similarly, if released into water, DINP
31 binds to suspended solids and sediment Biodegradation is expected to occur in both soil and water
32 over of period of days to months, depending on environmental conditions. DINP has a low potential
33 for bioaccumulation given measured bioconcentration factor of 3 (HSDB, 2009].
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 ofDiisononyl Phthalate
1 1.1.3. Human Exposure Pathways
2 The ways that humans are exposed to phthalates along with the magnitude of the exposures
3 have changed over time as the quantities and uses of phthalates have changed. As noted above, the
4 Consumer Product Safety Improvement Act (CPSIA) of 2008 placed an interim ban on DINP in
5 children's toys and certain child care articles at concentrations greater than 0.1 percent and the
6 CHAP recommended that the interim ban on DINP be made permanent in children's toys and child
7 care products at level greater than 0.1% [CHAP. 20141. In December 2013, California EPA added
8 DINP to the Proposition 65 list as a carcinogen. These recommendations and statements reflect the
9 changing levels of phthalates in different products and exposure sources.
10 Diet is currently understood to be the greatest source of exposure to DINP. DINP has been
11 found in beverages, dairy, fish, grain, poultry, other meats, and vegetables [CHAP. 2014: Schecter et
12 al.. 20131 It was not detected in infant formula fSchecter etal.. 2013: Clark. 20101 Lesser
13 exposures to DINP may occur through inhalation and dermal contact with products containing
14 DINP. In background settings, DINP has been measured in dust and soil, but not found in air [CHAP,
15 2014]. In association with contaminated settings, it has been found in sludge and sludge amended
16 soil and in waste water [Clark. 2010: Vikels0e etal.. 1999].
17 Calafatetal. [2011] identified monocarboxyisooctyl phthalate [MCIOP] as the most
18 appropriate metabolite of DINP to characterize exposure to DINP. Zotaetal. [2014] looked at the
19 temporal trends of phthalate metabolites in NHANES from 2001 to 2010. For MCIOP, they found an
20 increasing trend in concentrations, with geometric means at about 5.1 ng/mL in the 2005/2006
21 cycle, 7.0 ng/mL in the 2007/2008 cycle, and 13.4 ng/mL in the 2009/2010 cycle.
22 Intake exposures can be estimated on a pathway-basis by combining exposure media
23 concentrations and contact rates. Using this approach, Clark etal. [2011] estimated a median
24 intake of DINP between 0.7 and 2.1 [ig/kg-day for various lifestages as defined by the author:
25 adults (20-70 years of age], teens (12-19 years of age], children (5-11 years of age], toddlers (ages
26 0.5-4 years of age], and infants (0-0.5 years of age]. Toddlers had the highest intake noted.
27 Pathways the authors assessed include ingestion of food, drinking water, dust/soil, and inhalation
28 of air. For the adult, teen, child, and toddler, ingestion of food accounts for 61-71% of intake,
29 depending on the age group. The remainder of the exposure for these age groups (and all of the
30 exposure to the infant] is due to ingestion of dust Infant and toddler intakes with toys and teethers
31 have been estimated to range from 1.7 to 120 [ig/kg-day by RIVM [1998]. Health Canada [1998].
32 Wormuthetal. [2006]. U.S. Consumer Product Safety Commission [CPSC. 1998] and [Babich etal..
33 2004]. Estimates of mean total intakes using a pathway-based approach were provided by the
34 CPSC [CHAP. 2014]: 5.1 [ig/kg-day for women, ages 15-45, and 20.7 [ig/kg-day for infants (0 - <1
35 yr], 30.8 [ig/kg-day for toddlers (1 to <3 yr], and 14.3 [ig/kg-day for children (3-12 yr]. For all age
36 categories, diet dominated the estimates, at over 90% for adult women to 67% for toddlers (with
37 "child care" products explaining most of the remainder].
38 An estimate of total exposure by all pathways can be determined based on urine
39 concentrations of phthalate metabolites. Kransler et al. [2012] reviewed the literature on general
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 ofDiisononyl Phthalate
1 population intakes of DINP and found reported mean intakes in the range of 1-2 |ig/kg-day. They
2 reviewed pathway-based estimates as well as intakes determined from surveys of MCOP in urine.
3 On a body weight basis, they found the highest intakes for children ages 6-11 at about 3 |ig/kg-day,
4 with all other ages in the 1-2 [ig/kg-day range. Oianetal. [20141: using NHANES 2007/2008, a
5 median intake of 1.1 |ig/kg-day and a 95th percentile intake of 9.4 |ig/kg-day was found.
6 Christensen et al. T20141 combined the data from NHANES 2005-2008 and found similar results to
7 Oianetal. [2014]. with a median over that time span of 1.3 |ig/kg-day and a 95th percentile intake
8 of 11.7 [ig/kg-day. The CPSC [CHAP. 2014] found median and 99% percentile intakes of 1.1 and
9 35.0 [ig/kg-day, respectively, for adults aged 15-45, using data from NHANES 2005-06.
10 1.2. SCOPE OF THE ASSESSMENT
11 The National Research Council has recommended, "Cumulative risk assessment based on
12 common adverse outcomes is a feasible and physiologically relevant approach to the evaluation of
13 the multiplicity of human exposures and directly reflects EPA's mission to protect human health
14 (NRC 2009, pi 2]." They envisioned facilitating the process by "defining the groups of agents that
15 should be included for a given outcome" (NRC 2009, p!3]. In humans, the NRC cited results from
16 the National Health and Nutrition Examination Survey that demonstrate exposure to multiple
17 phthalates in most people (NRC 2009, p23]. A recent review of human exposure to eight phthalates
18 estimated DINP to have the second-highest concentrations in dust and soil (CPSC 2014, p El-11].
19 Thus, an evaluation of the human health hazards of DINP is necessary to future cumulative risk
20 assessments that assess effects on human health outcomes that might be associated with DINP.
21 In order to evaluate the potential health effects resulting from exposure to DINP, the IRIS
22 Program is developing an IRIS assessment for this chemical. Once final, the assessment of DINP will
23 help to inform EPA programs and regions and other groups. DINP has not been assessed previously
24 by the IRIS Program.
25
26
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 ofDiisononyl Phthalate
1
2
3 2. METHODS FOR IDENTIFYING AND SELECTING
4 STUDIES
5 The NRG [2011] recommended that the U.S. Environmental Protection Agency (EPA)
6 develop a detailed search strategy utilizing a graphical display documenting how initial search
7 findings are narrowed to the final studies that are selected for further evaluation on the basis of
8 inclusion and exclusion criteria. Following these recommendations, a literature search and
9 screening strategy was applied to identify literature related to characterizing the health effects of
10 diisononyl phthalate (DINP). This strategy consisted of a search of online scientific databases and
11 other sources, casting a wide net in order to identify all potentially pertinent studies. In subsequent
12 steps, references were screened to exclude papers not pertinent to an assessment of the health
13 effects of DINP, and remaining references were sorted into categories for further evaluation.
14 Section 2.1 describes the literature search and screening strategy in detail. The NRG [2011] further
15 recommended that after studies are identified for review by utilizing a transparent search strategy,
16 the next step is to summarize the details and findings of the most pertinent studies in the evidence
17 tables. The NRC suggested that such tables should provide a link to the references, and include
18 details of the study population, methods, and key findings. This approach provides for a systematic
19 and concise presentation of the evidence. The NRC also recommended that the methods and
20 findings should then be evaluated with a standardized approach. The approach that was outlined
21 identified standard issues for the evaluation of epidemiological and experimental animal studies.
22 Section 2.2 describes the approach taken for DINP for selecting studies to be included in the
23 preliminary evidence tables and exposure-response arrays. Section 3 presents the selected studies
24 in preliminary evidence tables and exposure-response arrays, arranged by health effect
25 2.1. DRAFT LITERATURE SEARCH AND SCREENING STRATEGY
26 The literature search for DINP was conducted in four online scientific databases (PubMed,
27 Web of Science, Toxline, and TSCATS2] in June of 2013; the search was repeated in January 2014.
28 This document is complete through January 2014. Additional updates will be performed at regular
29 (e.g., 6-month] intervals. The detailed search approach, including the search strings and number of
30 citations identified per database, is presented in Table 2-1. This search of online databases
31 identified 542 citations (after electronically eliminating duplicates]. The computerized database
32 searches were also supplemented by a manual search of citations from other regulatory documents
33 (Table 2-2]; 85 citations were obtained using these additional search strategies. In total,
34 604 citations were identified using online scientific databases and additional search strategies.
35
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 ofDiisononyl Phthalate
Table 2-1. Database search strategy for DINP
Database
(search date)
Keywords3
PubMed
01/2014
06/2013
(28553-12-0 OR ("Diisononyl phthalate" OR "1,2-Benzenedicarboxylic acid diisononyl ester"
OR "Isononyl alcohol phthalate" OR "Phthalic acid diisononyl ester" OR "1,2-
Benzenedicarboxylic acid 1,2-diisononyl ester" OR "Di isononyl phthalate" OR
Diisononylphthalate OR "di-isononylphthalate") OR ("alpha-Dinonyl phthalate"[tw] OR " 1,2-
Benzenedicarboxylic acid bis(3,5,5-trimethylhexyl) ester"[tw] OR "Bis(3,5,5-trimethylhexyl)
phthalate"[tw] OR "Di-3,5,5-trimethylhexyl phthalate"[tw] OR "Phthalic acid bis(3,5,5-
trimethylhexyl) ester"[tw] OR "Di(C8-10, C9 rich) branched alkyl phthalates"[tw] OR ("1,2-
Benzenedicarboxylic acid" AND "di-C8-10-branched alkyl esters" AND "C9-rich")[tw] OR ("1,2-
Benzenedicarboxylic acid" AND "di-C8-C10-branched alkyl ester" AND "C9-rich")[tw] OR
"Branched dinonyl phthalate"[tw] OR "Di-(C9-branched alkyl) phthalate"[tw] OR "1,2-
Benzenedicarboxylic acid 1,2-dinonyl ester"[tw] OR "1,2-Benzenedicarboxylic acid dinonyl
ester"[tw] OR "Di(C8-C10) branched alkyl phthalate"[tw] OR "BIS(7-METHYLOCTYL)
PHTHALATE"[tw]) OR (("diisononyl phthalate"[Substance Name] OR "diisononyl phthalate"[AII
Fields]) OR (Palatinol[AII Fields] AND DN[AII Fields]) OR (Palatinol[AII Fields] AND N[AII Fields]))
OR (dinp AND (phthalic OR phthalate* OR isononyl* OR benzenedicarboxylic OR diisononyl))
Web of Science
01/2014
06/2013
TS="12 benzenedicarboxylic acid" OR TS="1 2 benzenedicarboxylic acid 1 2 dinonyl ester" OR
TS="12 benzenedicarboxylic acid 12 diisononyl ester" OR TS="1 2 benzenedicarboxylic acid
diisononyl ester" OR TS="12 benzenedicarboxylic acid dinonyl ester" OR TS="alpha dinonyl
phthalate" ORTS="baylectrol 4200" ORTS="branched dinonyl phthalate" ORTS="c9 rich" OR
TS="di 355 trimethylhexyl phthalate" ORTS="di c8 10 branched alkyl esters" OR
TS="diisononyl phthalate" ORTS="di isononylphthalate" ORTS="di isononyl phthalate" OR
TS="diisononylphthalate" ORTS="dinp" ORTS="dinp2" ORTS="dinp3" ORTS="enj 2065" OR
TS="isononyl alcohol phthalate" ORTS="palatinol dn" ORTS="palatinol n" ORTS="phthalic
acid diisononyl ester" ORTS="sansocizerdinp" ORTS="vestinol 9" ORTS=" vinylcizer 90" OR
TS="vestinol nn" ORTS="witamol 150" ORTS="28553-12-0" ORTS="68515-48-0" OR
TS="71549-78-5" OR TS="14103-61-8" OR TS="12 benzenedicarboxylic acid bis 3 5 5
trimethylhexyl ester" OR TS="bis 355 trimethylhexyl phthalate" OR TS="phthalic acid bis 3 5
5 trimethylhexyl ester" ORTS="di c8 10 c9 rich branched alkyl phthalates" ORTS="di c8 clO
branched alkyl phthalate" ORTS="di c9 branched alkyl phthalate" ORTS="bis 7 methyloctyl
phthalate") OR (TS="12 benzenedicarboxylic acid" AND TS="ester*" AND (TS="diisononyl" OR
TS="di isononyl" ORTS="branched" ORTS="dinonyl" ORTS="trimethylhexyl")) OR
(TS="phthalic acid" AND TS="ester*" AND (TS="diisononyl" OR TS="di isononyl" OR
TS="branched" ORTS="dinonyl" ORTS="trimethylhexyl"))
Toxline
01/2014
06/2013
(("diisononyl phthalate" OR "vestinol nn" OR "sansocizer dinp" OR "palatinol dn" OR
"palatinol n" OR dinp OR 28553-12-0 [rn]) OR (68515-48-0 [rn]) OR ( 71549-78-5 [rn]) OR (
"alpha dinonyl phthalate" OR 14103-61-8 [rn]) OR ("diisononyl phthalate" OR "12
benzenedicarboxylic acid diisononyl ester" OR "isononyl alcohol phthalate" OR "phthalic acid
diisononyl ester" OR "1 2 benzenedicarboxylic acid 12 diisononyl ester" OR "di isononyl
phthalate" OR diisononylphthalate OR "di isononylphthalate") OR ("alpha dinonyl phthalate"
OR" 1 2 benzenedicarboxylic acid bis ( 3 5 5 trimethylhexyl) ester" OR "bis (355
trimethylhexyl) phthalate" OR "di 355 trimethylhexyl phthalate" OR "phthalic acid bis ( 3 5 5
trimethylhexyl) ester" OR "di ( c8 10 c9 rich ) branched alkyl phthalates") OR ("12
benzenedicarboxylic acid" AND "di c8 10 branched alkyl esters" AND "c9 rich") OR ("branched
dinonyl phthalate" OR "di ( c9 branched alkyl) phthalate" OR "12 benzenedicarboxylic acid 1
2 dinonyl ester" OR "12 benzenedicarboxylic acid dinonyl ester" OR "di ( c8 clO ) branched
alkyl phthalate" OR "bis ( 7 methyloctyl) phthalate") OR ("enj 2065" OR "baylectrol 4200" OR
dinp OR dinp2 OR dinpS OR "palatinol dn" OR "palatinol n" OR "vestinol 9" OR "vestinol nn"
OR "vinylcizer 90" OR "witamol 150") OR ( di AND isononyl AND phthalate ) OR ("12
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 ofDiisononyl Phthalate
benzenedicarboxylic acid" AND ester* AND ( diisononyl OR "di isononyl" OR branched OR
dinonyl OR trimethylhexyl)) OR ("phthalic acid" AND ester* AND ( diisononyl OR "di
isononyl" OR branched OR dinonyl OR trimethylhexyl))) NOT PubMed [org] NOT pubdart
[org] NOT tscats [org]
TSCATS2
01/2014
10/2013
(2000-) 28553-12-0, 68515-48-0, 71549-78-5, 14103-61-8
1
2
3
aThe search strings did not include DINP metabolites; a PubMed search using metabolites of DINP did not capture
any additional pertinent studies.
Table 2-2. Summary of additional search strategies for DINP
Approach
used
Manual
search from
reviews
conducted by
other
international
and federal
agencies
Electronic
forward
Search
through Web
of Science1
References
obtained
during the
assessment
process
Source(s)
CPSC (2010). Toxicity review of Diisononyl Phthalate
(DINP). Bethesda, MD.
ECJRC (2003). European Union risk assessment report:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl
esters, C9-rich - and di-"isononyl" phthalate (DINP). (EUR
20784 EN). Luxembourg, Belgium: Office for Official
Publications of the European Communities.
http://bookshop.europa.eu/en/european-union-risk-
assessment-report-pbEUNA20784/.
CPSC (2001). Report to the U.S. Consumer Product Safety
Commission by the Chronic Hazard Advisory Panel on
diisononyl phthalate (DINP). Bethesda, MD.
NTP-CERHR (2003). NTP-CERHR monograph on the
potential human reproductive and developmental effects
of di-isononyl phthalate (DINP) (pp. i-MI90). Research
Triangle Park, NC: National Toxicology Program Center for
the Evaluation of Risks to Human Reproduction.
http://cerhr.niehs.nih.gov/chemicals/phthalates/dinp/DiN
P Monograph Final.pdf.
Lington et al. (1997). Chronic toxicity and carcinogenic
evaluation of diisononyl phthalate in rats. Fundam Appl
Toxicol 36: 79-89.
http://dx.doi.0rg/10.1093/toxsci/36.l.79.
Masutomi et al. (2003). Impact of dietary exposure to
methoxychlor, genistein, or diisononyl phthalate during
the perinatal period on the development of the rat
endocrine/reproductive systems in later life. Toxicology
192: 149-170. http://dx.doi.org/10.1016/S0300-
483X(03)00269-5.
DINP references obtained from submissions, full study
reports from HERO, or in previous assessment
Date
performed
08/2013
08/2013
08/2013
08/2013
08/2013
08/2013
08/2013
Number of additional
citations identified
17 citations
31 citations
7 citations added
0 citations added
0 citations
0 citations
15 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 ofDiisononyl Phthalate
Approach
used
Background
Check
Source(s)
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)
CalEPA OEHHA Toxicity Criteria Database
(http://www.oehha.ca.gov/tcdb/index.asp)
CalEPA Biomonitoring California-Priority Chemicals
(http://www.oehha.ca.gov/multimedia/biomon/pdf/Prior
ityChemsCurrent.pdf)
CalEPA Biomonitoring California-Designated Chemicals
(http://www.oehha.ca.gov/multimedia/biomon/pdf/Desi
gnatedChemCurrent.pdf)
CalEPA Cal/Ecotox database
(http://www.oehha.ca.gov/scripts/caLecotox/CHEMLIST.
ASP)
CalEPA OEHHA Fact Sheets
(http://www.oehha.ca.gov/publicjnfo/facts/index.html)
CalEPA 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/p
age.action?pagelD=9)
Environment Canada - Search entire site if not found
below:
(http://www.ee. 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-eng.php)
Date
performed
1/2013
Number of additional
citations identified
15 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 ofDiisononyl Phthalate
Approach
used
Source(s)
Health Canada Second Priority List Assessments
(http://www.hc-sc.gc.ca/ewh-
semt/pubs/contaminants/psl2-lsp2/index-eng.php)
IARC (http://monographs.iarc.fr/htdig/search.html)
HER (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)
NCTR
(http://www.fda.gov/AboutFDA/CentersOffices/OC/Offic
eofScientificandMedicalPrograms/NCTR/default.htm)
National Institute for Environmental Health Sciences
(NIEHS) http://www.niehs.nih.gov/
NICNAS (PEC only covered by eChemPortal)
(http://www.nicnas.gov.au/industry/aics/search.asp)
NIOSH (http://www.cdc.gov/niosh/topics/)
NIOSHTIC 2 (http://www2a.cdc.gov/nioshtic-2/)
NTP - RoC, status, results, and management reports
(http://ntpsearch.niehs.nih.gov/query.html)
OS HA
(http://www.osha.gov/dts/chemicalsampling/toc/toc_che
msamp.html)
RTECS http://www.ccohs.ca/search.html
Date
performed
Number of additional
citations identified
1
2
3
4
5
6
7
These citations were screened using the title, abstract, and in limited instances, full text for
pertinence to examining the health effects of DINP exposure. The citations were then screened
using inclusion criteria (Table 2-3) describing specific information to help identify primary source
health effect data and mechanistic and/or genotoxic data, as well as resources useful in preparation
of the DINP package. The process for screening the literature search is described below and is
shown graphically in Figure 2-1:
8 • 38 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 • 51 references were identified as supporting studies; of these, 9 were toxicokinetic studies
11 and 42 were mechanistic and genotoxicity studies.
12 • 75 references were identified as secondary literature (e.g., reviews and editorials, risk
13 assessments, and regulatory documents); these references were kept as additional
14 resources for development of the Toxicological Review.
15 • 416 references were excluded because these studies did not include the primary source
16 data evaluating DINP in relation to any kind of toxicity or health endpoint, and did not
17 provide either supporting information (e.g., toxicokinetic or mechanistic/genotoxic data) or
18 secondary literature information (see Figure 2-1 and Table 2-3 for inclusion categories and
19 criteria).
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
1
2
Database Searches
(see Table 2-1 for keywords and limits)
Pubmed
n = 189
Web of
Science
n =
Toxline
(incl. TSCATS)
n = 96
(After duplicates removed electronically)
n = 542
Additional Search
Strategies
(see Table 2-2 for
methods and results)
n = 85
Phthalates - Epidemiological
Studies Search
(see Table 2-4 for keywords and
limits)
Primary Source Human Data
n = 149
(See Table 2-5 for inclusion criteria)
Combined Dataset
(After all duplicates removed)
n = 604
Manual Screening For Pertinence
(Title/Abstract/Full Text)
(see Table 2-3 for inclusion criteria)
Excluded: No Primary Data on Toxic Effects
(n = 416)
26 Abstract only
236 Not chemical specific
21 Manufacture/use
7 Chemical treatment, disposal, remediation
29 Measurement methods
7 Miscellaneous
10 Ecosystem effect
55 Exposure levels
23 Fate and transport
3 Chemical/physical properties
Other Studies
Studies with Supporting Data (n = 51)
9 Toxicokinetic
42 Mechanistic and genotoxicity
Secondary Literature
(n = 75)
44 Reviews, editorials
19 Regulatory documents
12 Risk assessments studies
Animal Primary
Source Health
Effects Studies
{n = 38)
Human Primary
Source Health
Effects Studies
(n = 14)
(See Table 2-6 for a
listing of DINP-specific
epidemiological studies)
Figure 2-1. Literature search approach for DINP.
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl 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 DINP 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 DINP?
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 Eight human studies were also identified from the initial literature search using the search
9 strings presented in Table 2-1. However, work being done concurrently on the development of
10 other phthalate preliminary materials revealed that this set of DINP 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], DINP,
22 butyl benzyl phthalate [BBP], di(2-ethylhexyl)phthalate [DEHP], di-ethyl phthalate [DEP], dipentyl
23 phthalate [DPP], and diisobutyl phthalate [DIBP]). In contrast to animal toxicology studies, most of
24 the epidemiology studies examine more than one phthalate, resulting in considerable overlap in the
25 sets of studies identified using individual-phthalate search terms. Full text screening of the same
26 studies identified in multiple searches results in an inefficient use of resources.
27 For these reasons, EPA developed a process for identifying epidemiological studies
28 evaluating phthalates by performing a single broad search to create a listing of epidemiological
29 studies of all phthalates mentioned above, from which the selection of studies examining potential
30 health effects of an individual phthalate could be drawn. This list records each of the phthalates
31 included in the study, based on information in the methods section of the paper, and the outcome (s)
32 examined. This literature search for epidemiological studies examining phthalates in relation to
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
1
2
3
4
5
6
health-related endpoints (from which the DINP studies were drawn) was conducted in PubMed,
Web of Science, and ToxNet databases in June 2013, using keywords and limits described in
Table 2-4; the search was updated in December 2013. For this search, "phthalate" (and related
terms) rather than names of specific phthalates was used as the foundation of the search, along
with terms designed specifically to identify epidemiological studies. These terms 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" ORTS="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
Epidemiology articles meeting inclusion criteria
4,127
127
December 2013
search
PubMed
Web of Science
ToxNet
Merged Reference Set
Additional epidemiology articles meeting inclusion
criteria
155
249
114
350
22
8
9
10
11
12
13
14
15
More than 4,000 citations were identified through this search. These were then screened
using inclusion criteria describing specific population (i.e., human), exposure measures,
comparison, and health effects (Table 2-5). Note that other studies obtained in the search, for
example mechanistic and pharmacokinetic studies, are excluded from consideration with respect to
the specific objective of this search (i.e., identification of epidemiology studies), but could be
included in other steps in the assessment. Duplicate citations of the same article were excluded and
articles written in a language other than English were retained for subsequent review.
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 ofDiisononyl 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 tob...
- 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 DINP, metabolites would include MINP (monoisononyl phthalate), MCOP (mono-carboxyoctyl phthalate),
5 MCIOP (mono-carboxyisooctyl phthalate), MOINP (mono-oxoisononyl phthalate), and MHINP (mono-
6 hydroxyisononyl phthalate).
7
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
1 One hundred and forty-nine epidemiological studies examining one or more phthalate in
2 relation to one or more endpoints were identified by the searches conducted through December
3 2013 (127 in the initial search and 22 in the December 2013 update). Fourteen studies analyzed
4 one or more health effects in relation to a measure of DINP (Table 2-6; eight had been identified in
5 the DINP-specific search described in Table 2-1 and Figure 2-1).
6
7
Table 2-6. Primary source epidemiological studies examining health effects of
DINP
Outcome category
Sexual differentiation measures
Male reproductive
Female reproductive
Pregnancy-related outcomes
Male pubertal development
Female pubertal development
Thyroid hormones, children
Immune
Obesity
Reference3
Main et al. (2006)
Joensen etal. (2012)
Jurewiczetal. (2013)
Buck Louis etal. (2013)
Hart etal. (2013)
Philippatetal. (2012)
Meeker et al. (2009)
Mieritz et al. (2012)
Frederiksen etal. (2012)
Hart etal. (2013)
Boas etal. (2010)
Wu et al. (2013)b
Hoppinetal. (2013)
Bertelsen et al. (2013)
Bornehag et al. (2004)
Hart etal. (2013)
DINP measure
MINP (urine)
Sum 4 DINP metabolites (urine)
MINP (urine)
MINP (urine)
Sum 2 DINP metabolites (urine)
Sum 4 DINP metabolites (urine)
MINP (urine)
MINP (urine)
MINP (urine)
Sum 4 DINP metabolites (urine)
Sum 4 DINP metabolites (urine)
Sum 2 DINP metabolites (urine)
Sum 2 DINP metabolites (+ 2 others in
supplemental material) (urine)
Accidental contamination (with DEHP)
MCOP (urine)
MCOP (urine)
DINP (dust)
Sum 2 DINP metabolites (urine)
8
9
10
11
12
13
14
15
16
aSuzuki etal. (2010) and Weinberger et al. (2014), measured a DINP metabolite (MINP), but levels were reported to
be too low for analysis; these studies are not included in the listing of DINP-related studies.
bWu et al. (2013) is not included in the evidence tables because the exposure was characterized by food-
contamination with both DEHP and DINP, without separate measures of these exposures.
Additional strategies are also being used to supplement this broad search for epidemiology
studies of phthalates (Table 2-7); the screening process for the publications identified through
these methods is currently underway.
17
18
Table 2-7. Summary of additional search strategies for epidemiology studies
of phthalate exposure in relation to health-related endpoints
Approach used
Date
performed
Number of additional
citations identified
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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
June 2014
July 2014
July 2014
7: review in process
3: review in process
5: review in process
1
2
3
4
5
6
7
8
9
10
11
12
13
aThe following studies were used to conduct the forward searches: (Trasande et al. (2013); 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); Adibi et al. (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)).
The literature for both epidemiological and animal studies will be regularly monitored for
the publication of new studies; regular updates of the searches are planned at 6-month intervals.
The documentation and results for this supplementary search can be found on the Health and
Environmental Research On-line (HERO) website1 [http://hero.epa.gov/DINP] and
[http://hero.epa.gov/phthalates-humanstudies].
14 2.2. SELECTION OF CRITICAL STUDIES IN EARLY STAGES OF DRAFT
15 DEVELOPMENT
16 2.2.1. General Approach
17 Each study retained following the literature search and screen was evaluated for aspects of
18 design, conduct, or reporting that could affect the interpretation of results and the overall
19 contribution to the synthesis of evidence for determination of hazard potential. Much of the key
20 information for conducting this evaluation can generally be found in the study's methods section
21 and in how the study results are reported. Importantly, this evaluation does not consider study
22 results or, more specifically, the direction or magnitude of any reported effects. For example,
23 standard issues for evaluation of experimental animal data identified by the NRC and adopted in
24 this approach include consideration of the species and sex of animals studied, dosing information
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 DINP may
not match the numbers of references identified in Figure 2-1 (current through January 2014).
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1 (dose spacing, dose duration, and route of exposure), endpoints considered, and the relevance of
2 the endpoints to the human endpoints of concern. Similarly, observational epidemiologic studies in
3 this approach for evaluation should consider the following:
4 • Approach used to identify the study population and the potential for selection bias.
5 • Study population characteristics and the generalizability of findings to other populations.
6 • Approach used for exposure assessment and the potential for information bias, whether
7 differential (nonrandom) or nondifferential (random).
8 • Approach used for outcome identification and any potential bias.
9 • Appropriateness of analytic methods used.
10 • Potential for confounding to have influenced the findings.
11 • Precision of estimates of effect
12 • Availability of an exposure metric that is used to model the severity of adverse response
13 associated with a gradient of exposures.
14 To facilitate the evaluation outlined above, evidence tables are constructed that
15 systematically summarize the important information from each study in a standardized tabular
16 format as recommended by the NRG (2011). In general, the evidence tables include all studies that
17 inform the overall synthesis of evidence for hazard potential. At this early stage of study
18 evaluation, the goal is to be inclusive. Exclusion of studies may unnecessarily narrow subsequent
19 analyses by eliminating information that might later prove useful. Premature exclusion might also
20 give a false sense of the consistency of results across the database of studies by unknowingly
21 reducing the diversity of study results. However, there may be situations in which the initial review
22 of the available data will lead to a decision to focus on a particular set of health effects and to
23 exclude others from further evaluation.
24 2.2.2. Exclusion of Studies
25 After the literature search was manually screened for pertinence, studies were excluded if
26 fundamental flaws were identified in their design, conduct, or reporting. The DINP experimental
27 animal database consists of studies designed to examine repeat-dose oral toxicity (including
28 chronic, subchronic, and short-term duration studies) and endpoint-specific toxicities (including
29 reproductive and developmental toxicity). All studies involved administration of DINP in the diet
30 or via gavage administration. Acute studies are generally less pertinent for characterizing health
31 hazards associated with chronic exposure; there are 10 acute and short-term studies that are not
32 summarized in the preliminary evidence tables. Nevertheless, these studies will still be evaluated
33 as possible sources of supporting health effects information during assessment development.
34 Experimental animal studies that were sources of subchronic or chronic health effects were
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1 evaluated for potential flaws in their design, reporting, or conduct. As a result, one study was
2 removed from consideration in the assessment. Bio Dynamics [1982b] had a malfunction in
3 delivery of water to the rats (Sprague-Dawley) that resulted in water deprivation. The authors did
4 not provide information on the number of animals that may have been affected by this issue, and,
5 therefore, there is uncertainty in the results.
6 The remaining studies are all sources of health effects data that may be used in the
7 assessment The studies summarized in the evidence tables are considered the "critical" studies
8 from which the study methods and results are presented in preliminary evidence tables and
9 exposure-response arrays (Section 3).
10 2.3. STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE
11 FUTURE EVALUATION AND SYNTHESIS OF THE CRITICAL
12 EPIDEMIOLOGICAL STUDIES FOR DINP
13 Several considerations will be used in EPA's evaluation of epidemiological studies of human
14 health effects of DINP. The evaluation of these studies considered aspects of the study design
15 affecting the internal or external validity of the results (e.g., population characteristics and
16 representativeness, exposure and outcome measures, confounding, data analysis), focusing on
17 specific types of bias (e.g., selection bias; information bias due to exposure misclassification), and
18 other considerations that could otherwise influence or limit the interpretation of the data. A study
19 is externally valid if the study results for the study population can be extrapolated to external target
20 populations. An internally valid study is free from different types of biases, and is a prerequisite for
21 generalizing study results beyond the study population. These issues are outline in the IRIS
22 Preamble, and are described below.
2 3 Study Populotion
24 Evaluation of study population characteristics (including key socio-demographic variables
25 and study inclusion criteria) can be used to evaluate external validity (i.e., generalizability) and to
26 facilitate comparison of results across different study populations. Some aspects of the selection
27 process may also affect the interval validity of a study, resulting in a biased effect estimate.
28 The general considerations for evaluating issues relating to the study population include
29 adequate documentation of participant recruitment, including eligibility criteria and participation
30 rates, as well as missing data, and loss to follow-up. This information is used to evaluate internal
31 study validity related to selection bias. Several different types of selection bias that may occur
32 include the healthy worker effect, differential loss to follow up, Berkson's bias, and participation
33 bias. It is important to note that low participation rates, or differences in participation rates
34 between exposed and non-exposed groups or between cases and controls, are not evidence of
35 selection bias. Rather, selection bias arises from a differential pattern of participation with respect
36 to both the exposure and the outcome, i.e., patterns of participation that would result in a biased
37 effect estimate. This could occur, for example, if people with high exposure and the outcome of
38 interest are more likely to participate than people with low exposure and the outcome.
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1 The available DINP studies have generally examined metabolites from many different
2 phthalates within the context of research on environmental exposures. Most of these studies rely
3 on objective exposure measures (e.g., biomonitoring data), some of which are collected prior to
4 onset of the outcomes being examined (e.g., in the prospective pregnancy cohort studies). Study
5 participants also typically do not have knowledge of the study hypothesis or their exposure to DINP
6 and thus, knowledge of exposure or exposure level is unlikely to result in differential participation
7 with respect to outcomes. These study features should minimize the potential for selection bias.
8 However, EPA will consider the possibility that a particular concern about the specific sources of
9 DINP (e.g., polyvinyl chloride [PVC] applications including toys, flooring, wall coverings (ECHA,
10 2013)), in conjunction with knowledge of specific health outcomes, may motivate people to
11 participate in a study or to continue participation throughout a follow-up period. In the absence of
12 evidence that any of these scenarios is likely to occur in a study, EPA will not consider selection bias
13 as a limitation of a study.
14 Exposure Considerations
15 General considerations for evaluating exposure include: (1) how exposure and dose can
16 occur (e.g., exposure sources, routes and media); (2) appropriate critical exposure period(s) for the
17 outcomes under study; (3) variability in the exposure metrics of interest (e.g., temporal and spatial
18 variability for environmental measures or inter-individual variability for biomonitoring data)
19 which can impact the choice of exposure metric (e.g., cumulative, average, or peak exposure);
20 (4) analytical methodology employed (e.g., choice of biological matrix, sampling protocol,
21 quantification approach, etc.); (5) choice of exposure surrogate evaluated (e.g., constituent chemical
22 or group/mixture); and (6) classification of individuals into exposure categories. These
23 considerations help determine how accurate and precise the exposure estimates are, and how likely
24 measurement error is with respect to the exposure metrics that were used. Nondifferential
25 misclassification of exposure categories, for example, can also result from measurement error and
26 is expected to predominantly result in attenuated effect estimates.
27 Some common sources of exposure to DINP include PVC applications, children's toys,
28 flooring, and wall covering materials (Zota etal., 2014), with the primary route of exposure
29 occurring through ingestion and some exposure via inhalation and dermal routes (see Section
30 1.1.3). Exposure to DINP may be increasing, as it (along with DiDP) is increasingly being used as a
31 substitute for DEHP (Zota etal.. 2014: Koch and Angerer. 2007). Although temporal analyses based
32 on National Health and Nutrition Examination Survey (NHANES) biomonitoring data from the U.S.
33 general population are limited because repeated measures are not collected on the same
34 individuals, a recent study of the U.S. general population found that urinary concentrations of the
35 DINP metabolite, MCOP, have increased since 2005 (geometric mean concentration of MCOP was
36 13.4 ng/mL in 2009-2010 compared to 5.1 ng/mL in 2005-2006) (Zota etal.. 2014).
37 Urine provides an integrated measure of phthalate exposure from all sources.
38 Measurement of DINP metabolites, rather than the parent compound, is preferred because the
39 parent compound is metabolized very quickly. The most commonly reported DINP metabolites
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1
2
3
4
5
measured in epidemiology studies include the simple monoester metabolite MINP (monoisononyl
phthalate), and the oxidative metabolites, MCOP (mono-carboxyoctyl phthalate) and MCIOP (mono-
carboxyisooctyl phthalate); other less commonly measured metabolites may include MOINP (mono-
oxoisononyl phthalate) and MHINP (mono-hydroxyisononyl phthalate) [Silvaetal., 2006).
Table 2-8 shows synonyms for the most commonly measured DINP metabolites.
Table 2-8. DINP metabolites and their synonyms
Metabolite name
Simple monoester metabolite
MINP (monoisononyl phthalate)
MCOP (mono-carboxyoctyl phthalate)
MCiOP (mono-carboxyisooctyl phthalate)
MOINP (mono-oxoisononyl phthalate)
MHINP (mono-hydroxyisononyl phthalate)
Synonyms
MiNP
MCiOP
CX-MiNP
7cx-MMeHP (Mono(4-methyl-7-carboxyheptyl) phthalate)
Mono(2,6-dimethyl-6-carboxyhexyl) phthalate
OXO-MiNP
7oxo-MMeOP (Mono(4-methyl-7-oxo-octyl) phthalate)
OH-MiNP
7OH-MMeOP (Mono(4-methyl-7-hydroxyoctyl) phthalate)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
These metabolites vary in terms of validity as surrogates of DINP exposure in epidemiology
studies. Two controlled human dosing studies evaluated what fraction of the total DINP ingested
produced MINP (the simple monoester metabolite) via excretion. One was conducted in a single
volunteer [Koch and Angerer, 2007), and the other among 20 volunteers [Anderson etal., 2011):
both found that MINP represented only a small fraction of the total DINP ingested (2-3%), while
the secondary metabolites accounted for larger proportions (9-18%). MINP often falls below the
limit of detection, making accurate measurement difficult. The correlations among secondary DINP
metabolites are generally high, ranging from 0.73 to 0.83 for MCIOP, MHINP, and MOINP (Silva et
al.. 2006). while correlations between these secondary metabolites and MINP have not been
reported. The oxidative metabolites have been recommended for use as biomarkers in
epidemiology studies (Koch and Angerer, 2007: Silvaetal., 2006). Based on these considerations,
EPA considers measures of DINP based solely on MINP to be less informative (i.e., subject to greater
measurement error) than measures that include at least one of the oxidative metabolites. Although
a summation of two or more metabolites could offer some advantages over a single metabolite, EPA
does not consider use of a single oxidative metabolite to be a major limitation.
Although urine measures are most commonly used in epidemiological studies of phthalate
exposure, measures in serum, semen, and breast milk have also been used. One study reported that
none of the three secondary DINP metabolites examined were above the limit of detection in breast
milk samples from 30 women, and the detection rate in cord blood (n = 30) ranged from 3 to 13%;
the correlation when comparing the summation of DINP metabolites in maternal urine and breast
milk could not be calculated, and the correlation between maternal urine and cord blood was
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1 Pearson r = 0.35 [Linetal., 2011]. Another study conducted among 60 men ages 18-26 years found
2 that while 43.3% of serum samples had MCIOP concentrations above the limit of detection, only
3 10% of serum samples had detectable MINP concentrations, and both metabolites were detected at
4 low levels in semen samples (MINP: 12.1%, MCIOP: 1.7%) (Frederiksen etal.. 2010). In this latter
5 study, the Spearman correlation coefficient between MCIOP levels measured in urine and serum
6 was r = 0.37) [Frederiksenetal.. 2010). The lower detection rate in tissues other than urine
7 reduces EPA's confidence in DINP metabolite measures in these biological matrices.
8 Given their first-order kinetics with half-lives on the order of hours [~3-5 hours for MINP,
9 and ~5-18 hours for oxidized metabolites in [Koch and Angerer, 2007], ~4-8 hours for both MINP
10 and oxidized metabolites in [Anderson etal., 2011]], urinary phthalate metabolite concentrations
11 peak shortly after exposure. Thus, for single-time exposure scenarios (rather than multi-source,
12 multiple time exposure scenarios), urine sampled during this time of peak concentration could lead
13 to overestimates of average daily intake, and conversely, measurements made after concentrations
14 have peaked and declined could lead to underestimates of intake. One study conducted among
15 pregnant women in Puerto Rico included one of the DINP metabolites, however, and found that
16 sampling time was not a significant predictor of urinary MCOP concentrations; that is, there was
17 little difference in MCOP levels for women whose samples were collected in early morning,
18 morning, early afternoon, or evening time periods (Cantonwine etal.. 2014). Urinary measures of
19 DINP metabolite concentrations in epidemiological studies are generally conducted using spot
20 urine samples (i.e., collected at time of a clinic or study examination visit) rather than at a specified
21 time (e.g., first morning void) or in 24-hour urine samples. Although the time of sample collection
22 described above may affect the accuracy of an estimated intake for a single individual, studies of
23 other phthalates (e.g., DEHP) have demonstrated that on a group level, spot urine samples provide
24 a reasonable approximation of concentrations that would have been observed using full-day urine
25 samples (Christensen etal.. 2014] and that a single spot sample was reliable in ranking subjects
26 according to tertile (Teitelbaum etal.. 2008). Although neither of these studies included DINP
27 metabolites, the general conclusions are expected to be similar. Based on this information, EPA
28 does not consider the reliance on spot urine samples for exposure estimation (including ranking of
29 individuals into different DINP categories) to be a major limitation for epidemiological studies.
30 However because of the potential for greater inaccuracy of estimates in the "tails" of the
31 distribution, EPA will include additional considerations (e.g., discussion of analysis of residuals,
32 sample size, outliers) when evaluating analyses based on use of DINP metabolites as continuous
33 measures.
34 Another potential limitation of measurement of DINP metabolites in urine is the
35 reproducibility of phthalate metabolite concentrations over time; that is, how well does a single
36 measure reflect the key exposure metric (average, peak) for the critical exposure window of
37 interest For many short-lived chemicals, considerable temporal variability in exposure level is
38 expected, and thus, repeated measures in the critical exposure window are preferred over a single
39 measurement Reproducibility is usually evaluated with the intraclass correlation coefficient (ICC),
40 a measure of the 'between-individual' variance divided by the total variance (between and within
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1 individuals). A higher ICC indicates greater reproducibility (i.e., lower within-person variance).
2 Frederiksen et al. [2013) reported the ICC calculated for urine samples collected over a 3-month
3 period among young Danish men, using a summed measure of DINP metabolites (comprising MINP,
4 MHINP, MOINP, and MCIOP). This study reported ICCs of 0.26 for 24-hour urine samples and
5 0.25-0.29 for first-morning urine samples; the ICCs for spot samples were considerably lower
6 (0.08-0.13). In a study of pregnant women in Puerto Rico. Cantonwine etal. (2014) reported an
7 ICC of 0.29 for MCOP when comparing urine samples taken at 18, 22, and 26 weeks of gestation. No
8 studies have evaluated temporal variability of DINP metabolites in children, limiting the ability to
9 examine this source of uncertainty for certain endpoints such as timing of puberty. EPA considers
10 the available data pertaining to reproducibility of DINP measures to be very limited; these results
11 indicate a low level of reproducibility over periods of 1-3 months, and highlight the value of
12 repeated exposure measures collected during the appropriate critical period for the outcome(s)
13 under study.
14 EPA will also consider the potential for differential misclassification of biomarker measures
15 of exposure, for example in situations in which a health outcome (e.g., diagnosis with diabetes or
16 cancer) could result in changes in behavior that could affect DINP exposure. This type of scenario
17 adds an additional challenge to the interpretation of the DINP metabolites as valid measures of
18 exposure in a relevant time window(s) with respect to disease development.
19 Some researchers have hypothesized that the fraction of primary metabolites (i.e., percent
20 of the total metabolites accounted for by the primary monoester, MINP) is better than
21 concentration of a single (or summed) metabolite(s) as a measure of relevant exposure (Toensen et
22 al., 2012). Because this idea is not currently established, EPA will focus on results reflecting
23 measures of absolute metabolites concentrations rather than relative (percent of total)
24 concentrations.
25 EPA also considers the distribution of exposure in evaluating individual studies and when
26 comparing results among groups of studies. One consideration is the contrast of exposure levels
27 (i.e., the difference between "high" and "low"): a study with a very narrow contrast may not have
28 sufficient variability to detect an effect that would be seen over a broader range. Another
29 consideration is the absolute level of exposure, as different effect estimates may be expected in
30 studies examining different exposure levels even if they had similar exposure contrasts.
31 Prim ary Outcom e Measures
32 The general considerations for evaluating issues relating to accuracy, reliability, and
33 biological relevance of outcomes include adequate duration of exposure and follow-up in order to
34 evaluate the outcomes of interest, and use of appropriate ascertainment methods to classify
35 individuals with regard to the outcome (e.g., high sensitivity and specificity).
36 Issues relating to assessment of the specific primary health effects are discussed below and
37 summarized in Table 2-9 at the end of Section 2.3.
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1 Sexual differentiation
2 Cryptorchidism and hypospadias are two disorders of the development of the male
3 reproductive system. Cryptorchidism, or undescended testes, can be present at birth (congenital
4 Cryptorchidism) or can occur later during infancy and childhood (acquired Cryptorchidism).
5 Surgical correction (orchiopexy) is recommended in cases of Cryptorchidism that do not resolve
6 during infancy because long-term complications include impaired sperm production and increased
7 risk of testicular cancer (Virtanen et al., 2007). Retractile testes can move back and forth between
8 the scrotum and the abdomen; this condition usually resolves by puberty and is not associated with
9 reproductive or other complications. Classification criteria for Cryptorchidism that involve
10 testicular positioning are commonly used in clinical research (Tohn Radcliffe Hospital
11 Cryptorchidism Study Group. 1988: Scorer. 1964). EPA will consider the definition used and age
12 range in interpreting studies of Cryptorchidism or related outcomes.
13 In animal toxicology studies, anogenital distance (AGD) is a routine marker to assess
14 endocrine disruption; this marker has only recently been adapted for use in epidemiological
15 studies. One study in adult men reported associations between decreased AGD and measures
16 relating to infertility (Eisenbergetal., 2011): most studies have used this measure in infants,
17 however, as a marker of endocrine environment during development. It is important to consider
18 general size, in addition to sex, in the evaluation of AGD, for example by incorporating birth weight
19 or length (e.g., calculation of "anogenital index" by dividing anogenital distance by weight. With
20 regard to reproducibility of this measure, a low degree of between-observer variability was found
21 using a standardized protocol and trained observers (Romano-Riqueraetal., 2007: Salazar-
22 Martinez etal., 2004). Because of the importance of size and age in the interpretation of this
23 measure, EPA has greater confidence in studies with measures taken at birth rather than among a
24 group spanning a larger age range.
25 Reproductive (steroidal and gonadotropin) hormones
26 The details of the laboratory procedures, including information on the basic methods, level
27 of detection, and coefficient of variation, are important considerations for hormone assays and
28 measures of semen parameters. Timing within a menstrual cycle can also be an important
29 consideration for interpretation of reproductive hormone concentrations in pre-menopausal
30 women.
31 Much of the focus of the research on male steroidal and gonadotropin hormones in the DINP
32 database concerns testosterone. One issue with respect to these measures is the estimation method
33 used for free testosterone. Based on the analysis by Vermeulen et al. (1999). EPA will consider
34 estimates based on total testosterone divided by immunoassay-derived sex-hormone binding
35 globulin (SHBG) levels to be most reliable.
36 Other male reproductive outcomes
37 The World Health Organization (WHO) laboratory methods for analysis of sperm counts
38 and semen parameters (see, for example. WHO. 1999) are generally recognized as standards in this
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1 field. EPA will consider studies that reference these methods, regardless of which revision used, to
2 be reliable measures.
3 Other female reproductive outcomes
4 Endometriosis can be symptomless, or can lead to surgical intervention; it is often
5 diagnosed as part of a work-up for infertility. Variability in clinical presentation and in access and
6 use of health care services present considerable challenges to conducting epidemiological studies of
7 this condition [Holt and Weiss, 2000]. Confirmation of "case" and "control" status (i.e., presence or
8 absence of endometriosis) by ultrasound or clinical evaluation is recommended to reduce outcome
9 misclassification, and representation of the source population should be carefully considered.
10 Pregnancy outcomes
11 Gestational age and birth weight are two outcomes commonly used in reproductive
12 epidemiology studies. These variables are sometimes defined as dichotomous outcomes (e.g., low
13 birth weight, defined as <2,500 g or preterm birth, defined as <37 weeks of gestation). They can
14 also be examined as continuous variables, often in analyses in which preterm or low birthweight
15 births are excluded, so that the focus of the analysis is on variability within the "normal" range. EPA
16 considers both types of analyses to be informative with respect to hazard identification, but will
17 consider each separately as they address different issues. In the birth cohort studies included in the
18 DINP database, data pertaining to birth weight are generally taken directly from medical records.
19 EPA considers this to be a reliable source as this is a very accurate and precise measurement
20 Although more prone to measurement error than birth weight measures, gestational age can be
21 estimated from several approaches. Some of these include ultrasonography, estimates based on
22 date of last menstrual period based on maternal recall, or from clinical examination based on
23 antenatal or newborn assessments (which may include an ultrasound). None of the currently
24 available studies examined size for gestational age (e.g., small for gestational age) as an outcome;
25 this outcome accounts for both fetal growth and gestational duration, and would thus be preferred
26 over a measure of birthweight that includes preterm births.
27 Timing of male and female puberty, and conditions of unusual pubertal development
28 Pubertal development in humans is often assessed using timing of peak height velocity
29 ("growth spurt") and secondary markers of sexual development Secondary markers for females
30 include breast development (thelarche) and pubic hair development (pubarche), and age at first
31 period (menarche). Secondary markers for males include gonadal development (gonadarche) and
32 pubic hair development, and age at first sperm emission (spermarche).
33 Evaluation of breast, pubic hair, and gonadal development is frequently performed using
34 the Tanner stages (Marshall and Tanner, 1970,1969], which places the individual in one of five
35 stages, ranging from pre-pubertal (stage 1) to adult maturation (stage 5). However, the process of
36 this staging is not straightforward, and is most reliable when performed by trained personnel
37 (rather than by the individual or a parent, for example) (Slough etal.. 2013: Schlossberger et al..
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1 1992: Espelandetal., 1990]. Age at menarche is considered to more reliable when assessed via
2 self-report [Koprowskietal., 2001], although reliability may decrease with increasing time since
3 menarche [Cooper etal., 2006]. Additionally, hormone levels may sometimes be used to evaluate
4 pubertal development Individuals may vary widely in the timing of these developmental
5 milestones.
6 Several clinical syndromes are known to disrupt the timing and order of markers of
7 pubertal development Considerations in the diagnosis of either precocious or delayed puberty
8 include the diagnostic criteria used and the source of the information (e.g., whether collected from
9 medical records or from self- or parental report]. For females, precocious puberty is usually
10 defined as the onset of puberty before the age of 8 years, while delayed puberty is usually defined
11 as the lack of pubertal development by the age of 13 years [Marshall and Tanner, 1969]:
12 corresponding ages in male are before the age of 9 years for precocious puberty and lack of
13 pubertal development by the age of 14 years for delayed puberty [Marshall and Tanner. 1970].
14 Clinical evaluation would involve hormone assays to distinguish between gonadotropin dependent
15 ("central"], gonadotropin independent ("peripheral"], or a combination of both (Traggiai and
16 Stanhope, 2003] forms of these conditions.
17 Thyroid
18 Thyroid-related endpoints examined in epidemiological studies of DINP include thyroid
19 hormones (triiodothyronine, T3, and thyroxine, T4] and thyroid stimulating hormone (TSH] (or
20 thyrotropin] produced by the pituitary.
21 As with other hormone assays, the details of the laboratory procedures, including
22 information on the basic methods, limit of detection, and coefficient of variation, are important
23 considerations for the hormone assays. Thyroid hormones are generally measured in serum,
24 although they may also be measured in dried blood spots, such as are collected from newborn
25 infants in screening for congenital hypothyroidism as well as for genetic metabolic diseases such as
26 phenylketonuria. Studies in older age groups have also shown a very high correlation (r = 0.99]
27 between thyroid hormone levels measured in dried blood spots and levels in serum (Hofmanetal..
28 2003].
29 With respect to thyroid hormones, time of day and season of sampling are two main
30 potential sources of variability. For example, serum TSH measured shortly after midnight may be
31 as much as twice as high as the value measured in late afternoon (Brabant etal.. 1991: Weeke and
32 Gundersen. 1978]. The evidence with respect to seasonal variability is mixed (Plasqui etal.. 2003:
33 Nicolau etal.. 1992: Simonietal.. 1990: Behalletal.. 1984: Postmes etal.. 1974] and this effect is
34 likely to be smaller than that of time of day. The impact of these sources of variation will depend on
35 whether they are also related to DINP (i.e., whether DINP levels vary diurnally or seasonally]. If
36 this is the case, failure to address these factors in the design or analysis could result in confounding
37 of the observed association, with the direction of this bias determined by the direction of the
38 association between these factors and DINP. If this is not the case, the lack of consideration of time
39 of day or seasonality would result in greater variability in the hormone measures, and would thus
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1 result in more imprecise (but not biased) estimates was located. EPA has not found evidence of a
2 seasonal variation in DINP levels, and only one study with information on diurnal variability
3 [Cantonwine etal., 2014]: in this study, MCOP levels did not vary by sampling time (e.g., early
4 morning, morning, early afternoon, or evening time periods (Cantonwine etal., 2014]. Based on
5 these data, EPA does not consider the lack of consideration of time of day or season in the analysis
6 of thyroid outcomes to be a likely source of bias, but recognizes the limited nature of the available
7 data.
8 Immune
9 Skin prick testing is a standard method for assessing atopy (allergic disease] used in some
10 epidemiologic studies. Other studies use an assessment protocol based on reported history of
11 symptoms (e.g., rhinitis, hay fever] or specific types of allergies. These can be considered
12 complementary types of measures: skin prick tests provide information on a defined set of
13 potential antigens to which a person may be exposed, and symptom-based evaluations provide
14 information on experiences of individuals and the variety of exposures they encounter. Studies
15 comparing questionnaire responses with skin prick tests in children have reported relatively high
16 specificity (89-96%] and positive predictive value (69-77%] for self-reported history of pollen or
17 pet dander allergy or for answers to a combination of questions incorporating itchy eyes with nasal
18 congestion in the absence of a cold or flu (Braun-Fahrlander etal.. 1997: Dotterudetal.. 1995]. The
19 validity was somewhat lower for a more restricted set of questions (nasal congestion in the absence
20 of a cold or flu; specificity 83%, positive predictive value 52%] (Braun-Fahrlander etal.. 1997].
21 Based on these data, EPA considers allergy history based only on rhinitis symptoms to have a
22 greater likelihood of outcome misclassification compared with those based on a combination of
23 symptoms.
24 Epidemiologic studies of asthma typically use a questionnaire-based approach to define
25 asthma based on symptoms relating to wheezing episodes or shortness of breath, reported history
26 of asthma attacks, or use of asthma medication, usually for a period defined as "current" or in the
27 past year. Much of this work is based upon the American Thoracic Society questionnaire (Ferris.
28 1978] or subsequent instruments that built upon this work, including the International Society of
29 Arthritis and Allergies in Children Questionnaire and the European Community Respiratory Health
30 Survey. These questionnaire-based approaches have been found to have an adequate level of
31 specificity and positive predictive value for use in etiologic research (Ravault and Kauffmann. 2001:
32 Pekkanen and Pearce. 1999: Burneyetal.. 1989: Burney and Chinn. 1987]. EPA considers
33 outcomes defined over a recent time period (e.g., symptoms in the past 12 months] to be more
34 relevant within the context of concurrent exposure measurements compared with outcomes
35 defined over a lifetime (e.g., ever had asthma].
36 Obesity
37 The study of obesity measures in the DINP database is based on body mass index (BMI]
38 using measurements taken as part of the data collection protocol. Although not relevant for the set
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1 of studies currently available, EPA notes that use of self-reported weight (e.g., report of pre-
2 pregnancy weight) would not be considered to be as reliable as actual measurements.
3 Confounding
4 The general considerations for evaluating issues relating to potential confounding include
5 consideration of which factors may be potential confounders (i.e., those which are strongly related
6 to both the exposure and the outcome under consideration, and are not intermediaries on a causal
7 pathway), adequate control for these potential confounders in the study design or analysis, and
8 where appropriate, quantification of the potential impact of mismeasured or unmeasured
9 confounders. Uncontrolled confounding by factors that are positively associated with both the
10 exposure (e.g., DINP) and health endpoint of interest, and those that are inversely associated with
11 both exposure and health endpoint, will result in an upward bias of the effect estimate.
12 Confounding by factors that are positively associated with either exposure or the health endpoint,
13 and inversely associated with the other axis, will result in a downward bias of the effect estimate.
14 Potential confounding by other phthalates
15 DINP has been used as a substitute for DEHP, and available data indicate a moderate
16 correlation between metabolites of these two phthalates. In an analysis conducted by EPA of
17 5,109 samples from the 2005-2008 National Health and Nutrition Examination Survey (NHANES)
18 participants aged >6 years, the pairwise Spearman correlation coefficient between MCOP (the only
19 DINP metabolite measured in the NHANES) and DEHP metabolites (mono-2-ethyl-5-hydroxyhexyl
20 phthalate [MEHHP], mono-2-ethyl-oxohexyl phthalate [MEOHP], or mono-2-ethyl-carboxypentyl
21 phthalate [MECCP]) ranged from 0.40 to 0.60. The correlations between DINP metabolites and
22 those of other phthalates are generally lower than seen with DEHP metabolites, with correlation
23 coefficients between -0.1 and 0.2 reported for MEP, and correlation coefficients between 0.01 and
24 0.3 for monobutyl phthalate (MBP), monoisobutyl phthalate (MIBP), and mono-benzyl phthalate
25 (MBzP) (Buck Louis etal.. 2013: Hart etal.. 2013: lurewicz etal.. 2013). Thus, EPA does not
26 consider lack of adjustment for these other phthalate metabolites to be a limitation of a study; an
27 exception would be a situation in which an association with DEHP metabolites was considerably
28 stronger than the association seen with DINP metabolites.
29 Potential confounding by demographic factors
30 Age, race/ethnicity, and sex are considered important explanatory factors for most types of
31 outcomes measured in epidemiological research. In NHANES 2009-2010 data, urinary MCOP levels
32 were similar among children ages 6-11 (geometric mean of 15.0 ug/L) and teenagers ages
33 12-19 (geometric mean of 16.1 |J.g/L), and both groups had higher levels compared to adults
34 >20 years (geometric mean of 11.9 ug/L) (CDC, 2013). Variability by sex and by race or ethnicity
35 was also observed, with higher levels in men compared with women (geometric means of 14.0 and
36 11.4 ug/L, respectively, in women and men) and lower levels in Mexican Americans (geometric
37 mean of 10.0 ug/L) compared with non-Hispanic whites and non-Hispanic blacks (geometric means
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1 of 13.4 and 12.6 |ig/L, respectively). EPA will consider these differences in assessing the potential
2 influence of demographic factors on observed effect estimates for DINP.
3 Potential confounding by other factors
4 Some of the health effects under consideration may have strong associations with other risk
5 factors. For example, smoking is associated with increased risk of low birth weight and preterm
6 births, and with infertility. Abstinence time is strongly related to sperm concentration measures.
7 In evaluating the potential for confounding by any of these factors, EPA will review evidence
8 pertaining to the strength and direction of its association with DINP (or its metabolites).
9 Data Analysis
10 The general considerations for evaluating issues relating to data analysis include adequate
11 documentation of statistical assumptions and analytic approach (including addressing skewness of
12 exposure or outcome variable and shape of exposure-response), consideration of sample size and
13 statistical power, and use of appropriate statistical methods for the study design.
14 One other issue specific to much of the DINP literature concerns the optimal approach to
15 addressing urinary volume or dilution in the analysis of spot urine or first morning void samples.
16 Options include use of creatinine- or specific-gravity-adjusted metabolite concentrations, or use of
17 unadjusted concentrations. Although use of some kind of correction factor has been advocated for
18 studies of obesity (Goodman etal.. 2014). a simulation study reported that creatinine-adjusted
19 exposure measures may produce biased effect estimates for outcomes that are strongly related to
20 factors affecting creatinine levels, of which obesity is a prime example (Christensenetal., 2014).
21 EPA recognizes the lack of consensus atthis time, as well as the need for continued research into
22 the potential bias introduced by different analytic approaches. Based on current understanding of
23 this issue, EPA prefers results using unadjusted concentration for outcomes strongly related to
24 creatinine levels; for other outcomes, EPA does not have a basis for preferring one type of analysis
25 over another.
26 Table 2-9. General and outcome-specific considerations for DINP study
27 evaluation
General considerations
Study population
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-
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Exposure
Analysis
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, range), proportion < LOD
• Consideration of data distribution including skewness of exposure and
outcome measures
• Consideration of influence of "tails" in analysis based on continuous exposure
measure
• Consideration of analytic approaches exploring different shapes of exposure-
response
• Consideration of values below LOD or LOQ
• Consideration of creatinine or other approach to adjust for urine volume.
Presentation of effect estimates, rather than statement regarding presence or
absence of statistical significance
Outcome-specific considerations
Sexual differentiation
Measures
Consideration of
confounding
Relevant exposure
time window(s)
Steroidal and
gonadotropin
hormones (adults; sex-
specific)
Measures
Consideration of
confounding
Relevant exposure
time window(s)
Sperm parameters
Measures
Consideration of
confounding
Relevant exposure
time window(s)
• AGO: protocol, training procedures, standardization and inter-rater reliability
• Cryptorchidism: definition
• AGO: variability by size (e.g., birth weight), sex, age; temporal trends in DINP
exposure if study spans several years and includes a wide age range
• Cryptorchidism, preterm birth
• In utero for outcomes assessed in infancy; for acquired Cryptorchidism, other
time window(s) during childhood may also be relevant
• 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 mo preceding hormone sample collection
• Type of assay (e.g., WHO protocol)
• Age, smoking, BMI, abstinence time (consider if these are related to
exposure)
• Up to 6 mo preceding semen sample collection; could also consider cycle-
specific (or lagged cycle-specific) window
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Infertility
Measures
Consideration of
confounding
Relevant exposure
time window(s)
• Definition, source of data
• Age, smoking, alcohol use, heavy metal exposure, radiation time (consider if
these are related to exposure)
• Time preceding attempt to become pregnant
Gestational age
Measures
Consideration of
confounding
Relevant exposure
timewindow(s)
• Source of data (e.g., birth certificate) 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; particularly third trimester
Birth weight
Measures
Consideration of
confounding
Relevant exposure
timewindow(s)
• Source of data (e.g., medical records, birth certificate)
• Gestational age, maternal age, ethnicity, infections, pregnancy complications
(e.g., pre-eclampsia), nutritional intake, smoking, alcohol/drug use, weight
gain during pregnancy; maternal height/BMI, heavy metal exposures
(consider if these are related to exposure)
• In utero; particularly third trimester
Timing of puberty
Measures
Consideration of
confounding
Relevant exposure
timewindow(s)
• Source of data (e.g., measures of sexual maturation [menarche; spermarche;
breast, pubic hair, axillary hair, and genital development]; self-report,
physician assessment, or other)
• Age, sex, ethnicity, body size, nutritional status (consider if these are related
to exposure)
• In utero? Up to 12 mo preceding transition from one stage to another
stage?
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
• Biological sample used (e.g., serum, dried whole blood spots)
• 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)
• Lifestage considerations (i.e., adults, children, etc)
Immune
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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Relevant exposure
time window(s)
• For current conditions (e.g., asthma in past 12 mo): up to 12 mo preceding
outcome assessment
Obesity
Measures
Consideration of
confounding
Relevant exposure
time window(s)
• Source of data (e.g., measures of weight and height, if BMI used; self-report
• Age, sex, ethnicity, caloric intake, physical activity (consider if these are
related to exposure)
• Not established (likely to be more than one)
2
3
4
5
6
7
8
9
10
11
12
2.4. STUDY CHARACTERISTICS THAT WILL BE CONSIDERED IN THE
FUTURE EVALUATION AND SYNTHESIS OF THE CRITICAL
EXPERIMENTAL STUDIES FOR DINP
Beyond the initial methodological screening described above in Section 2.2.2,
methodological aspects of a study's design, conduct, and reporting will be considered again in the
overall evaluation and synthesis of the pertinent data that will be developed for each health effect.
Some general questions that will be considered in evaluating experimental animal studies are
presented in Table 2-10. These questions are, for the most part, broadly applicable to all
experimental studies.
Table 2-10. 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?
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 2-10 such as
exposure, is likely to be relatively independent of outcome. Other methodological features, in
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1 particular those related to experimental setup and endpoint evaluation procedures, are generally
2 outcome specific (i.e., reproductive and developmental toxicity). In general, experimental animal
3 studies will be compared against traditional assay formats (e.g., those used in guideline studies),
4 with deviations from the protocol evaluated in light of how the deviations could alter interpretation
5 of the outcome in question. A full evaluation of all critical studies will be performed as part of the
6 critical review and synthesis of evidence for hazard identification for each of the health endpoints
7 identified in the evidence tables presented in Section 3.
8
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1
2 3. PRELIMINARY EVIDENCE TABLES AND
3 EXPOSURE-RESPONSE ARRAYS
4 3.1. DATA EXTRACTION FOR EPIDEMIOLOGICAL AND ANIMAL STUDIES:
5 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 also provide the specific formulation of diisononyl phthalate (DINP) in the
10 reference design column if this information is available. Evidence tables will serve as an additional
11 method for presenting and evaluating the suitability of the data to inform hazard identification for
12 DINP during the analysis of hazard potential and utility of the data for dose-response evaluation.
13 For each critical study selected, key information on the study design, including characteristics that
14 inform study quality, and study results pertinent to evaluating the health effects from subchronic
15 and chronic oral exposure to DINP are summarized in preliminary evidence tables.
16 Epidemiological studies are presented first where each study per table is listed in reverse
17 chronological order. Animal studies are then presented where each study per health endpoint is
18 presented in alphabetical order by study author, followed by species and strain. Most results are
19 presented as the percent change from the control group; an asterisk (*) indicates a result that has
20 been calculated and reported by study authors to be statistically significant compared to controls
21 (p < 0.05). Unless otherwise noted in a footnote, doses presented in the animal evidence tables
22 were those reported by the study authors.
23 The information in the preliminary evidence tables is also displayed graphically in
24 preliminary exposure-response arrays. In these arrays, a significant effect (indicated by a filled
25 circle) is based on statistical significance by the study authors. The complete list of references
26 considered in preparation of these materials can be found on the HERO website at
27 [http://hero.epa.gov/DINP) and [http://hero.epa.gov/phthalates-humanstudies).
28
29
30
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l 3.2, EPIDEMIOLOGICAL STUDIES
2 3,2,1. Sexual Differentiation Measures
3
4
Table 3-1. Evidence pertaining to DINP metabolite(s) and measures of sexual
differentiation in humans
Reference and study design
Results
Cryptorchidism or testicular position
Main et al. (2006) (Denmark and Finland)
Population: 62 cases, 68 controls from two pregnancy
cohorts, born 1997-2001, age 3 mo
Outcome: Cryptorchidism, at birth and/or 3 mo
Exposure: Breast milk sample collected 1-3 mo of age
MINP in breast milk (u.g/L), all samples:
Median (range)
Denmark 101 (27-469)
Finland 89 (28-230)
Analysis: Mann-Whitney U test for comparison of MINP
concentrations in boys with and without Cryptorchidism
Median MINP in breast milk (u.g/L)
Controls Cases
91.75 98.52
(p>0.4)
Infant hormone levels
Main et al. (2006) (Denmark and Finland)
Population: 130 male infants from two pregnancy
cohorts (Cryptorchidism cases and controls combined for
this analysis), born 1997-2001, age 3 mo
Outcome: Serum steroidal and gonadotropin hormone
levels in infants, sample collected when breast milk
sample delivered to hospital
Exposure: Breast milk sample collected 1-3 mo of age
MINP in breast milk (u.g/L), all samples:
Median (range)
Denmark 101 (27-469)
Finland 89 (28-230)
Analysis: Cases and controls combined for analysis of
association between metabolite concentration and
hormone level using partial Spearman correlation
coefficients adjusted for country of birth; hormone ratios
evaluated using linear regression considering gestational
age, weight for gestational age, parity, smoking,
diabetes, and country of origin as potential covariates
Spearman correlation coefficient (p-value), MINP
(u.g/L) and serum hormone level (n = 96 boys)
Testosterone (nmol/L)
Free testosterone (nmol/L)
SHBG (nmol/L)
LH (IU/L)
FSH (IU/L)
Inhibin B
0.184(0.078)
0.070 (0.51)
0.187(0.076)
0.243 (0.019)
-0.043 (0.68)
-0.004 (0.97)
Estimated percentage increase (95% Cl) in LH level
with 10-fold increase in MINP = 97% (23, 214%) based
on regression analysis (adjusted covariates were not
reported). Regression results for other hormones
were not reported.
The magnitude of the association between LH and
MINP was greater than that observed for the other
metabolites evaluated (correlation coefficients ranged
from 0.001 to 0.185, all p-values > 0.05).
5
6
7
Cl = confidence interval; FSH = follicle-stimulating hormone; LH = luteinizing hormone; MINP = monoisobutyl
phthalate; SHBG = sex-hormone binding globulin
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3,2.2. Pregnancy Related Outcomes
Table 3-2. Evidence pertaining to DINP metabolite(s) and pregnancy
outcomes in humans
Reference and study design3
Results
Birth weight, birth length, head circumference, and gestational age
Philippatetal. (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)
gestational wks
MCIOP in urine (u.g/L):
Median 95th percentile
Measured 2.7 17.2
Standardized* 3.9 25.8
Analysis: Cases and controls combined for analysis;
weighted linear regression using tertiles or
In-transformed urine concentrations, adjusting for
variables shown in the 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 outcome
by MCIOP tertile and per unit change in In-MClOP
(standardized, ng/mL) (adjusted for gestational
duration, maternal pre-pregnancy weight and height,
maternal smoking, maternal education, parity,
recruitment center, and urine creatinine; head
circumference model also adjusted for mode of
delivery)
MCIOP
tertile
(Mg/U
1 (<2.4)
2 (2.4-5.9)
3 (>5.9)
(trend
p-value)
Ln (MCIOP)
Birth
weight (g)
(cm)
0 (referent) 0 (referent)
Head
Birth length circumference
(cm)
-40
-0.2
(-192, 110) (-0.9, 0.4)
-27
(-200, 147)
(0.87)
-8
(-72, 55)
0.4 (-0.5,
1.2)
(0.19)
0.1
(-0.2, 0.4)
0 (referent)
-0.1
(-0.7, 0.4)
0.0
(-0.6, 0.6)
(0.79)
0.0
(-0.2, 0.3)
Preterm birth (<37 wks)°
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
MCIOP in urine, unadjusted (ng/L):
Median 75th percentile
Term births 0.80 1.2
Preterm births 1.2 1.7
MCIOP in urine, SG-adjusted (ng/L):
Median 75th percentile
Term births 0.49 1.3
Preterm births 1.0 1.5
MCIOP in urine, Cr-adjusted (ng/g Cr):
Median 75th percentile
Term births 0.68 1.8
Preterm births 0.90 1.7
Analysis: Logistic regression, considering maternal age,
OR (95% Cl) for preterm birth by MCIOP above
compared with below the median (adjusted for marital
status, maternal education, infant sex, and gestational
age at time of urine sample)
Unadjusted (ng/L)
SG-adjusted (ng/L)
Cr-adjusted (ng/g Cr)
4.3 (1.2,14.9)
1.3 (0.5, 3.9)
2.0 (0.7, 6.0)
The unadjusted association between MCIOP and
preterm birth was similar or smaller in magnitude
compared to that for DEHP metabolites (ORs from
2.8 to 7.1), MBP (OR of 10.7), MIBP (OR of 3.6), or
MCPP (OR of 6.3). It was greater in magnitude
compared to that for MBzP (OR of 2.5), MCNP (OR of
1.3) or MEP (OR of 2.3).
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1
2
3
4
5
6
Reference and study design3
prepregnancy BMI, parity, education, marital status,
infant's sex, and gestational age at urine sample as
potential covariates
Results
DEHP = di(2-ethylhexyl)phthalate; MBP = monobutyl phthalate; MBzP = mono-benzyl phthalate; MCIOP = mono-
carboxyisooctyl phthalate; MCNP = monocarboxyisononyl phthalate; MCPP = mono(S-carboxypropyl) phthalate;
MEP = monoethyl phthalate; MIBP = methyl isobutyl phthalate; OR = odds ratio
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3,2,3. Male Reproductive Effects in Humans
Table 3-3. Evidence pertaining to DINP metabolite(s) and male reproductive
effects in humans
Reference and study design
Results
Reproductive hormones
Jurewicz et al. (2013) (Poland)
Population: 269 men from infertility clinic with normal
sperm concentration (20-300 million/mL) or slight
oligozoospermia (15-20 million/mL), mean age 32 yrs;
MINP measured in 113 samples.
Outcome: Plasma testosterone, E2, FSH
Exposure: Urine sample collected at same time as
plasma sample
MINP: unadjusted Cr-adjusted
geometric mean (SD) 1.4 (1.9) u.g/L 1.2 (1.9) u.g/g Cr
Analysis: Linear regression, adjusting for age, smoking,
medical history (mumps, cryptorchidism, testes
surgery, testes trauma), abstinence time, and urinary
creatinine
Adjusted regression coefficient (P) for increase in hormone
in relation to In-transformed MINP (adjusted for age,
smoking, medical history (mumps, cryptorchidism, testes
surgery, testes trauma), abstinence time, and urinary
creatinine)
Hormone
Testosterone
(ng/mL)
E2(pg/mL)
FSH (IU/L)
Beta
0.30
0.96
0.53
(p-value)
(0.37)
(0.61)
(0.38)
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
Unadjusted DINP metabolites in urine (ng/mL):
Median 95th percentile
MINP 0.6 4.7
MHINP 4.5 23
MOINP 2.3 12
MCIOP 7.7 41
ZDINP metabolites 21 107
%MINP 5% 15%
(%MINP calculated as percentage of total ZDINP
metabolites excreted as MINP)
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)
No association between ZDINP metabolites and
testosterone or other hormone measures (quantitative
results not reported by study authors.
Additional analyses focused on %MINP as exposure
measure, adjusting for age, BMI, smoking, alcohol
consumption, and time of blood sampling (and assay type
for inhibin-B only). Inverse associations were seen between
%MINP and measures of testosterone. For example,
comparing highest with lowest quartile %MINP, regression
coefficient for differences in In-transformed hormones:
Hormone Beta (95% Cl) trend p-value
Total testosterone -0.05 (-0.12, 0.01) 0.11
(nmol/L)
FAI -0.15 (-0.23, <0.001
-0.08)
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 ofDiisononyl Phthalate
Reference and study design
Results
Sperm parameters
Jurewicz et al. (2013) (Poland)
Population: 269 men from infertility clinic with normal
sperm concentration (20-300 million/mL) or slight
oligozoospermia (15-20 million/mL), mean age 32 yrs;
MINP measured in 113 samples
Outcome: Semen analysis
Exposure: Urine sample collected at same time as
semen sample
MINP: unadjusted cr-adjusted
Geometric mean (SD) 1.4 (1.9) ug/L 1.2 (1.9) ug/g Cr
Analysis: Linear regression, adjusting for age, smoking,
medical history (mumps, cryptorchidism, testes
surgery, testes trauma), abstinence time, and urinary
creatinine
Adjusted regression coefficient (P) for change in semen
measure in relation to In-transformed MINP (adjusted for
age, smoking, medical history (mumps, cryptorchidism,
testes surgery, testes trauma), abstinence time, and urinary
creatinine)
Parameter Beta
Concentration -0.31
(million/mL)
Motility (%) -9.05
Abnormal 6.21
morphology (%)
(p-value)
(0.19)
(0.033)
(0.060)
With additional adjustment for MEHP and 5OH-MEHP, Beta
for motility = -4.00 (p = 0.39).
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
Unadjusted DINP metabolites in urine (ng/mL):
Median 95th percentile
MINP 0.6 4.7
MHINP 4.5 23
MOINP 2.3 12
MCIOP 7.7 41
ZDINP metabolites 21 107
%MINP 5% 15%
(%MINP calculated as percentage of total ZDINP
metabolites excreted as MINP)
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, recent use of medication, abstinence time, and
time from ejaculation to analysis as potential covariates
No association between ZDINP metabolites and
testosterone or other hormone measures (quantitative
results not reported by study authors.
Additional analyses focused on %MINP as exposure
measure and semen volume, sperm concentration, and
sperm count (adjusted for abstinence time), motility
(adjusted for time from ejaculation to analysis), and
morphology (unadjusted). Associations were not observed
with these variables (trend p-values ranged from 0.18 to
0.99), with negative Beta coefficients (indicating inverse
associations) comparing highest with lowest quartile
%MINP seen only with sperm concentration (Beta = -0.03,
95% Cl -0.27, 0.31) and % normal morphology
(Beta = -0.06, 95% Cl -0.27, 0.15).
1
2
3
4
BMI = body mass index; MEHP = mono-(2-ethylhexyl) phthalate; MHINP = mono-hydroxyisononyl phthalate;
MOINP = oxo-(mono-oxoisononyl) phthalate; SD = standard deviation
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 ofDiisononyl Phthalate
3,2,4. Male Pubertal Development in Humans
Table 3-4. Evidence pertaining to DINP metabolite(s) and the timing of male
puberty in humans
Reference and study design
Results
Mieritzetal. (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 collected at clinical
evaluation
DINP metabolites in urine (ng/mL), Group 3:
Median 95th percentile
MINP 0.65 3.59
MHINP 5.6 22.92
MOINP 3.29 14.02
MCIOP 7.66 31.10
ZDINP metabolites 23.48 90.93
(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
ZDINP metabolites (ng/mL) by group
Group 1 = boys with palpable gynecomastia
Group 2 = boys without palpable gynecomastia (age-
matched)
Group 3 = boys without palpable gynecomastia (all ages)
MINP Median
95th percentile
Group 1
(n = 38)
23.55
112.6
Group 2
(n = 189)
20.14
84.53
Group 3
(n = 517)
23.48
90.93
No association between DINP metabolite concentration and
timing of puberty or serum testosterone level (quantitative
results not reported).
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 ofDiisononyl Phthalate
3,2.5. Female Reproductive Effects in Humans
Table 3-5. Evidence pertaining to DINP metabolite(s) and gynecological
conditions or reproductive and steroidal hormones in humans
Reference and study design
Results
Endometriosis
Buck Louis et al. (2013) (California and Utah, United States)
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, collected at time of surgery
Cr-adjusted MINP in urine (ng/mL):
Geometric mean (95% Cl)
Operative cohort-Controls 0.16 (0.14, 0.18)
Population cohort-Controls 0.16 (0.12, 0.21)
Analysis: Student's t-test or Wilcoxon test for continuous
data; logistic regression, adjusting for variables shown in
results column; 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-MINP concentration, by cohort (adjusted for age,
BMI, and creatinine)
Operative cohort
Population cohort
0.85 (0.68,1.06)
0.90 (0.50, 1.63)
OR (95% Cl) for endometriosis per unit increase in
In-MINP 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)
Note: Concentrations were log transformed and
rescaled by their SDs for analysis.
0.99 (0.76-1.28)
0.93 (0.70, 1.25)
0.84(0.64,1.11)
Polycystic ovary and hormones in adolescence
Hart etal. (2013) (Australia)
Population: 121 girls from pregnancy cohort study
(Western Australian Pregnancy Cohort), born 1989-1991;
follow-up at ages 14-16 yrs
Outcome: Uterine volume, ovarian volume, and antral
follicle count by ultrasound, polycystic ovarian morphology
defined as >1 ovary more than 10 cm3 or >12 follicles
between 2 and 9 mm in diameter; two definitions of
polycystic ovarian syndrome (1: presence of at least two of:
polycystic ovarian morphology, clinical or biochemical
hyperandrogenism, or oligo-anovulation; 2) oligo-
anovulatory menstrual cycles with either clinical or
biochemical hyperandrogenism); reproductive and
gonadotropin hormones; all measures on d 2-5 of
menstrual cycle, blinded to phthalate measures
Exposure: Maternal serum samples (n = 123) collected at
18 and 34-36 wks of gestation (combined aliquot from
both time periods)
Unadjusted DINP metabolite in serum (ng/mL):
Median 90th percentile
MINP 0.29)
(p>0.19)
No association with polycystic ovarian syndrome
using either definition (quantitative results not
reported by authors).
No association with SHBG, FSH, total testosterone,
free androgen index, anti-Mullerian hormone, or
inhibin B (quantitative results not 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 ofDiisononyl Phthalate
Reference and study design
Results
0.44
1.13
£DINP metabolites (molar sum)
*LOD for MiNP = 0.20 ng/mL
Analysis: Correlation between log-transformed DINP
metabolites and uterine volume, ovarian volume, and
antral follicle counts; associations between DINP
metabolites and PCOS were calculated using t-tests or
Mann-Whitney U tests
Maternal hormones during pregnancy
Hart etal. (2013) (Australia)
Population: 123 mothers from pregnancy cohort (Western
Australian Pregnancy Cohort), 1989-1991
Outcome: Serum androgens, samples collected at 18 and
34-36 wks of gestation
Exposure: Maternal serum samples collected at 18 and
34/36 wks of gestation (combined aliquot from both time
periods)
Unadjusted DINP metabolite in serum (ng/mL):
Median 90th percentile
MINP 0.10)
(p>0.10)
(p>0.10)
(p>0.10)
*Text states negative correlation, but Table 4
displays positive correlation; email (May 30, 2014)
from study authors confirmed negative correlation is
correct.
Correlation between log-transformed JDINP
metabolites at 34-36 gestation wks (n = 114) and
Androstenedione
(nmol/L)
DHEAS (u.mol/L)
Testosterone
(pmol/L)
SHBG (nmol/L)
Free testosterone
(pmol/L)
Free testosterone
index
r = -0.09
r = -0.12
r = 0.02
r = 0.10
r = -0.04
r = -0.04
(p>0.10)
(p>0.10)
(p>0.10)
(p>0.10)
(p>0.10)
(p>0.10)
1
2
3
4
5
LOD = level of detection; PCOS = polycystic ovarian syndrome
DHEAS= Dehydroepiandrosterone
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 ofDiisononyl Phthalate
3,2.6. Female Pubertal Development in Humans
Table 3-6. Evidence pertaining to DINP metabolite(s) and the timing of female
puberty in humans
Reference and study design
Results
Precocious puberty and premature thelarche
Frederiksen etal. (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
Outcome: Precocious puberty, early normal puberty, or
premature thelarche, defined based on clinical
standards
Exposure: Urine sample (child's), collected at clinical
evaluation
ZDINP metabolites (MINP, MHINP, MOINP, and MCIOP)
in urine (ng/mL), controls:
Median (range)
Unadjusted 30(1.0-214)
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) ZDINP metabolites in urine (ng/mL) in
cases and controls:
Controls
30 (1.0-214)
Precocious
puberty
34 (7.9-575)
(p-value)
(>0.05)
Pubertal development (general population)
Hart etal. (2013) (Australia)
Population: 121 girls from pregnancy cohort study
(Western Australian Pregnancy Cohort), born
1989-1991; follow-up at ages 14-16 years
Outcome: Age at menarche (questionnaire) (blinded to
phthalate measures)
Exposure: Maternal serum samples (n = 123) collected
at 18 and 34-36 wks of gestation (combined aliquot
from both time periods)
Unadjusted DINP metabolite in serum (ng/mL):
Median 90th percentile
MINP
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Results
Frederiksen etal. (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;
Serum steroid and gonadotropin hormones
Exposure: Urine sample (child's), collected at time of
pubertal stage assessment
Unadjusted DINP metabolite in urine (ng/mL), all
725 participants:
Median 95th percentile
MINP 0.7 4.8
MHINP 6.1 26
MOINP 3.6 17
MCIOP 8.7 35
ZDINP metabolites not reported
Analysis: Probit analysis, results verified using Pool-
Adjacent-Violators algorithm
Mean age (95% Cl) (yrs) at entry into breast stage 2 or
pubic hair stage 2, by quartile of JDINP metabolites:
IDINP
metabolite
quartile
1 (low)
2
3
4 (high)
Breast stage 2
(n = 394)
Pubic hair stage 2
(n not reported)
9.78 (9.29, 10.26) 10.84 (10.54, 11.14)
9.94 (9.47, 10.41) 11.05 (10.76, 11.35)
10.15 (9.69, 10.63) 11.46* (11.15, 11.78)
9.87 (9.42, 10.33) 11.15 (10.86, 11.47)
*Significantly different from quartile 1, p < 0.05
Levels of FSH, LH, estradiol, and testosterone were
similar across DINP metabolite exposure groups when
adjusted for age distribution (quantitative results not
reported).
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 ofDiisononyl Phthalate
3,2,7. Thyroid Effects in Humans
Table 3-7. Evidence pertaining to DINP metabolite(s) and thyroid effects in
humans
Reference and study design3
Boas et al. (2010) (Denmark)
Population: 758 children who were participants in
longitudinal cohort study, examined 2006-2007 at ages
4-9 yrs
Outcome: Serum thyroid hormone levels (nonfasting
sample)
Exposure: Urine sample (child's) collected same day as
serum sample
Cr-unadjusted DINP metabolites in urine (ng/L):
Median 75th percentile
MINP Boys 0.6 1.8
Girls 0.5 1.7
MCIOP Boys 7.2 12
Girls 6.5 12
Cr-adjusted DINP metabolites in urine (ng/g Cr):
Median 75th percentile
MINP Boys 1.0 2.7
Girls 1.1 3.3
MCIOP Boys 10 18
Girls 12 18
MHINP and MOINP also analyzed in 250 randomly
selected samples.
Analysis: Linear regression, adjusting for variables
shown in results column. Statistical analysis was not
performed on metabolites detected in <50% of samples
(included MINP)
Results
Regression coefficient (p-value) for change in hormone
level with unit change in In-MClOP (adjusted for sex and
age) (0.0 = no effect)
Cr-unadjusted Cr-adjusted
T3 (nmol/L) -0.07 (0.017) -0.01 (0.84)
Free T3 -0.18 (0.002) -0.04 (0.58)
(pmol/L)
T4 (nmol/L) -0.31(0.84) 1.14(0.57)
FreeT4 0.03(0.86) -0.01(0.97)
(pmol/L)
TSH (mU/L) -0.02 (0.25) 0.00 (0.96)
Similar patterns seen in analyses stratified by gender,
except that Cr-adjusted MCIOP was significantly
negatively associated with TSH in girls (P = -0.08,
p = 0.048). Inverse association with Free T3 also seen in
analyses of Cr-unadjusted and MOINP (Beta = -0.17,
p = 0.05).
The association between MCIOP and T3 and the
Cr-unadjusted association between MCIOP (and MOINP)
and free T3 were similar in magnitude to the associations
seen with the summed DEHP metabolites.
4
5
6
7
8
9
aWu et al. (2013) also contains data on thyroid effects, but the analysis focuses on DEHP (although contamination
with DINP also occurred).
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 ofDiisononyl Phthalate
3.2.8. Immune Effects in Humans
Table 3-8. Evidence pertaining to DINP metabolite(s) and immune effects in
humans
Reference and study design3
Results
Boasetal. (2010) (Denmark)
Population: 758 children from birth cohort study, born
1997-2001; examined 2006-2007, ages 4-9 yrs
Outcome: Serum thyroid hormone levels (nonfasting
sample)
Exposure: Urine sample (child's) collected same day as
serum sample
Unadjusted DINP metabolites in urine (ng/L):
Median 75th percentile
MINP Boys 0.6 1.8
Girls 0.5 1.7
MCIOP Boys 7.2 12
Girls 6.5 12
Cr-adjusted DINP metabolites in urine (ng/g Cr):
Median 75th percentile
MINP Boys 1.0
Girls 1.1
MCIOP Boys 10
Girls 12
2.7
3.3
18
18
MHINP and MOINP also analyzed in 250 randomly
selected samples.
Analysis: Linear regression, adjusting for variables
shown in results column. Statistical analysis was not
performed on metabolites detected in <50% of samples
(included MINP)
Regression coefficient (p-value) for change in hormone
level with unit change in In-MClOP (adjusted for sex and
age) (0.0 = no difference in hormone level per unit
change in In-MClOP exposure)
T3 (nmol/L)
Free T3
(pmol/L)
T4 (nmol/L)
Free T4
(pmol/L)
TSH (mU/L)
Unadjusted DINP
-0.07 (0.017)
-0.18(0.002)
-0.31 (0.84)
0.03 (0.86)
-0.02 (0.25)
Cr-adjusted DINP
-0.01 (0.84)
-0.04 (0.58)
1.14 (0.57)
-0.01 (0.97)
0.00 (0.96)
Similar patterns seen in analyses stratified by gender,
except that a statistical significant inverse association was
detected between Cr-adjusted MCIOP with TSH among
girls (P = 0.08, p = 0.048). Inverse association with Free
T3 also seen in analyses of Cr-unadjusted and MOINP
(P = -0.17, p = 0.05) for boys and girls.
The association between MCIOP and T3 and the
Cr-unadjusted association between MCIOP (and MOINP)
and free T3 were similar in magnitude to the associations
seen with the summed DEHP metabolites.
4
5
6
7
aWu et al. (2013) also contains data on thyroid effects, but the analysis focuses on DEHP (although contamination
with DINP also occurred).
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 ofDiisononyl Phthalate
3,2,9. Immune Effects in Humans
Table 3-9. Evidence pertaining to DINP metabolite(s) and immune effects in
humans
Reference and study design
Results
Hoppinetal. (2013) (United States, NHANES)
Population: 2,325 participants in population-based
survey (NHANES), 2005-2006; ages >6 yrs
Outcome: Self-reported (self-administered
questionnaire) current allergy symptoms (asthma,
wheeze, hay fever, allergy, itchy rash, rhinitis) in past
year; allergic sensitization as measured by serum IgE
(19 allergen specific IgEs)
Exposure: Urine sample collected same day as serum
sample (data reported in Ferguson et al., 2011)
MCOP in urine (ng/L):
75th 95th
Median percentile percentile
Cr-adjusted 4.98 10.86 52.74
Analysis: Logistic regression, adjusting for age,
race/ethnicity, gender, BMI, creatinine, and cotinine;
separate analyses for children (ages 6-17 yrs) and adults
(>17 yrs)
Prevalence (weighted by sampling weights) and OR per 1
unit change (log 10) in urinary MCOP level
Children (n = 779)
Asthma 8.4%
Wheeze 10.7%
Hay fever 3.6%
Rhinitis 27.6%
IgE sensitization 46.1%
(any)
Adults (n = 1,546)
Asthma 7.4%
Wheeze 16.6%
Hay fever 7.4%
Rhinitis 35.4%
IgE sensitization 44.0%
(any)
0.74 (0.36, 1.52)
1.16(0.65,2.07)
0.54(0.11,2.56)
1.40 (0.83, 2.37)
0.69 (0.40, 1.18)
0.96 (0.73, 1.25)
0.83 (0.58, 1.18)
0.64(0.37,1.11)
0.97 (0.76, 1.25)
1.21 (0.95, 1.54)
Bertelsen etal. (2013) (Norway)
Population: 623 children from birth cohort (Environment
and Childhood Asthma study), 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, collected at study
examination
MCOP in urine (ng/L):
Median 75th percentile 95th percentile
Unadjusted 6.0 10.2 21.2
SG-adjusted 6.2 10.2 21.9
Analysis: Logistic regression, potential confounders
considered included: sex, BMI, allergic sensitization in
the child, parental smoking at home [between the school
age of the child (6-7 yrs) and the 10-yr follow-up],
parental asthma (at child's birth), maternal education (at
child's birth), and household income (at the 10-yr follow-
up)
OR (95% Cl) for current asthma by quartile of MCOP
(Hg/L) (adjusted for urine specific gravity, sex, parental
asthma, and household income)
1: <3.5 (referent)
2: >3.5-6.0
3: >6.0-10.2
4: >10.2
1 (referent)
1.0 (0.60,1.9)
1.2 (0.67, 2.3)
1.9 (1.0, 3.3)
Increase in odds of current asthma per logio interquartile
range MCOP (95% Cl) = 1.3 (0.98,1.7)
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 ofDiisononyl Phthalate
Reference and study design
Results
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 2-7 yrs
Outcome: Eczema, wheezing, or rhinitis (cases report at
least two incidents of eczema, or wheezing or rhinitis
without a cold, in the preceding year, and at follow-up
1.5 yrs later)
Exposure: Surface dust sample from children's bedrooms
DINP in dust (mg/g):
Median
All homes 0.041
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)
Controls
Cases (all)
Median, all
homes
(n = 346)
0.047
0.000
p>0.8in both tests
Geometric mean (95% Cl),
homes with phthalate >
detection limit (n = 175)
0.446 (0.351, 0.566)
0.453 (0.352, 0.583)
1
2
3
IgE = immunoglobulin E; NHANES = National Health and Nutrition Examination Survey
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 ofDiisononyl Phthalate
3.2,10. Obesity Effects in Humans
Table 3-10. Evidence pertaining to DINP metabolite(s) and obesity in humans
Reference and study design
Results
Hart etal. (2013) (Australia)
Population: 121 girls from pregnancy cohort study
(Western Australian Pregnancy Cohort), born
1989-1991; follow-up at ages 14-16 yrs
Outcome: BMI (height and weight measured at clinic
visit)
Exposure: Maternal serum samples (n = 123) collected
at 18 and 3,436 wks of gestation (combined aliquot from
both time periods)
Unadjusted DINP metabolite in serum (ng/mL):
Median 90th percentile
MINP
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
l 3.3. ANIMAL STUDIES
2 3.3,1, Liver Effects
3 Table 3-11. Evidence pertaining to liver effects in animals following oral
4 exposure to DINP
Reference and study design3
Results
Liver weight change
Bio Dynamics (1986)
Rat (Sprague-Dawley); 70/sex/dose
0, 500, 5,000, 10,000 ppm (0, 27,
271, 553 mg/kg-day in males;
0, 33, 331, 672 mg/kg-day in
females)
Diet (Santicizer 900)
2 years (interim sacrifice at 1 year)
Lington et al. (1997)
Rat (F344); 110/sex/dose
0, 0.03, 0.3, 0.6% (0, 15, 152,
307 mg/kg-day in males; 0, 18, 184,
375 mg/kg-day in females)
Diet(DINP-l)
2 years (interim sacrifices at 6, 12,
and 18 months)
Covance Laboratories (1998b)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm
(0, 29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442,
885 mg/kg-day in females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in
males, 774 mg/kg-day in females)
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Liver weight at terminal sacrifice
compared to control)
Doses (M) 0
absolute weight 0%
liver/body weight 0%
Doses (F) 0
absolute weight 0%
liver/body weight 0%
Liver weight at terminal sacrifice
compared to control)
Doses (M) 0
absolute weight
liver/body weight 0%
Doses (F) 0
absolute weight
liver/body weight 0%
Liver weight at terminal sacrifice
compared to control)
Doses (M) 0 29
absolute weight 0% -5%
liver/body weight 0% -4%
Doses (F) 0 36
absolute weight 0% 4%
liver/body weight 0% 7%
(n = 26-47/sex/dose) (percent change
27
0%
0%
33
-2%
-3%
(n - 48-
15
6%
18
3%
(n - 37-
88
-4%
1%
109
3%
3%
271
5%
1%
331
15%
16*%
553
27*%
27*%
672
14*%
26*%
-65/sex/dose) (percent change
152
Data not reported
19*
184
Data not reported
16*%
307
31*%
375
29*%
-45/sex/dose) (percent change
359 733
28*% 47*%
35*% 61*%
442 885
23*% 57*%
26*% 71*%
Recovery
5%
10%
Recovery
3%
8%
This document is a draft for review purposes only and does not constitute Agency policy,
3-17 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Covance Laboratories (1998a)
Mouse (B6C3Fi); 70/sex/dose
0, 500, 1,500, 4,000, 8,000 ppm (0,
90, 276, 742, 1,560 mg/kg-day in
males; 0, 112, 336, 910,
1,888 mg/kg-day in females)
Recovery group (55/sex/dose):
8,000 ppm (1,377 mg/kg-day in
males; 1,581 mg/kg-day in
females)
Diet
Main study: 2 years (interim
sacrifice at 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Hazleton Laboratories (1991)
Rat (F344); 10/sex/dose
0, 2,500, 5,000, 10,000,
20,000 ppm (0, 175.8, 354.6, 719.6,
1,544.7 mg/kg-day in males; 0,
218.9, 438, 823.8,
1,687.1 mg/kg-day in females)
Diet(DINP-2/3)
13 weeks
Bio Dynamics (1982a)
Rat (F344); 15/sex/dose
0, 0.1, 0.3, 0.6, 1.0, 2.0% (0, 67,
210, 410, 730, 1,500 mg/kg-day in
males;
0, 77, 230, 480, 830,
1,600 mg/kg-day in females)b
Diet
13 weeks
Results
Liver weight at terminal sa
compared to control)
Doses (M) 0
absolute weight 0%
liver/body weight 0%
Doses (F) 0
absolute weight 0%
liver/body weight 0%
90 276 742
1% 1% 13*%
4% 4% 25*%
112 336 910
8% 23% 18%
8% 30% 24%
) (percent change
1,560
33*%
60*%
1,888
35%
48%
Recovery
16%
32*%
Recovery
34%
39%
Liver weight (percent change compared to control)
Doses (M) 0
absolute weight 0%
liver/body weight 0%
Doses (F) 0
absolute weight 0%
liver/body weight 0%
176 355
7% 29*%
11*% 27*%
219 438
12% 20*%
7% 18*%
720
47*%
54*%
824
35*%
37*%
1,545
86*%
110*%
1,687
77*%
103*%
Liver \Ne\ght( percent change compared to control)
Doses (M) 0
absolute weight 0%
liver/body weight 0%
Doses (F) 0
absolute weight 0%
liver/body weight 0%
67 210 410
-1% 8% 23*%
38% 50*% 73*%
77 230 480
2% 5% 21*%
3% 9% 24*%
730
33*%
92*%
830
39*
48*%
1,500
58*%
158*%
1,600
77*%
103*%
This document is a draft for review purposes only and does not constitute Agency policy,
3-18 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Hall etal. (1999)
Marmoset; 4/sex/dose
0, 100, 500, 2,500 mg/kg-day
Gavage in 1% methylcellulose and
0.5%Tween
13 weeks
Boberg et al. (2011)
Rat (Wistar); 1-7 litters/dose;
18-35 males/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
CDs 7-21
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 25 control dams
(litters)
0, 760, 3,800, 11,400 ppm (0, 109,
555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Clewell et al. (2013a)
Rat (Sprague-Dawley);
4-9 dams/timepoint/dose;
8 litters/dose and 9 control litters
0, 50, 250, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GD 12-19; dams sacrificed 0.5, 1,
2, 6, 12, and 24 hours after final
dose
Results
Liver weight (percent change compared to control)
Doses (M) 0
absolute weight 0%
liver/body weight 0%
Doses (F) 0
absolute weight 0%
liver/body weight 0%
100 500 2,500
58% 25% 19%
47% 17% 20%
100 500 2,500
18% 30% 3%
8% 18% -1%
Liver weight in males, PND 90
Doses 0
300 600 750 900
absolute 0% 4% 8% -2% -5%
weight
Note: Study authors did not examine this endpoint in females. Relative
weights not reported by study authors.
Liver weight in males, PNDs 49-50
Doses 0
absolute 0%
weight
liver/body 0%
weight
109 555 1,513
4% -1% -2%
3% -0.4% 2%
Liver weight in dams, GD 19 (percent change compared to control)
Doses 0
absolute weight 0%
liver/body weight 0%
50 250 750
-1% 17*% 15*%
2% 12*% 12*%
This document is a draft for review purposes only and does not constitute Agency policy,
3-19 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Hellwigetal. (1997)
Rat (Wistar), 8-10 dams
(litters)/dose per DINP formulation
0, 40, 200, 1,000 mg/kg-day
Gavage in olive oil (DINP- 1,2,3)
GDs 6-15; dams sacrificed on
GD 20
Waterman et al. (2000); one-
generation study
Rat (Sprague-Dawley), 30 breeding
pairs/dose
0, 0.5, 1, 1.5%
(0, 446, 889.5, 1,321 mg/kg-day in
males;
0, 493.5, 951.5, 1,404 mg/kg-day in
premating females;
0, 390.5, 768.5, 1,136.5 mg/kg-day
during gestation in females;
0, 706.5, 1,384, 1,760 mg/kg-day
during lactation in females)
Diet(DINP-l)
10 weeks prior to mating and
through mating (M) or PND 21 (F)
Waterman et al. (2000); two-
generation study
Rat (Sprague-Dawley), 30 breeding
pairs/dose
0, 0.2, 0.4, 0.8%
PI animals
0, 165, 331, 665 mg/kg-day in
males;
0, 182, 356, 696 mg/kg-day in
premating females;
0, 146, 287, 555 mg/kg-day during
gestation in females;
0, 254, 539, 1,026 mg/kg-day
during lactation in females
P2 (Fl) animals
0, 189, 379, 779 mg/kg-day in
males;
0, 197, 397, 802 mg/kg-day in
premating females;
Results
Liver weight in dams (percent change compared to control)
Doses 0
D\NP-1 absolute weight 0%
D\NP-2 absolute weight 0%
D\NP-3 absolute weight 0%
Note: Relative weight not reported
Liver weight in PO animals (percent
Doses (M) 0
absolute weight 0%
liver/body weight
Doses (F) 0
absolute weight 0%
liver/body weight
Liver weight in PI animals (percent
Doses (M) 0
absolute weight 0%
liver/body weight
Doses (F) 0
40 200 1,000
0% -2% 6%
-1% 2% 5%
-2% 3% 11*%
by study authors.
change compared to control)
446 889.5 1,321
13*% 27*% 34*%
Data not reported
493.5 951.5 1,404
26*% 44*% 52*%
Data not reported
change compared to control)
165 331 665
1% 6% 16*%
Data not reported
182 356 696
absolute weight 0% 11% 20*% 22*%
liver/body weight Data not reported
Liver weight in P2 (Fl) animals
(percent change compared to control)
Doses (M) 0
absolute weight 0%
liver/body weight
Doses (F) 0
absolute weight 0%
189 379 779
4% 1% 6%
Data not reported
197 397 802
9% 13% 18*%
This document is a draft for review purposes only and does not constitute Agency policy,
3-20 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
0, 143, 288, 560 mg/kg-day during
gestation in females;
0, 285, 553, 1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21 (F)
Results
liver/body weight
Data
not reported
Serum clinical chemistry
Bio Dynamics (1986)
Rat (Sprague-Dawley); 70/sex/dose
0, 500, 5,000, 10,000 ppm (0, 27,
271, and 553 mg/kg-day in males;
0, 33, 331, and 672 mg/kg-day in
females)
Diet (Santicizer 900)
2 years (interim sacrifice at 1 year)
Lington et al. (1997)
Rat (F344); 110/sex/dose
0, 0.03, 0.3, 0.6% (0, 15, 152, or
307 mg/kg-day in males; 0, 18, 184,
or 375 mg/kg-day in females)
Diet(DINP-l)
2 years (interim sacrifices at 6, 12,
and 18 months)
Serum liver enzyme levels at terminal sacrifice
change compared to control)
Doses (M) 0
ALT 0%
AST 0%
ALP 0%
Doses (F) 0
ALT 0%
AST 0%
ALP 0%
27
6%
15%
-25%
33
-3%
-39%
-36%
Serum liver enzyme levels at terminal sacrifice
change compared to control)
Doses (M) 0
ALT 0%
AST 0%
ALP 0%
Doses (F) 0
ALT 0%
AST 0%
ALP 0%
15
7%
1%
15%
18
7%
45%
38%
(n = 10/sex/dose
271
6%
11%
-10%
331
8%
-25%
-41%
(n = 20/sex/dose
152
112*%
22%
59*%
184
29%
33%
55%
) (percent
553
218%
111%
33%
672
63%
-11%
38%
) (percent
307
76%
124%
183*%
375
145%
123%
66%
This document is a draft for review purposes only and does not constitute Agency policy,
3-21 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Covance Laboratories (1998b)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm
(0, 29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442, 885 mg/kg-
day in females)
Recovery group (55/sex):
12,000 ppm (637 mg/kg-day in
males, 774 mg/kg-day in females)
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Covance Laboratories (1998a)
Mouse (B6C3Fi); 70/sex/dose
0, 500, 1,500, 4,000, 8,000 ppm (0,
90, 276, 742, 1,560 mg/kg-day in
males; 0, 112, 336, 910,
1,888 mg/kg-day in females)
Recovery group (55/sex/dose):
8,000 ppm (1,377 mg/kg-day in
males; 1,581 mg/kg-day in
females)
Diet
Main study: 2 years (interim
sacrifice at 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Bio Dynamics (1982a)
Rat (F344); 15/sex/dose
0, 0.1, 0.3, 0.6, 1.0, 2.0% (0, 67,
210, 410, 730, 1,500 mg/kg-day in
males; 0, 77, 230, 480, 830, or
1,600 mg/kg-day in females)
Diet
13 weeks
Results
Serum liver enzyme levels at terminal sacrifice (10/sex/dose) (percent change
compared to control)
Doses (M) 0
ALT 0%
AST 0%
ALP
Doses (F) 0
ALT 0%
AST 0%
ALP
29
13%
9%
36
-10%
-6%
88 359
-4% 123%
-12% 136*%
Not evaluated
109 442
-6% 137*%
-5% 165*%
Not evaluated
733 Recovery
113% 123%
103% 162*%
885 Recovery
73% 16%
57% 13%
Serum liver enzyme levels at terminal sacrifice (10/sex/dose) (percent change
compared to control)
Doses (M) 0
ALT 0%
AST 0%
ALP
Doses (F) 0
ALT 0%
AST 0%
ALP
90
-12%
8%
112
-26%
-12%
Serum liver enzyme levels at ter
change compared to control)
Doses (M) 0
ALT 0%
AST 0%
ALP 0%
Doses (F) 0
ALT 0%
AST 0%
ALP 0%
67
-13%
-17%
3%
77
17%
5%
-4%
276 742
-8% 20%
24% 30%
Not evaluated
336 910
134% 6%
83% 9%
Not evaluated
minal sacrifice (n = 10-
210 410
0% -8%
-9% -21%
9% 9%
230 480
3% 0%
-2% 0%
7% 13%
1,560 Recovery
960% 742%
473% 343%
1,888 Recovery
-2% 118%
7% 31%
-13/dose) (percent
730 1,500
26% 38*%
14% 14%
27*% 49*%
830 1,600
11% 11%
0% -8%
27% 70*%
This document is a draft for review purposes only and does not constitute Agency policy,
3-22 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Results
Hall et al. (1999)
Marmoset; 4/sex/dose
0,100, 500, or 2,500 mg/kg-day
Gavage in 1% methylcellulose and
0.5%Tween
13 weeks
Blood chemistry was analyzed at weeks 4 and 13. No treatment-related effects
were observed (quantitative data not reported by study authors)
HistopathologyQ
Bio Dynamics (1986): CPSC (2001)
Rat (Sprague-Dawley); 70/sex/dose
0, 500, 5,000,10,000 ppm (0, 27,
271, 553 mg/kg-day in males;
0, 33, 331, 672 mg/kg-day in
females)
Diet (Santicizer 900)
2 years (interim sacrifice at 1 year)
Doses(M)
0
27
271
553
Hepatic necrosis (all animals)c
incidence 5/70 17/69
percentage 1% 25%
Spongiosis hepatis (all animals)c
incidence 16/70 11/69
percentage 23% 16%
11/69
16%
30/69**
43%
23/70
33%
32/70**
46%
Doses (F)
0
33
331
672
Hepatic necrosis (all animals)c
incidence 10/70 15/70 7/70
percentage 14% 21% 10%
Spongiosis hepatis (all animals)c
incidence 4/70 3/70 6/70
percentage 6% 4% 9%
10/70
14%
11/70**
16%
(EPLU999): Lington et al. (1997))
Rat (F344); 110/sex/dose
0, 0.03, 0.3, 0.6% (0, 15, 152,
307 mg/kg-day in males; 0,18,184,
375 mg/kg-day in females)
Diet(DINP-l)
2 years (interim sacrifices at 6,12,
and 18 months)
Doses(M)
0
15
152
307
Hepatocellular enlargement
incidence 1/81 1/80
percentage 1% 1%
Hepatic necrosis
incidence 10/81 9/80
percentage 12% 11%
Spongiosis hepatisd
incidence 22/81 24/80
percentage 27% 30%
1/80
1%
16/80
20%
51/80**
64%
9/80**
11%
26/80
33%
62/80**
78%
Doses (F)
0
18
184
375
Hepatocellular enlargement
incidence 1/81 0/81
percentage 1% 0%
0/80
0%
11/80**
14%
This document is a draft for review purposes only and does not constitute Agency policy,
3-23 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Covance Laboratories (1998b); EPL
(1999)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm
(0, 29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442,
885 mg/kg-day in females)
Recovery group (55/sex):
12,000 ppm (637 mg/kg-day in
males, 774 mg/kg-day in females
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Results
Hepatic necrosis
incidence 13/81
percentage 16%
Spongiosis hepatisd
incidence 4/81
Percentage 5%
Doses (M) 0 29
Hepatocellular enlargement
incidence 0/55 0/55
percentage 0% 0%
Hepatic necrosis
incidence 0/55 0/55
percentage 0% 0%
Spongiosis hepatisd
incidence 6/55 6/50
percentage 11% 12%
Increased cytoplasmic eosinophilic
incidence 0/55 0/55
percentage 0% 0%
Doses (F) 0 36
Hepatocellular enlargement
incidence 0/55 0/55
percentage 0% 0%
Hepatic necrosis
11/81
14%
1/81
1%
88
0/55
0%
0/55
0%
19/80
24%
3/80
4%
359 733
0/55 17/556
0% 31%
1/55 5/55e
2% 9%
3/50 18/55** 26/55**
6% 33% 47%
hypertrophy of hepatocytes
0/55 0/55 31/556
0%
109
0/55
0%
Evaluated but data not
Spongiosis hepatisd
incidence 0/55 0/50
percentage 0% 0%
Increased cytoplasmic eosinophilic
incidence 0/55 0/55
percentage 0% 0%
0/50
0%
0% 56%
442 885
0/55 27/55e
0% 49%
reported
1/55 2/55
2% 4%
21/80
26%
4/80
5%
Recovery
0/55
0%
0/55
0%
10/55
20%
0/55
0%
Recovery
0/55
0%
0/50
0%
hypertrophy of hepatocytes
0/55
0%
0/55 35/55e
0% 64%
0/55
0%
This document is a draft for review purposes only and does not constitute Agency policy,
3-24 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Covance Laboratories (1998a)
Mouse (B6C3Fi); 70/sex/dose
0, 500, 1,500, 4,000, 8,000 ppm (0,
90, 276, 742, 1,560 mg/kg-day in
males; 0, 112, 336, 910,
1,888 mg/kg-day in females)
Recovery group (55/sex/dose):
8,000 ppm (1,377 mg/kg-day in
males; 1,581 mg/kg-day in
females)
Diet
Main study: 2 years (interim
sacrifice at 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Hazleton Laboratories (1991)
Rat (F344); 10/sex/dose
0, 2,500, 5,000, 10,000,
20,000 ppm (0, 175.8, 354.6, 719.6,
1,544.7 mg/kg-day in males; 0,
218.9, 438, 823.8, 1,687.1 mg/kg-
day in females)
Diet(DINP-2/3)
13 weeks
Note: Study authors did not
perform statistical analysis on
histopathological findings.
Results
Doses (M) 0 90 276
Hepatocellular enlargement
742 1,560
incidence 0/46 1/41 0/36 1/35 32/32
percentage 0% 2% 0% 3% 100%
Spongiosis hepatis
Evaluated but data not reported
Increased cytoplasmic eosinophilic hypertrophy of hepatocytes
incidence 0/46 0/41 0/36 0/35 32/32
percentage 0% 0% 0%
Doses (F) 0 112 336
Hepatocellular enlargement
incidence 0/42 0/36 0/37
percentage 0% 0% 0%
Spongiosis hepatis
Evaluated but data not
0% 100%
910 1,888
0/29 40/40
0% 100%
reported
Recovery
0/38
0%
0/38
0%
Recovery
0/35
0%
Increased cytoplasmic eosinophilic hypertrophy of hepatocytes
incidence 0/42 0/36 0/37
percentage 0% 0% 0%
Doses (M) 0 176
Hepatocellular enlargement
incidence minimal 0/10 0/10
percentage 0% 0%
incidence slight 0/10 0/10
percentage 0% 0%
Hepatic necrosis
incidence minimal 0/10 0/10
percentage 0% 0%
incidence slight 0/10 1/10
percentage 0% 10%
0/29 40/40
0% 100%
355 720
0/10 0/10
0% 0%
0/10 0/10
0% 0%
1/10 0/10
10% 0%
1/10 0/10
10% 0%
Doses (F) 0 2,199 438 824
Hepatocellular enlargement
incidence minimal 0/10 0/10
percentage 0% 0%
incidence slight 0/10 0/10
percentage 0% 0%
0/10 1/10
0% 10%
0/10 0/10
0% 0%
0/35
0%
1,545
3/10
30%
7/10
70%
0/10
0%
0/10
0%
1,687
0/10
0%
10/10
100%
This document is a draft for review purposes only and does not constitute Agency policy,
3-25 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Results
Hepatic necrosis No incidence of necrosis
Increased cytoplasmic eosinophilic hypertrophy of hepatocytes
Evaluated but data not reported for males or females
Bio Dynamics (1982a)
Rat (F344); 15/sex/dose
0, 0.1, 0.3, 0.6,1.0, 2.0% (0, 67,
210, 410, 730,1,500 mg/kg-day in
males; 0, 77, 230, 480, 830,
1,600 mg/kg-day in females)b
Diet
13 weeks
Note: Study authors did not
perform statistical analysis on
histopathological findings.
Increased cytoplasmic eosinophilic hypertrophy of hepatocytes
Doses(M)
0
67
210
410
730
1,500
incidence
0/13 12/12 13/13 12/12 13/13
13/13
Doses (F)
0
77
230
480
830
1,600
incidence
0/13 13/13 12/12 13/13 13/13
13/13
Waterman et al. (2000)
One-generation study
Rat (Sprague-Dawley), 30 breeding
pairs/dose
0, 0.5,1,1.5%
(0, 446, 889.5,1,321 mg/kg-day in
males;
0, 493.5, 951.5,1,404 mg/kg-day in
premating females;
0, 390.5, 768.5,1,136.5 mg/kg-day
during gestation in females;
0, 706.5,1,384,1,760 mg/kg-day
during lactation in females)b
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21 (F)
Increased cytoplasmic eosinophilic hypertrophy of hepatocytes
Minimal to moderately increased cytoplasmic eosinophilia in males and
females from all treatment groups (quantitative data not reported by study
authors)
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 ofDiisononyl Phthalate
Reference and study design3
Waterman et al. (2000); two-
generation study
Rat (Sprague-Dawley), 30 breeding
pairs/dose
0, 0.2, 0.4, 0.8%
PI (or Fl) animals"
0, 165, 331, 665 mg/kg-day in
males
0, 182, 356, 696 mg/kg-day in
premating females
0, 146, 287, 555 mg/kg-day during
gestation in females
0, 254, 539, 1,026 mg/kg-day
during lactation in females
P2 (or F2) animals"
0, 189, 379, 779 mg/kg-day in
males
0, 197, 397, 802 mg/kg-day in
premating females
0, 143, 288, 560 mg/kg-day during
gestation in females
0, 285, 553, 1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21 (F)
Results
Increased cytoplasmic eosinophilic hypertrophy of hepatocytes
Minimal to moderately increased cytoplasmic eosinophilia in males and
females from all treatment groups (quantitative data not reported by study
authors)
Hepatocellular adenoma and carcinoma
Bio Dynamics (1986); CPSC (2001)
Rat (Sprague-Dawley); 70/sex/dose
0, 500, 5,000, 10,000 ppm (0, 27,
271, 553 mg/kg-day in males;
0, 33, 331, 672 mg/kg-day in
females)
Diet (Santicizer 900)
2 years (interim sacrifice at 1 year)
Doses (M) 0 27 271
Neoplastic nodules (all animals)c
incidence 2/70 5/69 6/69
percentage 3% 7% 9%
Carcinomas (all animals)c
incidence 2/70 2/69 6/69**
percentage 3% 3% 9%
Doses (F) 0 33 331
Neoplastic nodules (all animals)c
incidence 1/70 1/70 5/70
percentage 1% 1% 7%
Carcinomas (all animals)c
incidence 0/70 0/70 5/70**
percentage 0% 0% 7%
553
5/70
7%
4/70
6%
672
2/70
3%
7/70**
10%
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 ofDiisononyl Phthalate
Reference and study design3
(EPLU999): Lington et al. (1997))
Rat (F344); 110/sex/dose
0, 0.03, 0.3, 0.6% (0, 15, 152, 307
mg/kg-day in males;
0, 18, 184, 375 mg/kg-day in
females)
Diet(DINP-l)
2 years (interim sacrifices at 6, 12,
and 18 months)
Covance Laboratories (1998b); EPL
(1999)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm
(0, 29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442,
885 mg/kg-day in females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in
males, 774 mg/kg-day in females)
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Results
Doses (M)
0
15
152
307
Ademonas at terminal sacrificed
incidence 3/81
percentage 4%
Carcinomas at terminal sacrifice"1
incidence
percentage
Combinedd
incidence
percentage
Doses (F)
0/81
0%
3/81
4%
0
1/80
1%
1/80
1%
2/80
3%
18
2/80
3%
0/80
0%
2/80
3%
184
1/80
1%
3/80
4%
4/80
5%
375
Adenomas at terminal sacrifice"1
incidence
percentage
0/81
0%
4/81
5%
0/80
0%
2/80
3%
Carcinomas at terminal sacrifice"1
incidence
percentage
Combinedd
incidence
percentage
Doses (M)
1/81
1%
1/81
1%
0 29
0/81
0%
4/81
5%
88
0/80
0%
0/80
0%
359 733
1/80
1%
2/80
2.5%
Recovery
Adenomas at terminal sacrifice"1
incidence 2/55 4/50
percentage 4% 8%
Carcinomas at terminal sacrifice"1
incidence 1/55 0/50
percentage 2% 0%
Combinedd
incidence
percentage
Doses (F)
3/55 4/50
5% 8%
0 36
1/50
2%
0/50
0%
1/50
2%
109
4/55 7/55
7% 13%
3/55 11/55
5% 20%
7/55 17/55
13% 31%
442 885
6/50
12%
3/50
6%
9/50
18%
Recovery
Adenomas at terminal sacrifice"1
This document is a draft for review purposes only and does not constitute Agency policy,
3-28 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Note: PWG did not perform
statistical analysis on
histopathological findings.
Covance Laboratories (1998a);
CPSC (2001)
Mouse (B6C3Fi); 70/sex/dose
0, 500, 1,500, 4,000, 8,000 ppm (0,
90, 276, 742, 1,560 mg/kg-day in
males; 0, 112, 336, 910,
1,888 mg/kg-day in females)
Recovery group (55/sex/dose):
8,000 ppm (1,377 mg/kg-day in
males; 1,581 mg/kg-day in
females)
Diet
Main study: 2 years (interim
sacrifice at 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Results
incidence
percentage
1/55
2%
1/50
2%
0/50
0%
1/55
2%
1/55
2%
1/50
2%
Carcinomas at terminal sacrifice"1
incidence
percentage
Combinedd
incidence
percentage
Doses (M)
Adenomas (all
incidence
percentage
0/55
0%
1/55
2%
0
animals)'
10/70
14%
0/50
0%
1/50
2%
90
7/60
10%
0/50
0%
0/50
0%
276
8/60
12%
1/55
2%
2/55
4%
742
15/60
23%
7/55
11%
8/55
14.5%
1,560
13/70
19%
2/50
4%
2/50
4%
Recovery6
8/50
16%
Carcinomas (all animals)c
incidence
percentage
Combined (all
incidence
percentage
Doses (F)
Adenomas (all
incidence
percentage
10/70
14%
animals)c
16/70
23%
0
animals)'
2/70
3%
8/67
12%
13/67
19%
112
4/61
6%
10/66
15%
18/66
27%
336
5/60
7%
17/65**
26%
28/65**
43%
910
4/60
6%
20/70**
29%
31/70**
44%
1,888
18/70*
26%
12/50
24%
Recovery6
8/50*
16%
Carcinomas (all animals)c
incidence
percentage
Combined (all
incidence
percentage
1/70
1%
animals)c
3/70
4%
2/68
3%
5/68
7%
5/68
7%
10/68**
15%
7/67**
10%
11/67**
16%
19/70**
27%
33/70**
47%
13/50*
26%
1
2 aDINP formulation referenced when the study authors provided the specific formulation.
3 bCalculated as follows: [% in diet x intake food/water (mg)] -f body weight (kg) = mg/kg-day.
4 "Incidence data as reported by Chronic Hazard Advisory Panel (CPSC, 2001).
5 Incidence data as reported by Pathology Working Group reanalysis (EPL, 1999).
6 6Recovery group incidence data from study authors; Chronic Hazard Advisory Panel (CPSC, 2001) did not evaluate
7 these data.
8 'Incidence data from study authors; Chronic Hazard Advisory Panel (CPSC, 2001) did not evaluate these data.
This document is a draft for review purposes only and does not constitute Agency policy.
3-29 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
1 gResults shown are at terminal sacrifice unless otherwise stated.
2 *Statistically significant from control group, as reported by study authors.
3 **Statistically significant, as reported by Chronic Hazard Advisory Panel (CPSC, 2001).
4 Percent change compared to control = ([treated value - control value] -f control value) x 100
5
6 ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; GD = gestational
7 day; PND = postnatal day
8
This document is a draft for review purposes only and does not constitute Agency policy.
3-30 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Q.
CD
X
op
'CD
Hazelton, 1991
13 wks; F344 rat (male)
Hazelton, 1991
13 wks; F344 rat (female)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Bio Dynamics, 1982a
13 wks; F344 rat (male)
Bio Dynamics, 1982a
13 wks; F344 rat (female)
Halletal., 1999
13 wks; Marmoset
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Covance, 1998a
2 years; B6C3F1 mice (male)
Covance, 1998a
2 years; B6C3F1 mice (female)
Boberget al., 2010
GD7-PND 17; Wistar rat
Clewelletal.2013b
GD 12- PND 14; Sprague-Dawley rat
Clewelletal.2013a
GD 12-19; Sprague-Dawley rat
Hellwigetal., 1997
GD 6-15; Wistar rats
Waterman et al., 2000
PND 21; Sprague-Dawley rats (male)
Waterman et al., 2000
PND 21; Sprague-Dawley rats (female)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (P2) (male)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (PI) (female)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (P2) (male)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (P2) (female)
• statistically significant
O not statistically significan
<
On
,c
*A A
t
©A
• A A
• A
©A
G — eee
• — •
•— 4
G/~\ A
-•
-•
1 10
Doses (mg/kg-day)
100
1000
10000
2
3
Figure 3-1. Exposure-response array of liver weight effects following oral
exposure to DINP.
This document is a draft for review purposes only and does not constitute Agency policy,
3-31 DRAFT—DO NOT CITE OR QUOTE
-------
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Bio Dynamics, 1982a
13wks; F344 rat (male)
Bio Dynamics, 1982a
13wks; F344 rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Covance, 1998a
2 years; B6C3F1 mice (male)
Covance, 1998a
2 years; B6C3F1 mice (female)
Bio/Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio/Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Bio Dynamics, 1982a
13wks; F344 rat (male)
Bio Dynamics, 1982a
13wks; F344 rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Covance, 1998a
2 years; B6C3F1 mice (male)
Covance, 1998a
2 years; B6C3F1 mice (female)
Bio/Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio/Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Bio Dynamics, 1982a
13wks; F344 rat (male)
Bio Dynamics, 1982a
13wks; F344 rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
• statistically significant
O not statistically significant
-6 6-
-e—©•
-©
-• e
-e ©
-•
-e
-e
-e
10
100
1000
10000
Doses (mg/kg-day)
3
4
Figure 3-2. Exposure-response array of liver serum chemistry enzyme levels
following oral exposure to DINP.
This document is a draft for review purposes only and does not constitute Agency policy.
3-32 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Q.
O
Hazelton, 1991
13 wks; F344 rat (male)
Hazelton, 1991
13 wks; F344 rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Covance, 1998a
2 years; B6C3F1 mice (male)
Covance, 1998a
2 years; B6C3F1 mice (female)
Hazelton, 1991
13 wks; F344 rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Bio Dynamics, 1982a
13 wks; F344 rat (male)
Bio Dynamics, 1982a
13 wks; F344 rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Covance, 1998a
2 years; B6C3F1 mice (male)
Covance, 1998a
2 years; B6C3F1 mice (female)
• statistically significant
O not statistically significant
0 — 9 — ©
o o o
o o
— ©
10
100
1000
10000
Doses (mg/kg-day)
2
3
Figure 3-3. Exposure-response array of liver histopathological effects
following oral exposure to DINP.
This document is a draft for review purposes only and does not constitute Agency policy.
3-33 DRAFT—DO NOT CITE OR QUOTE
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2
3
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
3,3.2, Kidney Effects
Table 3-12. Evidence pertaining to kidney effects in animals following oral
exposure to DINP
Reference and study design3
Results
Kidney weight change
Bio Dynamics (1986)
Rat (Sprague-Dawley);
70/sex/dose;
0, 500, 5,000, 10,000 ppm (0, 27,
271, 553 mg/kg-day in males;
0, 33, 331, 672 mg/kg-day in
females)
Diet (SANTICIZER 900)
2 years (interim sacrifice at
1 year)
Lington et al. (1997)
Rat (F344); 110/sex/dose;0, 0.03.
0.3. 0.6% (0, 15, 152, 307 mg/kg-
day in males; 0, 18, 184, 375
mg/kg-day in females)
Diet(DINP-l)
2 years (interim sacrifices at 6,
12, and 18 months)
Covance Laboratories (1998b)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm
(0, 29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442,
885 mg/kg-day in females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in
males; 733 mg/kg-day in females)
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and
79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Kidney weight at terminal
compared to control)
Doses (M)
absolute weight
kidney/body weight
Doses (F)
absolute weight
kidney/body weight
Kidney weight at terminal
compared to control)
Doses (M)
absolute weight
kidney/body weight
Doses (F)
absolute weight
kidney/body weight
Kidney weight at terminal
compared to control)
Doses (M) 0
absolute weight 0%
kidney/body 0%
weight
Doses (F) 0
absolute weight 0%
kidney/body 0%
weight
sacrifice (n = 25-47/sex/dose) (percent c
0 27 271
0% 5% -2%
0% 4% -6%
0 33 331
0% -3% 9*%
0% -5% 10%
sacrifice (n = 48-65/sex/dose) (percent c
0 15 152
Data not reported
0% 7% 10*
0 18 184
Data not reported
0% -1% 7*%
sacrifice (n = 27-40/sex/group) (percent
29 88 359 733
0% 3% 6% 15*%
0% 7% 8% 25*%
36 109 442 885
1% 2% 10*% 10*%
5% 6% 14* 22*%
hange
553
13*%
12*%
672
3%
14*%
hange
307
20*%
375
10*%
change
Recovery
3%
8%
Recovery
2%
4%
This document is a draft for review purposes only and does not constitute Agency policy,
3-34 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Covance Laboratories (1998a)
Mouse (B6C3Fi); 70/sex/dose
0, 500, 1,500, 4,000, 8,000 ppm
(0, 90, 276, 742, 1,560 mg/kg-day
in males; 0, 112, 336, 910,
1,888 mg/kg-day in females)
Recovery group (55/sex/group):
8,000 ppm (1,377 mg/kg-day in
males; 1,581 mg/kg-day in
females)
Diet
Main study: 2 years (interim
sacrifice at 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Hazleton Laboratories (1991)
Rat (F344); 10/sex/dose
0, 2,500, 5,000, 10,000,
20,000 ppm (0, 175.8, 354.6,
719.6, 1,544.7 mg/kg-day in
males; 0, 218.9, 438, 823.8,
1,687.1 mg/kg-day in females)
Diet(DINP-2/3)
13 weeks
Bio Dynamics (1982a)
Rat (F344); 15/sex/dose
0, 0.1 0.3, 0.6, 1.0, 2.0% (0, 67,
210, 410, 730, 1,500 mg/kg-day in
males;
0, 77, 230, 480, 830,
1,600 mg/kg-day in females)b
Diet
13 weeks
Waterman et al. (2000); one-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.5, 1, 1.5%
(0, 446, 889.5, 1,321 mg/kg-day
in males
Results
Kidney weight at terminal sacrifice (n = 24-42/sex/dose) (percent change
compared to control)
Doses (M) 0 90 276 742 1,560 Recovery
absolute weight 0% -4% -11*% -24*% -27*% -17*%
kidney/body 0% -1% -7% -13*% -9% -8%
weight
Doses (F) 0 112 336 910 1,888 Recovery
absolute weight Study authors did not observe a change compared to
controls (quantitative data not reported by study
authors)
kidney/body Study authors did not observe a change compared to
weight controls (quantitative data not reported by study
authors)
Kidney weight (percent change compared to control)
Doses (M) 0 176 355 720 1,545
absolute weight 0% 2% 11*% 16*% 15*%
kidney/ body weight 0% 5*% 9*% 21*% 29*%
Doses (F) 0 220 438 824 1,687
absolute weight 0% 8*% 10*% 11*% 8*%
kidney/ body weight 0% 3% 7*% 13*% 24*%
Kidney weight (percent change compared to control)
Doses (M) 0 67 210 410 730 1,500
absolute weight 0% -4% -3% 5% 9*% 7%
kidney/body weight 0% 0% 3% 7% 13*% 25*%
Doses (F) 0 77 230 480 830 1,600
absolute weight 0% 2% 7*% 12*% 15*% 7*%
kidney/body weight 0% 4% 10*% 14*% 19*% 17*%
Kidney weight in PO animals (percent change compared to control)
Doses (M) 0 446 889.5 1,321
absolute 0% 25*% 28*% 28*%
weight
liver/body Data not reported
weight
Doses (F) 0 493.5 951.5 1,404
This document is a draft for review purposes only and does not constitute Agency policy,
3-35 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
0, 493.5, 951.5, 1,404 mg/kg-day
in premating females
0, 390.5, 768.5,
1,136.5 mg/kg-day during
gestation in females
0, 706.5, 1,384, 1,760 mg/kg-day
during lactation in females) d
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21 (F)
Waterman et al. (2000); two-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.2, 0.4, 0.8%
PI (or Fl) animalsb
0, 165, 331, 665 mg/kg-day in
males
0, 182, 356, 696 mg/kg-day in
premating females
0, 146, 287, 555 mg/kg-day
during gestation in females
0, 254, 539, 1,026 mg/kg-day
during lactation in females
P2 (or F2) animals"
0, 189, 379, 779 mg/kg-day in
males
0, 197, 397, 802 mg/kg-day in
premating females
0, 143, 288, 560 mg/kg-day
during gestation in females
0, 285, 553, 1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21 (F)
Boberg et al. (2011)
Rat (Wistar); 1-7 litters/dose;
18-35 males/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
CDs 7-21
Results
absolute
weight
liver/body
weight
Kidney weight
Doses (M)
absolute
weight
liver/body
weight
Doses (F)
absolute
weight
liver/body
weight
Kidney weight
Doses (M)
absolute
weight
liver/body
weight
Doses (F)
absolute
weight
liver/body
weight
Kidney weight
Doses
absolute
weight
0% 13*% 8*% 0.4%
Data not reported
in PI animals (percent change compared to control)
0 165 331 665
0% 8% 14*% 20*%
Data not reported
0 182 356 696
0% 8*% 10*% 8*%
Data not reported
in P2 (F2) animals (percent change compared to control)
0 165 331 665
0% 6% 7% 14*%
Data not reported
0 182 356 696
0% 5% 4% 3%
Data not reported
in males at PND 90 (percent change compared to control)
0 300 600 750 900
0% -1% -2% -1% -3%
Note: Study authors did not examine this endpoint in females. Relative
weights not reported by study authors.
This document is a draft for review purposes only and does not constitute Agency policy,
3-36 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Hellwig et al. (1997)
Rat (Wistar), 8-10 dams
(litters)/dose per DINP
formulation
0, 40, 200, 1,000 mg/kg-day
Gavage in olive oil (DINP- 1,2,3)
GDs 6-15; dams sacrificed on
GD20
Results
Kidney weight in dams (percent change compared to control)
Doses 0 4C
) 200 1,000
D^P'\ . _ 0% 5% 8% is*%
absolute weight
DINP-2 0% 10*% 4% 7%
absolute weight
D)NP,"3 . . 0% 6% 7% 9%
absolute weight
Note: Relative weight not reported by study authors.
Serum clinical chemistry; kidney function
Covance Laboratories (1998b)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm
(0, 29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442,
885 mg/kg-day in females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in
males; 733 mg/kg-day in females)
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and
79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
BUN levels at terminal sacrifice (n = 10/sex/dose) (percent change compared
to control)
Doses (M) 0 29
BUN 0% -7%
88 359 733 Recovery
0% -13% 40*% 57%
Doses (F) 0 36 109 442 885 Recovery
BUN 0% 0%
0% 31% 25% -6%
Renal histopathology
Bio Dynamics (1986)
Rat (Sprague-Dawley);
70/sex/dose
0, 500, 5,000, 10,000 ppm (0, 27,
271, 553 mg/kg-day in males; 0,
33, 331, 672 mg/kg-day in
females)
Diet (SANTICIZER 900)
2 years (interim sacrifice at
1 year, 10/sex/group)
Note: Study authors did not
perform statistical analysis on
Papillary mineralization
Doses (M) 0
incidence 3/70
(unilateral)
percentage 4%
incidence (bilateral) 0/70
percentage 0%
Doses (F) 0
incidence 6/70
(unilateral)
percentage 9%
27 271 553
NE NE 9/70
NE NE 13%
NE NE 16/70
NE NE 23%
33 331 672
NE NE 4/70
NE NE 6%
This document is a draft for review purposes only and does not constitute Agency policy,
3-37 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
histopathological findings.
Lington et al. (1997)
Rat (F344); 110/sex/dose
0, 0.03. 0.3. 0.6% (0, 15, 152,
307 mg/kg-day in males; 0, 18,
184, 375 mg/kg-day in females)
Diet(DINP-l)
2 years (interim sacrifices at 6,
12, and 18 months; 10/sex/dose)
Covance Laboratories (1998b)
Rat (F344); 70 or 85/sex/dose 0,
500, 1,500, 6,000, 12,000 ppm (0,
29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442,
885 mg/kg-day in females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in
males; 733 mg/kg-day in females)
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and
79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Note: Study authors did not
perform statistical analysis on
histopathological findings.
Results
incidence (bilateral) 8/70
percentage 11%
Renal tubule pigmentation
NE
NE
NE
NE
Increase noted in high dose males at the 18-month interim
(quantitative data not reported by study authors)
sacrifice
11/70
16%
Histopathological lesions at terminal sacrifice
Doses (M) 0
Renal tubule pigmentation
incidence 34/36
percentage 94%
severity 1.2
Tubule dilation
incidence 0/36
percentage 0%
Papillary mineralization
incidence 6/36
percentage 17%
severity 0.2
Doses (F) 0
Renal tubule pigmentation
incidence 36/37
percentage 97%
severity 1.4
Tubule dilation
incidence 0/37
percentage 0%
Papillary mineralization
incidence 7/37
percentage 19%
severity 0.2
29
35/35
100%
1.5
0/35
0%
11/35
31%
0.3
36
38/38
100%
1.3
0/38
0%
7/38
18%
0.2
88
39/39
100%
1.5
0/39
0%
9/39
23%
0.2
109
40/40
100%
1.2
0/40
0%
1/40
3%
0
359
31/31
100%
2.3
0/31
0%
30/31
97%
1.7
442
33/33
100%
2.0
1/33
3%
8/33
24%
0.2
733
27/27
100%
2.9
1/27
4%
25/27
93%
2.6
885
32/32
100%
2.4
0/32
0%
8/32
25%
0.3
Recovery
29/29
100%
2.1
1/29
3%
29/29
100%
2.9
Recovery
34/34
100%
2.0
0/34
0%
5/34
15%
0.1
This document is a draft for review purposes only and does not constitute Agency policy,
3-38 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Hazleton Laboratories (1991)
Rat (F344); 10/sex/dose
0, 2,500, 5,000, 10,000,
20,000 ppm (0, 175.8, 354.6,
719.6, 1,544.7 mg/kg-day in
males; 0, 218.9, 438, 823.8,
1,687.1 mg/kg-day in females)
Diet(DINP-2/3)
13 weeks
Note: Study authors did not
perform statistical analysis on
histopathological findings.
Bio Dynamics (1982a)
Rat (F344); 15/sex/dose; kidneys
examined microscopically in
12-13/sex/dose
0, 0.1 0.3, 0.6, 1.0, 2.0% (0, 67,
210, 410, 730, 1,500 mg/kg-day in
males; 0, 77, 230, 480, 830,
1,600 mg/kg-day in females)b
Diet
13 weeks
Note: Study authors did not
perform statistical analysis on
histopathological findings.
Results
Doses (M) 0
Granular casts/dilation
incidence minimal 0/10
percentage 0%
in cidence slight 0/10
percentage 0%
Tubular regeneration
incidence minimal 10/10
percentage 100%
incidence slight 0/10
percentage 0%
incidence
moderate
percentage 0%
Doses (F) 0
176
0/10
0%
0/10
0%
7/10
70%
3/10
30%
0/10
0%
220
355
6/10
60%
0/10
0%
0/10
0%
9/10
90%
1/10
10%
438
720
0/10
0%
10/10
100%
0/10
0%
9/10
90%
1/10
10%
824
1,545
0/10
0%
10/10
100%
0/10
0%
2/10
20%
8/10
80%
1,687
Granular casts/dilation: no incidence
Tubular regeneration
incidence minimal 1/10
percentage 10%
Doses (M) 0
Nephrosis (incidence)
incidence minimal 0/13
percentage 0%
incidence slight 0/13
percentage 0%
incidence 0/13
moderate
percentage 0%
Granular casts (incidence)
incidence minimal 0/13
percentage 0%
incidence slight 0/13
percentage 0%
incidence Q
moderate
percentage 0%
0/10
0%
67
0/12
0%
0/12
0%
0/12
0%
0/12
0%
0/12
0%
0/12
0%
0/10
0%
210 410
4/13 3/12
31% 25%
0/13 6/12
0% 50%
0/13 3/12
0% 25%
0/13 4/12
0% 33%
0/13 2/12
0% 17%
0/13 0/12
0% 0%
0/10
0%
730
0/13
0%
7/13
54%
5/13
38%
2/13
15%
9/13
69%
2/13
15%
2/10
20%
1,500
3/13
23%
5/13
38%
1/13
8%
1/13
8%
4/13
31%
4/13
31%
This document is a draft for review purposes only
3-39
and does not constitute Agency policy,
DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Results
Tubular regeneration
incidence minimal 4/13
percentage 31%
incidence slight 7/13
percentage 54%
incidence Q
moderate
percentage 0%
Doses (F) 0
Nephrosis: no incidence
Granular casts: no incidence
6/12 0/13 0/12 1/13
50% 0% 0% 8%
6/12 13/13 4/12 9/13
50% 100% 33% 69%
0/12 0/13 8/12 3/13
0% 0% 67% 23%
77 230 480 830
3/13
23%
7/13
54%
3/13
23%
1,600
Tubular regeneration: no incidence
incidence minimal 2/13
percentage 15%
0/13 1/12 0/13 1/13
0% 8% 0% 8%
0/13
0%
Chronic progressive nephropathy
Covance Laboratories (1998a)
Mouse (B6C3F1); 70/sex/dose
0, 500, 1,500, 4,000, 8,000 ppm
(0, 90, 276, 742, 1,560 mg/kg-day
in males; 0, 112, 336, 910,
1,888 mg/kg-day in females)
Recovery group (55/sex/group):
8,000 ppm (1,377 mg/kg-day in
males; 1,581 mg/kg-day in
females)
Diet
2 years (18-month interim
sacrifice; 15/sex/group)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Note: Study authors did not
perform statistical analysis on
histopathological findings.
Doses (M) 0
90 276 742 1,560
Recovery
Not observed
Doses (F) 0
incidence 40/60
percentage 67%
severity 0.8
112 336 910 1,888
36/61 39/60 39/60 61/62
59% 65% 65% 98%
0.7 0.8 0.8 1.8
Recovery
39/50
78%
0.9
Renal carcinoma
Lington et al. (1997)
Rat (F344); 110/sex/dose
0, 0.03. 0.3. 0.6 % (0, 15, 152, 307
mg/kg-day in males; 0, 18, 184,
375 mg/kg-day in females)
Diet(DINP-l)
Doses (M) 0
15 152
307
Renal tubular cell carcinoma at terminal sacrifice
incidence 0/81
percentage 0%
1/80 0/80
1% 0%
2/80
3%
Renal transitional cell carcinoma at terminal sacrifice
This document is a draft for review purposes only and does not constitute Agency policy,
3-40 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
2 years (interim sacrifices at 6,
12, and 18 months; 10/sex/dose)
Covance Laboratories (1998b):
CPSC (2001)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm
(0, 29, 88, 359, 733 mg/kg-day in
males; 0, 36, 109, 442, 885
mg/kg-day in females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in
males; 733 mg/kg-day in females)
Diet
Main study: 2 years (interim
sacrifices at 1, 2, 13, and
79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Results
incidence 0/81 0/80 3/80
percentage 0% 0% 4%
Doses (F) 0 18 184
Renal tubular cell carcinoma: no incidence
Renal transitional cell carcinoma: no incidence
Doses (M) 0 29 88 359 733
Renal tubular cell carcinoma at terminal sacrifice0
incidence 0/65 0/55 0/55 0/65 2/65**
percentage 0% 0% 0% 0% 3%
Doses (F) 0 36 109 442 885
Renal tubular cell carcinoma
No incidence
0/80
0%
375
Recovery
4/50**
8%
Recovery
1
2
3
4
5
6
7
8
9
10
11
*Statistically significant (p < 0.05) based on analysis of data conducted by study authors.
**Statistically significant difference from control group (p < 0.05), as reported by Chronic Hazard Advisory Panel
(CPSC, 2001).
aDINP formulation referenced when the study authors provided the specific formulation.
Calculated as follows: [% in diet x intake food (mg)] 4- body weight (kg) = mg/kg-day
incidence data as reported by Chronic Hazard Advisory Panel (CPSC, 2001).Percent change compared to control =
([treated value - control value] 4 control value) x 100
BUN = blood urea nitrogen; NE = not examined
This document is a draft for review purposes only and does not constitute Agency policy,
3-41 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Leeetal., 2006a,b
GD 15-PND 21; Wistar rat
Waterman et al. 1999
GD6-15; Sprague-Dawley rat • statistically significant <
Hazelton, 1991 O not statistically significant
Hazelton, 1991
13 wks; F344 rat (female)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Bio Dynamics, 1982a
13 wks; F344 rat (male)
Bio Dynamics, 1982a
13 wks; F344 rat (female)
Lingtonetal., 1997
2 years; F344, rat (male)
Lington etal., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Covance, 1998a
2 years; B6C3F1 mice (male)
Boberg et al., 2010
GD7-PND 17; Wistar rat
Hellwig etal., 1997
GD 6-15; Wistar rats
Waterman etal., 2000
PND 21; Sprague-Dawley rats (male)
Waterman et al., 2000
PND 21; Sprague-Dawley rats (female)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (PI) (male)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (PI) (female)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (P2) (male)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (P2) (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
• •
• 1
)
-•
— e
10
100
1000
10000
Doses (mg/kg-day)
2
3
Figure 3-4. Exposure-response array of kidney weight effects following oral
exposure to DINP.
This document is a draft for review purposes only and does not constitute Agency policy,
3-42 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
S E
-------
2
3
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
3,3,3, Male Reproductive Effects
Table 3-13. Evidence pertaining to male reproductive effects in animals
following oral exposure to DINP
Reference and study design
Results
Anogenital distance (AGD)b
Boberg et al. (2011)
Rat (Wistar); AGO assessed in
9-10 litters/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 25 control dams
(litters)
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Clewell et al. (2013a)
Rat (Sprague-Dawley);
4-9 dams/timepoint/dose; AGO
assessed in 8 litters/dose and
9 control litters
0, 50, 250, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 12-19; dams sacrificed 0.5,
1, 2, 6, 12, and 24 hours after
final dose
Lee et al. (2006b)c
Rat (Wistar-lmamichi); number
of dams/dose not reported;
16-47 pups/sex/dose
0, 40, 400, 4,000, 20,000 ppm (0,
4, 40, 400, 2,000 mg/kg-day)c
Diet (DINP-2)
GD15toPND21
AGD/BW1/3 (percent change compared to control)
Doses 0 300 600 750
900
PND1 0% -1% -2% -3% -5*%
Note: When more than one pup per litter was examined, statistical analysis
was adjusted using litter as an independent, random and nested factor.
Author sent original data for this endpoint.
AGD/BW1/3 (percent change compared to control)
Doses 0 109 555
PND2 0% 2% 2%
PND14 0% -1% -2%
Note: The litter was the statistical unit of comparison.
1,513
-1%
-7*%
AGD/BW1/3 (percent change compared to control)
Doses 0 109 555
GD 20 0% -3% -2%
Note: The litter was the statistical unit of comparison.
1,513
0.7%
AGD/BW1/3 (percent change compared to control)
Doses 0 4 40 400
PND1 0% 4*% 5*% 6*%
Note: The individual was the statistical unit of comparison.
2,000
9*%
This document is a draft for review purposes only and does not constitute Agency policy,
3-44 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Masutomi etal. (2003)
Rats (Sprague-Dawley);
5 dams/dose; AGO was assessed
in 5 litters/dose
0, 400, 4,000, 20,000 ppm
Gestation: 0, 30.7, 306.7,
1,164.5 mg/kg-day
Lactation: 0, 66.2, 656.7,
2,656.7 mg/kg-day
Diet(DINP-2)
GD15-PND10
Results
Absolute AGO (percent change compared to control)
Doses
PND2
Note: The litter was the
0 66.2 656.7 2,656.7
0% -3% -9% -9%
statistical unit of comparison.
Nipple retention
Boberg et al. (2011)
Rat (Wistar); nipple retention
assessed in 9-10 litters/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 24 control dams
(litters)
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Fetal testicular testosterone prodi
Adamsson et al. (2009)c
Rat (Sprague-Dawley);
7-8 dams/dose; fetal
testosterone production
assessed in 5-8 litters/dose
0, 250, 750 mg/kg-day
Gavage in corn oil
EDs 13.5-17.5
Nipple retention (percent change compared to control in litters)
Doses 0
300 600 750 900
PND13 0% 1% 47% 59*% 60*%
Note: When more than one pup per litter was examined, statistical analysis
was adjusted using litter as an independent, random and nested factor.
Author sent original data for this endpoint.
Nipple retention (percent change compared to control in litters)
Doses
PND14
Note: The litter was the
jction
0 109 555 1,513
0% -6% 6% 17%
statistical unit of comparison.
Intratesticular testosterone content (percent change compared to control
litters)
Doses
ED 19.5
Note: The litter was the
0 250 750
0% 3% -16%
statistical unit of comparison.
in
This document is a draft for review purposes only and does not constitute Agency policy,
3-45 DRAFT—DO NOT CITE OR QUOTE
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Boberg et al. (2011)
Rat (Wistar); fetal testosterone
production assessed in
3-4 litters/dose
(1-2 testes/litter)
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
CDs 7-21
Borch et al. (2004)c
Rat (Wistar); 8 dams/dose; fetal
testosterone production
assessed in 7-8 litters/dose
(2 testes/litter)
0, 750 mg/kg-day
Gavage in peanut oil (DINP-2)
CDs 7-21
Clewell et al. (2013a)
Rat (Sprague-Dawley);
4-9 dams/timepoint/dose;
Assessed in 8 litters/dose and
9 control litters
0, 50, 250, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 12-19; dams sacrificed 0.5,
1, 2, 6, 12, and 24 hours after
final dose
Hannasetal. (2011)
Rat (Sprague-Dawley);
3-6 dams/group, 3-6 litters
DINP1, 3 dams/group, 1-3 litters
DINP2
0, 500, 750, 1,000,
1,500 mg/kg-day
Gavage in corn oil (DINP-1 and
DINP-2)
GDs 14-18
Results
Fetal testicular testosterone
in litters)
Doses 0
production (percent change compared to control
300 600 750 900
GD 21 0% -51% -75% -69% -76%
Note: When more than one pup per litter was examined, statistical analysis
was adjusted using litter as an independent, random and nested factor.
Fetal testicular testosterone
in litters)
Doses
production (percent change compared to control
0 750
GD 21 0% -73*%
Note: The litter was the statistical unit of comparison.
Fetal testicular testosterone
in litters)
Doses 0
production (percent change compared to control
109 555 1,513
2 hours following 0% 4% -50*% -65*%
final dose
24 hours following 0% -16% 61% 22%
final dose
Note: The litter was the statistical unit of comparison.
Fetal testicular testosterone
in litters)
Doses 0
production (percent change compared to control
500 750 1,000 1,500
GDIS 0% -30*% -45*% -57*% -69*%
Note: The litter was the statistical unit of comparison. Litter means from DINP-
1- and DINP-2-treated rats were combined for statistical analysis.
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 ofDiisononyl Phthalate
Reference and study design
Adamsson et al. (2009)c
Rat (Sprague-Dawley);
7-8 dams/dose; fetal
testosterone production
assessed in 5-8 litters/dose
0, 250, 750 mg/kg-day
Gavage in corn oil
EDs 13.5-17.5
Sperm motility
Boberg et al. (2011)c
Rat (Wistar); semen quality
analysis in 1-3 males/litter
(6-10 males/dose)
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Malformations
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 24 control dams
(litters)
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Gray et al. (2000)
Rat (Sprague-Dawley);
dams
0, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GD14-PND3
Results
Intratesticular testosterone content (percent change compared to control in
litters)
Doses 0 250 750
ED 19.5 0% 3% -16%
Note: The litter was the statistical unit of comparison.
Sperm motility at PND 90 (percent change compared to control in litters)
Doses 0 300 600 750 900
PND 90 0% -4% -13*% -19*% -20*%
Note: When more than one pup per litter was examined, statistical analysis
was adjusted using litter as an independent, random and nested factor.
Author sent original data for this endpoint.
Hypospadias, PNDs 49-50 (incidence/total pups)
Doses 0 109 555 1,513
incidence 1/111 0/87 0/83 2/84
percent 0.9% 0% 0% 2%
Note: Study authors listed positive effect as very slight/borderline
hypospadias. Other effects were evaluated (epididymal agenesis, incomplete
epididymis, flaccid epididymis, undescended testes, unilateral enlarged testis,
atrophic testis, absent seminal vesicles) but no effects were observed by study
authors (quantitative data reported but not presented in evidence tables).
Epididymal agenesis
Doses 0 750
incidence 0/80 4/52*
percent 0* 8%
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 ofDiisononyl Phthalate
Reference and study design
Results
Histopathological changes
Bio Dynamics (1986)
Rat (Sprague-Dawley);
70/sex/dose
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
0, 500, 5,000, 10,000 ppm (0, 27,
271, 553 mg/kg-day in males;
0, 33, 331, 672 mg/kg-day in
females)
Diet(DINP-l)
Diet (Santicizer 900)
2 years (interim sacrifice at
1 year)
Boberg et al. (2011)
Rat (Wistar); 3-4 litters/dose;
one testis section evaluated
from 1-4 males/litter
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
CDs 7-21
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 24 control dams
(litters)
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Clewell et al. (2013a)
Rat (Sprague-Dawley);
4-9 dams/timepoint/dose;
Assessed in 8 litters/dose and
9 control litters
0, 50, 250, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 12-19; dams sacrificed 0.5,
1, 2, 6, 12, and 24 hours after
final dose
No hyperplasia at interim sacrifice
Doses 0
Unilateral interstitial cell hyperplasia
incidence 3/69
percent 4%
Bilateral interstitial cell hyperplasia
incidence 1/69
553
9/70
13%
13/70
Multinucleated gonocytes (affected litters/total number of litters)
Doses 0 300 600
750 900
incidence 0/3 2/4 3/3 3/3 3/3
percent 0% 50% 100*% 100*% 100*%
Multinucleated germ cells (affected animals/total number of animals PND 2)
Doses 0 109
incidence y24 2/20
percent 4% 10%
Leydig cell aggregates
incidence 4/24 4/20
percent 17% 20%
555 1,513
7/20* 18/19*
35*% 95*%
8/20 19/19*
40% 100%*
Multinucleated gonocytes (24 hours following final dose) (affected
animals/total number of litters)
Doses 0 50
incidence 0/25 0/8
Leydig cell aggregates (24 hours following final
number of litters)
incidence 2/25 3/8
250 750
2/8 6/7*
dose) (affected animals/total
1/8 7/7*
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 ofDiisononyl Phthalate
Reference and study design
Results
Testes weight change
Boberg et al. (2011)
Rat (Wistar); testes weighed in
6-10 litters/group
(1-7 males/litter,
18-35 males/group)
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Clewell et al. (2013b)
Rat (Sprague-Dawley);
20 dams(litters)/dose; 24
control dams (litters); testes
weighed in 1 pup/litter
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Masutomi et al. (2003)
Rats (Sprague-Dawley);
5 dams/dose; testes weighed in
5 male pups/dose
0, 400, 4,000, 20,000 ppm
Gestation: 0, 30.7, 306.7,
1,164.5 mg/kg-day
Lactation: 0, 66.2, 656.7,
2,656.7 mg/kg-day
Diet (DINP-2)
GD 15-PND 10
Testes weight at PND 90 (percent change compared to control)
Doses 0 300 600 750
900
absolute weight (right) 0% -1% 4% -4% 0%
absolute weight (left) 0% -1% 2% -3% 3%
Note: When more than one pup per litter was examined, statistical analysis
was adjusted using litter as an independent, random and nested factor.
Testes weight at PND 2 (percent change compared to control)
Doses 0 109 555
absolute 0% 2% 2%
weight (right)
absolute 0% 0% 3%
weight (left)
1,513
-2%
-2%
Testes weight at PND 27 (percent change compared to control)
Doses 0 30.7 306.7
1,164.5
absolute weight 0% 4% -21% -54*%
Note: There was no significant treatment-related effect on testes weight at
PNW 11.
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Results
Covance Laboratories (1998a)
Testes weight at terminal sacrifice (percent change compared to control)
Mouse (B6C3Fi); 70/sex/dose
(35-40/dose used for this
endpoint)
0, 500, 1,500, 4,000, 8,000 ppm
(0, 90, 276, 742,
1,560 mg/kg-day in males;
0,112, 336, 910,
1,888 mg/kg-day in females)
Recovery group (55/sex/group):
8,000 ppm (1,377 mg/kg-day in
males; 1,581 mg/kg-day in
females
Diet
Main study: 2 years (interim
sacrifice at 79 weeks)
Recovery group: 78 weeks,
followed by a 26-week recovery
period with basal diet alone
Doses
0
90
276
742
1,560 Recovery
absolute
weight
0%
0%
-3%
-10*% -21*%
-10*%
Waterman et al. (2000): one-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.5,1,1.5%
(0, 446, 889.5,1.321 mg/kg-day
in males
0, 493.5, 951.5,1.404 mg/kg-day
in premating females
0, 390.5, 768.5,
1.136.5 mg/kg-day during
gestation in females
0, 706.5,1.384,1.760 mg/kg-day
during lactation in females)d
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Testes weight in PI males (percent change compared to control)
Doses
446
889.5
1,321
absolute weight
(left)
absolute weight
(right)
0%
0%
3%
1%
5%
4%
11*%
9*%
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Waterman et al. (2000); two-
generation study
Rat (Sprague-Dawley),
30 breeding
pairs/dose/generation
0, 0.2, 0.4, 0.8%
Planimalsd
0, 165, 331, 665 mg/kg-day in
males
0, 182, 356, 696 mg/kg-day in
premating females
0, 146, 287, 555 mg/kg-day
during gestation in females
0, 254, 539, 1,026 mg/kg-day
during lactation in females
P2(Fl)animalsd
0, 189, 379, 779 mg/kg-day in
males
0, 197, 397, 802 mg/kg-day in
premating females
0, 143, 288, 560 mg/kg-day
during gestation in females
0, 285, 553, 1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Results
Testes weight in PI males (percent change compared to control)
Doses 0 165 331
absolute 0% 1% 2%
weight (left)
absolute 0% 2% 3%
weight
(right)
665
2%
2%%
P2 (Fl) males
Doses 0 189 379
absolute 0% 0% -1.5%
weight (left)
absolute 0% 3% 1%
weight
(right)
779
3%
4%
Prostate weight
Boberg et al. (2011)
Rat (Wistar); 6-10 litters/group
(1-7 males/litter,
18-35 males/dose)
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 24 control dams
(litters); testes weighed in
1 pup/litter
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
Prostate weight at PND 90 (percent change compared to control)
Doses 0 300 600 750
900
absolute weight 0% 0% 2% -4% -12%
Note: When more than one pup per litter was examined, statistical analysis
was adjusted using litter as an independent, random and nested factor
Ventral prostate at PNDs 49-50 (percent change compared to control)
Doses 0 109 555
absolute weight 0% 8% 0%
1,513
-8%
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Results
GD12-PND14
Waterman et al. (2000): one-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.5,1,1.5%
(0, 446, 889.5,1,321 mg/kg-day
in males
0, 493.5, 951.5,1,404 mg/kg-day
in premating females
0, 390.5, 768.5,
1,136.5 mg/kg-day during
gestation in females
0, 706.5,1,384,1,760 mg/kg-day
during lactation in females)d
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Prostate weight (percent change compared to control)
Doses
446
889.5
1,321
absolute weight 0%
5%
5%
-7%
Waterman et al. (2000): two-
generation study
Rat (Sprague-Dawley),
30 breeding
pairs/dose/generation
0, 0.2, 0.4, 0.8%
Planimalsd
Prostate weight in PI males (percent change compared to control)
Doses
165
331
665
0,165, 331, 665 mg/kg-day in
males
0,182, 356, 696 mg/kg-day in
premating females
0,146, 287, 555 mg/kg-day
during gestation in females
0, 254, 539,1,026 mg/kg-day
during lactation in females
P2(Fl)animalsd
0,189, 379, 779 mg/kg-day in
males
0,197, 397, 802 mg/kg-day in
premating females
0,143, 288, 560 mg/kg-day
during gestation in females
0, 285, 553,1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
absolute weight
P2 (Fl) males
absolute weight
0%
0%
2%
-2%
-8%
-2%
0%
-4%
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 ofDiisononyl Phthalate
Reference and study design
Results
Epididymis weight
Boberg et al. (2011)
Rat (Wistar); 6-10 litters/group
(1-7 males/litter,
18-35 males/dose)
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Left epididymis weight at PND 90 (percent change compared to control)
Doses
0
300
600
750
900
absolute weight
0%
-3.4%
0%
-5.2%
0%
Note: When more than one pup per litter was examined, statistical analysis
was adjusted using litter as an independent, random and nested factor.
Clewell et al. (2013b)
Rat (Sprague-Dawley);
20 dams(litters)/dose;
24 control dams (litters); testes
weighed in 1 pup/litter
0, 760, 3,800,11,400 ppm (0,
109, 555,1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Epididymis weight at PNDs 49-50 (percent change compared to control)
Doses 0 109 555 1,513
absolute weight 0%
(right)
absolute weight 0%
(left)
10%
5%
5%
0%
0%
-5%
Waterman et al. (2000): one-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.5,1,1.5%
(0, 446, 889.5,1,321 mg/kg-day
in males
0, 493.5, 951.5,1,404 mg/kg-day
in premating females
0, 390.5, 768.5,
1,136.5 mg/kg-day during
gestation in females
0, 706.5,1,384,1,760 mg/kg-day
during lactation in females)d
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Epididymis weight in PI males (percent change compared to control)
Doses
0
446
889.5
1,321
absolute weight
(right)
absolute weight
(left)
0%
0%
-1%
3%
3%
4%
7%
7%
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 ofDiisononyl Phthalate
Reference and study design
Results
Waterman et al. (2000): two-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose/
generation
0, 0.2, 0.4, 0.8%
Planimalsd
0,165, 331, 665 mg/kg-day in
males
0,182, 356, 696 mg/kg-day in
premating females
0,146, 287, 555 mg/kg-day
during gestation in females
0, 254, 539,1,026 mg/kg-day
during lactation in females
P2(Fl)animalsd
0,189, 379, 779 mg/kg-day in
males
0,197, 397, 802 mg/kg-day in
premating females
0,143, 288, 560 mg/kg-day
during gestation in females
0, 285, 553,1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Epididymis weight in PI males (percent change compared to control)
Doses
0
165
331
665
absolute weight
(right)
absolute weight
(left)
P2 (Fl) males
0%
0%
-2%
2%
0%
1%
1%
4%
Doses
0
189
379
779
absolute weight
(right)
absolute weight
(left)
0%
0%
2%
2%
1%
0%
7%
6%
1
2
3
4
5
6
7
8
9
10
*Statistically significant (p < 0.05) based on analysis of data conducted by study authors.
aDINP formulation referenced when the study authors provided the specific formulation.
bl\lormalized to the cube root of body weight
°Values reported by the study authors were estimated from published graphs using "Grab It!", a Microsoft Excel
based free software application used to digitizes data from image files. Publisher: www.datatrendsoftware.com.
Calculated as follows: [% in diet x intake food/water (mg)] 4- body weight (kg) = mg/kg-day
Percent change compared to control = ([treated value - control value] -f control value) x 100
ED = estrous day; PNW = postnatal week
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 ofDiisononyl Phthalate
Boberg et al., 2010
GD7-PND17; Wistar rat
Clewelletal., 2013b
GD 12- PND 14; Sprague-Dawley rat
Clewelletal., 2013a
GD 12-19; Sprague-Dawley rat
Lee et al., 2006a,b
GD15-PND21; Wistar rat
Masutomi et al., 2003
GD 15- PND 10; Sprague-Dawley rat
Boberg et al., 2010
GD7-PND17; Wistar rat
Clewelletal., 2013b
GD 12- PND 14; Sprague-Dawley rat
• statistically significant
O not statistically significant
10 100
Doses (mg/kg-day)
1000
10000
3
4
Figure 3-6. Exposure-response array of male reproductive puberty effects
following oral exposure to DINP.
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 ofDiisononyl Phthalate
o
e-
ra
£
Boberg et al., 2010
GD7-PND17; Wistar rat
• statistically significant
O not statistically significant
Borchetal., 2004
GD7-21; Wistar rat
Hannaset al., 2011
GD 14-18; Sprague-Dawley rat
Adamsson et al. 2009
ED 13.5 to 17.5; Sprague-Dawley rat
Clewell etal., 2013a
GD 12-19; Sprague-Dawley rat
10
100
Doses (mg/kg-day)
1000
10000
3
4
Figure 3-7. Exposure-response array of male reproductive testosterone
effects following oral exposure to DINP.
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 ofDiisononyl Phthalate
o
fr
ra
E
E
Boberg et al., 2010
GD7-PND17; Wistar rat
• statistically significant
O not statistically significant
Borchetal., 2004
GD7-21; Wistar rat
Hannaset al., 2011
GD 14-18; Sprague-Dawley rat
Adamsson et al. 2009
ED 13.5 to 17.5; Sprague-Dawley rat
Clewelletal., 2013b
GD 12-19; Sprague-Dawley rat
10
100
Doses (mg/kg-day)
1000
10000
2
3
Figure 3-8. Exposure-response array of male reproductive histopathological
effects following oral exposure to DINP.
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 ofDiisononyl Phthalate
o
e-
ra
E
Clewelletal.2013b
GD 12- PND 14; Sprague-Dawley rat
Covance, 1998a
2 years; B6C3F1 mice
Boberg et al., 2010
GD7-PND17;Wistarrat
Masutomi et al., 2003
GD 15- PND 10; Sprague-Dawley rat
Waterman et al., 2000
PND 21; Sprague-Dawley rats (PI)
Waterman etal., 2000
two-generation; Sprague-Dawley rats (Fl)
Waterman etal., 2000
two-generation; Sprague-Dawley rats (PI)
Boberg et al., 2010
GD7-PND17;Wistarrat
Clewelletal.2013b
GD 12- PND 14; Sprague-Dawley rat
Waterman etal., 2000
PND 21; Sprague-Dawley rats (PI)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (Fl)
Waterman etal., 2000
two-generation; Sprague-Dawley rats (PI)
Boberg etal., 2010
GD7-PND17;Wistarrat
Clewelletal.2013b
GD 12- PND 14; Sprague-Dawley rat
Waterman et al., 2000
PND 21; Sprague-Dawley rats (PI)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (Fl)
Waterman etal., 2000
two-generation; Sprague-Dawley rats (PI)
• statistically significant
O not statistically significant
o—e—o
G—e—e
e—e—o
G—e—e
10
0—e e
G—e—e
100 1000
10000
Doses (mg/kg-day)
2
3
Figure 3-9. Exposure-response array of male reproductive organ weight
effects following oral exposure to DINP.
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 ofDiisononyl Phthalate
3,3,4. Female Reproductive Effects
Table 3-14. Evidence pertaining to female reproductive effects in animals
following oral exposure to DINP
Reference and study design3
Results
Fertility
Boberg et al. (2011)
Rat (Wistar); 12 dams/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Note: 16 dams/dose were used
overall, however 4 dams/dose
were only exposed GDs 7-21 and
sacrificed at GD 21 for fetal
testosterone assessment.
Hellwig et al. (1997)
Rat (Wistar), 8-10 dams/dose
per DINP formulation
0, 40, 200, 1,000 mg/kg-day
Gavage in olive oil (DINP- 1,2,3)
GDs 6-15; dams sacrificed on
GD 20
(Leeetal. (2006b); Leeetal.
(2006a))
Rat (Wistar-lmamichi);
6-12 females/dose, four litters
per group
0, 40, 400, 4,000, 20,000 ppm (0,
4, 40, 400, 2,000 mg/kg-day)c
Diet (DINP-2)
GD15-PND21
Post implantation loss (resorptions plus dead fetuses, mean %)
Doses 0 300 600 750
percent 23% 15% 14% 10%
900
19%
(Percent change compared to control)
Doses 0 40 200
1,000
Implantations (mean/dam)
DINP-1 0% -16% -3%
DINP-2 0% -13%* -7%
DINP-3 0% -6% 0%
-13%
-3%
-9%
Resorptions (mean)
DINP-1 0% -57% 100%
DINP-2 0% 0% 57%
DINP-3 0% 29% 0%
-14%
71%
43%
Post implantation loss (resorptions plus dead fetuses, mean %)
DINP-1 4.1% 2.0% 9.0%
DINP-2 4.1% 4.5% 7.5%
DINP-3 4.1% 6.1% 4.3%
4.1%
7.8%
6.2%
Lordosis quotient at PNW 20
Doses 0 4 40 400
percent 75% -50*% -45*% -25*%
2,000
Not
reported
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Results
Waterman et al. (1999)
(percent change compared to control)
Rat (Sprague-Dawley);
23-25 dams/dose
0,100, 500,1,000 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 6-15; dams sacrificed at
GD21
Doses
0
100
500
1,000
Implantations (mean/dam) 0% -5% 1% -3%
Resorptions (mean/dam) 0% 25% -25% 50%
Post implantation loss 3.6% 5.0% 3.4% 5.5%
(resorptions plus dead
fetuses), mean (%)
Waterman et al. (2000): one-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.5,1,1.5%
(0, 446, 889.5,1,321 mg/kg-day
in males
0, 493.5, 951.5,1,404 mg/kg-day
in premating females
0, 390.5, 768.5,
1,136.5 mg/kg-day during
gestation in females
0, 706.5,1,384,1,760 mg/kg-day
during lactation in females)b
Diet (DINP-1)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Fertility
Doses
493.5
percent
Fecundity
percent
96.7%
89.7%
90%
81.5%
951.5
1,404
100%
90%
93.3%
89.3%
Waterman et al. (2000): two-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose/
generation
0, 0.2, 0.4, 0.8%
Planimalsb
Fertility, PI animals
Doses
182
356
696
Percent
93.3%
0,165, 331, 665 mg/kg-day in
males
0,182, 356, 696 mg/kg-day in
premating females
0,146, 287, 555 mg/kg-day
during gestation in females
0, 254, 539,1,026 mg/kg-day
during lactation in females
P2(F1) animals"
0,189, 379, 779 mg/kg-day in
males
0,197, 397, 802 mg/kg-day in
premating females
Fecundity, PI animals
Percent 92.9%
Fertility, P2 animals
93.1%
88.9%
90%
88.9%
93.3%
85.7%
Doses
0
197
397
802
Percent 90% 93.3% 83.3% 80%
Fecundity, P2 animals
Percent 77.8% 75% 80% 70.8%
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
0, 143, 288, 560 mg/kg-day
during gestation in females
0, 285, 553, 1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Ovary effects
Boberg et al. (2011)
Rat (Wistar); 12 dams/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Note: 16 dams/dose were used
overall, however 4 dams/dose
were only exposed GDs 7-21 and
sacrificed at GD 21 for fetal
testosterone assessment.
Hellwig et al. (1997)
Rat (Wistar), 8-10 dams/dose
per DINP formulation
0, 40, 200, 1,000 mg/kg-day
Gavage in olive oil (DINP- 1,2,3)
GDs 6-15; dams sacrificed on
GD20
Masutomi et al. (2003)
Rats (Sprague-Dawley);
5 dams/dose; ovaries examined
microscopically in 5 female
offspring/dose
0, 400, 4,000, 20,000 ppm
(Gestation: 0, 30.7, 306.7,
1,164.5 mg/kg-day
Lactation: 0, 66.2, 656.7,
2,656.7 mg/kg-day)
Diet (DINP-2)
GD 15-PND 10
Results
Ovarian weight (percent change compared to control)
Doses 0 300 600 750 900
0% 10% 9% 1% 17%
Number of corpora lutea, mean/dam (percent change compared to control)
Doses 0 40 200 1,000
DINP-1 0% -6% 0% -8%
DINP-2 0% -7% -7% -4%
DINP-3 0% -6% 0% -4%
Number of corpora lutea (in offspring at PNW 11)
Doses 0 30.7 306.7 1,164.5
percent change 0% -16% -16% -27*%
compared to control
Ovarian weight, PND 27 female pups
absolute weight 0% -13% -10% -30*%
(percent change
compared to control)
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 ofDiisononyl Phthalate
Reference and study design3
Waterman et al. (1999)
Rat (Sprague-Dawley);
23-25 dams/dose
0, 100, 500, 1,000 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 6-15; dams sacrificed at
GD 21
Waterman et al. (2000); one-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.5, 1, 1.5%
(0, 446, 889.5, 1,321 mg/kg-day
in males
0, 493.5, 951.5, 1,404 mg/kg-day
in premating females
0, 390.5, 768.5,
1,136.5 mg/kg-day during
gestation in females
0, 706.5, 1,384, 1,760 mg/kg-day
during lactation in females)b
Diet (DINP-1)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Waterman et al. (2000); two-
generation study
Rat (Sprague-Dawley),
30 breeding
pairs/dose/generation
0, 0.2, 0.4, 0.8%
Planimalsb
0, 165, 331, 665 mg/kg-day in
males
0, 182, 356, 696 mg/kg-day in
premating females
Results
Number of corpora lutea: mean/dam (percent change compared to control)
Doses 0 100 500
Mean/dam 0% -5% 0%
1,000
-2%
Ovarian weight (percent change compared to control)
Doses 0 493.5 951.5
Left 0% 8% -11%
Right 0% 4% -14%
1,404
-27*%
-36*%
Ovarian weight (percent change compared to control)
PI animals
Doses 0 182 356
Left 0% Q% 6o/o
Right 0% 5% 6%
P2(F1) animals
Doses 0 146 287
Left 0% -3% 12%
Right 0% -5% 10%
696
-17*%
fi%
555
-5%
-10%
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
0, 146, 287, 555 mg/kg-day
during gestation in females
0, 254, 539, 1,026 mg/kg-day
during lactation in females
P2(F1) animals"
0, 189, 379, 779 mg/kg-day in
males
0, 197, 397, 802 mg/kg-day in
premating females
0, 143, 288, 560 mg/kg-day
during gestation in females
0, 285, 553, 1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Results
Uterine weight
Boberg et al. (2011)
Rat (Wistar); 12 dams/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Note: 16 dams/dose were used
overall, however 4 dams/dose
were only exposed GDs 7-21 and
sacrificed at GD 21 for fetal
testosterone assessment.
Hellwig et al. (1997)
Rat (Wistar), 8-10 dams/dose
per DINP formulation
0, 40, 200, 1,000 mg/kg-day
Gavage in olive oil (DINP- 1,2,3)
GDs 6-15; dams sacrificed on
GD20
(Percent change compared to control)
Doses 0 300 600 750 900
0% 8% 5% 8% 4%
(Percent change compared to control)
Doses 0 40 200 1,000
DINP-1 0% -14% -7% -8%
DINP-2 0% -12% -10% -6%
DINP-3 0% -7% 2% -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 ofDiisononyl Phthalate
Reference and study design3
Masutomi et al. (2003)
Rats (Sprague-Dawley);
5 dams/dose; uterus weighed in
5 female pups/dose
0, 400, 4,000, 20,000 ppm
(Gestation: 0, 30.7, 306.7,
1,164.5 mg/kg-day
Lactation: 0, 66.2, 656.7,
2,656.7 mg/kg-day)
Diet(DINP-2)
GD 15-PND 10
Results
Female pups, PND 27 (percent change compared to control)
Doses 0 30.7 306.7 1,164.5
absolute weight 0% 7% -1% -48*%
PNW11
absolute weight 0% -9% 2% 2%
Maternal weight gain
Boberg et al. (2011)
Rat (Wistar); 12 dams/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Note: 16 dams/dose were used
overall; however, four
dams/dose were only exposed
GDs 7-21 and sacrificed at GD 21
for fetal testosterone
assessment.
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 25 control dams
(litters)
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Maternal body weight gain, GDs 7-21 (percent change compared to control)
Doses 0 300 600 750 900
0% 15% 9% 11% 12%
Maternal body weight gain (percent change compared to control)
Doses 0 109 555 1,513
GDs 10-20 0% -4% -6% -30*%
PNDs2-14 0% 23% 15% -35%
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design3
Clewell et al. (2013a)
Rat (Sprague-Dawley);
4-9 dams/timepoint/dose;
8 litters/dose and 9 control
litters
0, 50, 250, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GD 12-19; dams sacrificed 0.5, 1,
2, 6, 12, and 24 hours after final
dose
Gray et al. (2000)
Rat (Sprague-Dawley);
14 exposed dams, 19 control
dams
0, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GD14-PND3
Masutomi et al. (2003)
Rats (Sprague-Dawley);
5 dams/dose; uterus weighed in
5 female pups/dose
0, 400, 4,000, 20,000 ppm
(Gestation: 0, 30.7, 306.7,
1,164.5 mg/kg-day
Lactation: 0, 66.2, 656.7,
2,656.7 mg/kg-day)
Diet(DINP-2)
GD 15-PND 10
Waterman et al. (1999)
Rat (Sprague-Dawley);
23-25 dams/dose
0, 100, 500, 1,000 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 6-15; dams sacrificed at
GD21
Results
Maternal body weight gain,
Doses 0
0%
Maternal body weight gain
Doses
Maternal weight gain to
GD21
Note: 9 controls, 6 treated
Maternal weight gain to
PND3
Note: 10 controls, 8 treated
Maternal body weight gain
Doses 0
GDs 15-20 0%
PNDs 2-PND 10 0%
GDs 12-19 (percent change compared to control)
50 250 750
11% 11% 2%
(percent change compared to control)
0 750
0% -14*%
0% -32%
(percent change compared to control)
30.7 306.7 1,164.5
8% 21% -55*%
8% 13% -85*%
No significant treatment-related changes were observed in maternal body
weight gain during the overall gestation period (GDs 0-21). Compared with
controls, a significant reduction in maternal body weight was observed in the
1,000 mg/kg-day group during treatment (GDs 6-15). (Data reported
graphically).
1
2
3
4
5
6
*Statistically significant (p < 0.05) based on analysis of data conducted by study authors.
**Statistically significant difference from control group (p < 0.05), as reported by Chronic Hazard Advisory Panel
(CPSC, 2001).
aDINP formulation referenced when the study authors provided the specific formulation.
bCalculated as follows: [% in diet x intake food (mg)] -f body weight (kg) = mg/kg-day
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 ofDiisononyl Phthalate
1 "Values reported by the study authors were estimated from published graphs using "Grab It!", a Microsoft Excel
2 based free software application used to digitizes data from image files. Publisher: www.datatrendsoftware.com.
3 Percent change compared to control = ([treated value - control value] 4- control value) x 100
4
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 ofDiisononyl Phthalate
d
3
rc
1
£•
t
d
d
E
d
•s
£•
E
E
13
I/I £
0 -^
1 |
O
"o.
c
O
Q.
1
O
o
s
re
"o.
E
S
Q.
f
d
1
s
LJ_
Leeetal.,2006a,b
GD 15-PND 21; Wistar rat
Waterman etal. 1999 •statistically significant
GD6-15; Sprague-Dawley rat o not statistically significant
Hellwig etal., 1997
GD6-15; Wistar rat
Waterman etal. 1999
GD6-15; Sprague-Dawley rat
Hellwig etal., 1997
GD6-15; Wistar rat
Waterman etal. 1999
GD6-15; Sprague-Dawley rat
Boberg etal. ,2010
GD7-PND 17; Wistar rat
Hellwig etal., 1997
GD6-15; Wistar rat
Waterman et al., 2000
PND 21; Sprague-Dawley rats
Waterman etal., 2000
two-generation; Sprague-Dawley rats (PI)
Waterman etal., 2000
two-generation; Sprague-Dawley rats (P2)
Waterman et al., 2000
PND 21; Sprague-Dawley rats
Waterman et al., 2000
two-generation; Sprague-Dawley rats (PI)
Waterman etal., 2000
two-generation; Sprague-Dawley rats (P2)
Q
1
W {j UU
O (
G 6
(^^•^^
Go o
1 t? tJ
10 100 1000
Doses (mg/kg-d ay)
2
3
Figure 3-10. Exposure-response array of female reproductive fertility
measures following oral exposure to DINP.
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 ofDiisononyl Phthalate
.
c^
£
£
Masutomi etal., 2003
• statistically significant
Onot statistically significant
Bobergetal.,2010
GD 7-PND 17; Wistar rat
g Watermanet al., 2000
S PND 21; Sprague-Dawley rats
o
Waterman etal. ,2000
o two-generation; Sprague-Dawley rats (PI)
1
.1
S Waterman etal., 2000
two-generation; Sprague-Dawley rats (P2)
Masutomi et al., 2003
„ GDIS- PND 10; Sprague-Dawley rat
H
—
s-
0
g- Hellwig etal., 1997
" GD6-15; Wistar rat
^
1
c Waterman etal. 1999
GD6-15; Sprague-Dawley rat
Masutomi et al., 2003
GD 15- PND 10; Sprague-Dawley rat
£
GO
<: Bobergetal.,2010
£ GD 7-PND 17; Wistarrat
Oj
Hellwig etal., 1997
GD6-15; Wistar rat
Q
C
^
^
1 10 100 1000
Doses (mg/kg-day)
2
3
Figure 3-11. Exposure-response array of other female reproductive effects
following oral exposure to DINP.
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 ofDiisononyl Phthalate
Boberg et al., 2010
GD7-PND17;Wistarrat
Clewell et al. 2013b
GD 12- PND 14; Sprague-Dawley rat
Clewell etal. 2013a
GD 12-19; Sprague-Dawley rat
u o
£
Gray etal., 2000
GD14-PND3; Sprague-Dawley rat
Masutomi et al., 2003
GD 15- PND 10; Sprague-Dawley rat
Waterman et al. 1999
GD6-15; Sprague-Dawley rat
• statistically significant
O not statistically significant
10
100
Doses (mg/kg-day)
1000
10000
2
3
Figure 3-12. Exposure-response array of maternal weight gain effects
following oral exposure to DINP.
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2
3
Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
3,3.5. Developmental Effects
Table 3-15. Evidence pertaining to developmental effects in animals following
oral exposure to DINP
Reference and study design
Results
Skeletal and soft tissue variations
Hellwig et al. (1997)
Rat (Wistar), 8-10 dams
(litters)/dose per DINP
formulation
0, 40, 200, 1,000 mg/kg-day
Gavage in olive oil (DINP- 1,2,3)
GDs 6-15; dams sacrificed on
GD20
(NTP-CERHR (2003): Waterman
et al. (1999))b
Rat (Sprague-Dawley),
23-25 dams (litters)/dose
0, 100, 500, 1,000 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 6-15; dams sacrificed at
GD21
DINP-1: variations
Doses
% fetuses/litter
percent change
compared to control
DINP-2: variations
% fetuses/litter
percent change
compared to control
DINP-3: variations
% fetuses/litter
percent change
compared to control
0 40 200
35.3%% 41.5% 29.5%
0% 18% -16%
35.3% 37.5% 40.3%
0% 6% 14%
35.3% 29.6% 39.5%
0% 16% 12%
1,000
58.4*%
65%
36.6%
4%
60.7*%
72%
Skeletal variations
Doses
% fetuses/litter
percent change
compared to control
Visceral variations
% fetuses/litter
percent change
compared to control
0 100 500
16.4% 15% 28.3**%
0% -9% 73%
0.5% 3.3**% 3.7**%
0% 560% 640%
1,000
43.4**%
165%
5.8**%
1,060%
Pup weight
Adamsson et al. (2009)
Rat (Sprague-Dawley);
7-8 dams/dose
0, 250, 750 mg/kg-day
Gavage in corn oil
EDs 13.5-17.5; dams sacrificed
on ED 19.5
Pup weight, ED 19.5
Doses
M
F
(percent compared to control)
0 250
0% 6*%
0% 6%
750
3%
1%
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Boberg et al. (2011)
Rat (Wistar); 12 dams/dose
0, 300, 600, 750, 900 mg/kg-day
Gavage in corn oil (DINP-2)
GD7-PND17
Note: 16 dams/dose were used
overall, however 4 dams/dose
were only exposed GDs 7-21 and
sacrificed at GD 21 for fetal
testosterone assessment.
Clewell et al. (2013b)
Rat (Sprague-Dawley); 20 dams
(litters)/dose; 25 control dams
(litters)
0, 760, 3,800, 11,400 ppm (0,
109, 555, 1,513 mg/kg-day)
Diet(DINP-l)
GD12-PND14
Clewell et al. (2013a)
Rat (Sprague-Dawley);
4-9 dams/timepoint/dose;
8 litters/dose and 9 control
litters
0, 50, 250, 750 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 12-19; dams sacrificed 0.5,
1, 2, 6, 12, and 24 hours after
final dose
Hellwig et al. (1997)
Rat (Wistar), 8-10 dams (litters)/
dose per DINP formulation
0, 40, 200, 1,000 mg/kg-day
Gavage in olive oil (DINP- 1,2,3)
GDs 6-15; dams sacrificed on
GD20
Results
Pup weight, PND 13 (percent change compared to control)
Doses 0 300 600 750
M 0% -0.1% -4% -8%
F 0% -5% -10% -17*%
Male pup weight (percent change compared to control)
Doses 0 109 555
PND 2 0% -1% -6%
PND 14 0% -2% -5*%
Note: The litter was the statistical unit of comparison.
Fetal body weight (percent change compared to control)
Doses 0 50 250
GD19 0% -2.5% -1.5%
GD20 0% -2.5% -1.5%
Note: The litter was the statistical unit of comparison.
Fetal body weight (percent change compared to control)
Doses 0 40 200
DINP-1 0% 3% 3%
DINP-2 0% 5% 3%
DINP-3 0% 3% 5%
900
-11*%
-16%
1,513
-12*%
-16*%
750
0.7%
0.7%
1,000
5%
0%
-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 ofDiisononyl Phthalate
Reference and study design
Results
Lee et al. (2006b)
Pup weight, PND1 (percent change compared to control)
Rat (Wistar-lmamichi); number
of dams/dose not reported;
16-47 pups/sex/dose
0, 40, 400, 4,000, 20,000 ppm (0,
4, 40, 400, 2,000 mg/kg-day)c
Diet(DINP-2)
GD15-PND21
Doses
0
40
400
2,000
M
F
0% -4*% -5*% -8*% -16*%
0% -2*% -1% -5*% -18*%
Masutomi et al. (2003)
Pup weight gain, PNDs 2-10 (percent change compared to control)
Rats (Sprague-Dawley); 5 dams
(litters)/dose
0, 400, 4,000, 20,000 ppm
(Gestation: 0, 30.7, 306.7,
1,164.5 mg/kg-day
Lactation: 0, 66.2, 656.7,
2,656.7 mg/kg-day)
Diet(DINP-2)
GD 15-PND 10
Doses
0
30.7
306.7
1,164.5
M 0% -11% -22% -56*%
F 0% -11% -22% -56*%
Pup weight, PND 2 (percent change compared to control)
M 0% 1% -9% -16%
F 0% 6% -7% -11%
Pup weight, PND 27 (n = 5/sex/dose) (percent change compared to control)
M 0% -5% -18*% -43*%
F 0% 4% -2% -39*%
Waterman et al. (1999)
Fetal body weight, litter data (percent change compared to control)
Rat (Sprague-Dawley),
23-25 dams (litters)/dose
0,100, 500,1,000 mg/kg-day
Gavage in corn oil (DINP-1)
GDs 6-15; dams sacrificed at
GD21
Doses
0
100
500
1,000
M
F
0%
0%
4*%
5*%
2%
2%
4*%
3%
Waterman et al. (2000): one-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose
0, 0.5,1,1.5%
(0, 446, 889.5,1,321 mg/kg-day
in males
0, 493.5, 951.5,1,404 mg/kg-day
in premating females
0, 390.5, 768.5,
1,136.5 mg/kg-day during
gestation in females
0, 706.5,1,384,1,760 mg/kg-day
during lactation in females)0
Pup weight, PND 21 (percent change compared to control)
Doses
0
390.5
768.5
1,136.5
M
F
0%
0%
-10*
-8.5*
-26*
-27*
-46*%
-47*%
Note: Statistical analysis included a mixed model of covariance with pups
nested within dams, dams nested within dose, and total litter size as the
covariate.
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Preliminary Materials for the IRIS Toxicological Review ofDiisononyl Phthalate
Reference and study design
Results
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Waterman et al. (2000): two-
generation study
Rat (Sprague-Dawley),
30 breeding pairs/dose/
generation
0, 0.2, 0.4, 0.8%
PI (or Fl) animals"
0,165, 331, 665 mg/kg-day in
males
0,182, 356, 696 mg/kg-day in
premating females
0,146, 287, 555 mg/kg-day
during gestation in females
0, 254, 539,1,026 mg/kg-day
during lactation in females
P2(F2)animalsc
0,189, 379, 779 mg/kg-day in
males
0,197, 397, 802 mg/kg-day in
premating females
0,143, 288, 560 mg/kg-day
during gestation in females
0, 285, 553,1,229 mg/kg-day
during lactation in females
Diet(DINP-l)
10 weeks prior to mating, and
through mating (M) or PND 21
(F)
Pup weight,Fl offspring; PND 21 (percent change compared to control)
Doses
146
287
555
M
0%
-10*
-16*
-19*%
F 0% -9* -15* -17*%
Pup weight, F2 offspring; PND 21 (percent change compared to control)
Doses
0
143
288
560
M
F
0%
0%
-7
-7
-12*
-12*
-21*%
-22*%
1
2
3
4
5
6
7
8
9
10
11
*Statistically significant (p < 0.05) based on analysis of data conducted by study authors.
**Statistically significant difference from control group (p < 0.05), as reported by the National Toxicology Program
(NTP)-Center for the Evaluation of Risks to Human Reproduction (CERHR) to account for within-litter correlation
(NTP-CERHR, 2003).
aDINP formulation referenced only when the study authors provided the specific formulation.
Presented data from the reanalysis conducted by NTP-CERHR to account for within-litter correlation (NTP-CERHR,
2003).
Calculated as follows: [% or ppm in diet x intake food/water (mg)] 4- body weight (kg) = mg/kg-day
Percentage change compared to control = (treated value - control value) 4 control value x 100.
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 ofDiisononyl Phthalate
Hellwigetal., 1997
GD 6-15; Wistar rats
Waterman et al. 1999
GD6-15; Sprague-Dawley rat
Masutomi et al., 2003
GD 15- PND 10; Sprague-Dawley rat (male)
Masutomi et al., 2003
GD 15- PND 10; Sprague-Dawley rat (female)
Clewelletal. 2013b
GD 12- PND 14; Sprague-Dawley rat
Clewelletal.2013a
GD 12-19; Sprague-Dawley rat
Leeetal., 2006a,b
GD 15-PND 21; Wistar rat (male)
Leeetal., 2006a,b
GD 15-PND 21; Wistar rat (female)
Adamsson et al. 2009
ED 13.5 to 17.5; Sprague-Dawley rat (male)
Adamsson et al. 2009
ED 13.5 to 17.5; Sprague-Dawley rat (female)
Hellwigetal., 1997
GD 6-15; Wistar rats
Boberget al., 2010
GD 7-PND 17; Wistar rat (male)
Boberget al., 2010
GD 7-PND 17; Wistar rat (female)
Waterman etal. 1999
GD6-15; Sprague-Dawley rat (male)
Waterman etal. 1999
GD6-15; Sprague-Dawley rat (female)
Waterman et al., 2000
PND 21; Sprague-Dawley rats (PI)
Waterman etal., 2000
two-generation; Sprague-Dawley rats (PI)
Waterman et al., 2000
two-generation; Sprague-Dawley rats (P2)
• statistically significant
O not statistically significant
G — ee«
G — e«e
• — •-
)
>
•
10
Doses (mg/kg-day)
100
1000
10000
3
4
Figure 3-13. Exposure-response array of developmental effects following oral
exposure to DINP.
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 ofDiisononyl Phthalate
3,3,6. Hematopoietic Effects
Table 3-16. Evidence pertaining to hematopoietic effects in animals following
oral exposure to DINP
Study design and reference3
Hematology
Bio Dynamics (1986)
Rat (Sprague-Dawley); 70/sex/dose
0, 500, 5,000, 10,000 ppm (0, 27, 271,
553 mg/kg-day in males;
0, 33, 331, 672 mg/kg-day in females)
Diet (SANTICIZER 900)
2 years (interim sacrifice at 1 year)
Lington et al. (1997)
Rat (F344); 110/sex/dose
0, 0.03. 0.3. 0.6% (0, 15, 152, 307
mg/kg-day in males;
0, 18, 184, 375 mg/kg-day in females)
Diet(DINP-l)
2 years (interim sacrifices at 6, 12, and
io montns)
Covance Laboratories (1998b)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm (0, 29,
88, 359, 733 mg/kg-day in males;
0, 36, 109, 442, 885 mg/kg-day in
females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in males;
733 mg/kg-day in females)
Diet
Main study: 2 years (interim sacrifices
at 1, 2, 13, and 79 weeks)
Hematology at 2 years (n =
control)
Doses (M) 0
RBCs 0%
Hgb 0%
Hct 0%
Doses (F) 0
RBCs 0%
Hgb 0%
Hct 0%
Hematology at 2 years
compared to control)
Doses (M) 0
RBCs °%
Hgb 0%
Hct 0%
Doses (F) 0
RBCs 0%
Hgb 0%
Hct 0%
Hematology at 104 weeks (i
compared to control)
Doses (M) 0 29
RBCs 0% 4%
Hgb 0% 5%
Hct 0% 4%
Doses (F) 0 36
RBCs 0% -4%
Hgb 0% -4%
Hct 0% -4%
Results
10/sex/dose) (percent change compared to
27 271 553
-8% 4% -17*%
-13% 0% -18*%
-14% 0% -19*%
33 331 672
-20% -10% -15%
0% 7% 1%
0% 11% 3%
(n = 19-20/sex/dose) (percent change
15 152 307
0% -so/0 -14*%
-6% -8% -19*%
-5% -8% -19*%
18 184 375
-4% -14% -14%
-5% -15% -13%
-5% -14% -13%
i = 9-10/sex/dose) (percent change
88 359 733 Recovery
-3% -16% -21% -17%
-2% -15% -20*% -15%
-3% -15*% -19*% -12%
109 442 885 Recovery
-3% -18*% -26*% -3%
-3% -16% -25*% -1%
-2% -14% -24*% -1%
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 ofDiisononyl Phthalate
Study design and reference3
Results
Recovery group: 78 weeks, followed by
a 26-week recovery period with basal
diet alone
Spleen weight0
Lington et al. (1997)
Rat (F344); 110/sex/dose
0, 0.03. 0.3. 0.6 wt% (0, 15, 152, or
307 mg/kg-day in males;
0,18,184, or 375 mg/kg-day in
females)
Diet(DINP-l)
2 years
Spleen weight at terminal sacrifice (n = 48-65/sex/dose) (percent
change compared to control)
Doses (M)
0
15
152
307
spleen/body weight
0%
17%
61*%
61*%
Doses (F)
18
184
375
spleen/body weight
0%
29%
5%
57*%
Covance Laboratories (1998b)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm (0, 29,
88, 359, or 733 mg/kg-day (M);
0, 36,109, 442, or 885 mg/kg-day (F)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in males;
733 mg/kg-day in females)
Spleen weight at terminal sacrifice (n = 27-42/sex/dose) (percent
change compared to control)
Doses (M)
0
29
88 359 733
Recovery
absolute weight 0% -15% -31% 33% 33% 38%
spleen/body weight 0% -14% -30% 38% 53% 45%
Doses (F)
0
36
109 442 885 Recovery
Diet
Main study: 2 years (interim sacrifices
at 1, 2,13, and 79 weeks)
Recovery group: 78 weeks, followed by
a 26-week recovery period with basal
diet alone
absolute weight 0% 64% 3% 16*% 121*% 51%
spleen/body weight 0% 81% 18% 23% 150*% 61*%
Mononuclear cell leukemia (MNCL)
Bio Dynamics (1986)
Rat (Sprague-Dawley); 70/sex/dose
0, 500, 5,000,10,000 ppm (0, 27, 271,
553 mg/kg-day in males;
0, 33, 331, 672 mg/kg-day in females)
Diet (SANTICIZER 900)
2 years (interim sacrifice at 1 year)
Evaluated, but incidences were not reported by study authors
(EPLU999): Lington et al. (1997))
Rat (F344); 110/sex/dose
0, 0.03. 0.3. 0.6% (0, 15, 152,
307 mg/kg-day in males;
0,18,184, 375 mg/kg-day in females)
Diet(DINP-l)
104-week terminal sacrifice
Doses (M)
0
15
152
307
incidence11
percentage
32/81
40%
27/80
34%
48/80**
60%
49/80**
61%
Doses (F)
0
18
184
375
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 ofDiisononyl Phthalate
Study design and reference3
Results
2 years (interim sacrifices at 6,12, and
18 months)
incidence11
22/81
21/81
29/80
41/80*
Covance Laboratories (1998b): EPL
(1999)
Rat (F344); 70 or 85/sex/dose
0, 500, 1,500, 6,000, 12,000 ppm (0, 29,
88, 359, 733 mg/kg-day in males;
0, 36,109, 442, 885 mg/kg-day in
females)
Recovery group (55/sex/dose):
12,000 ppm (637 mg/kg-day in males;
733 mg/kg-day in females)
Diet
Main study: 2 years (interim sacrifices
at 1, 2,13, and 79 weeks)
Recovery group: 78 weeks, followed by
a 26-week recovery period with basal
diet alone
104-week terminal sacrifice
Doses (M)
0
29
88
359
733 Recovery
incidence11 21/55 23/50 21/50 32/55** 28/55** 30/50
percentage 38% 46% 42% 58% 51% 60%
Doses (F)
0
36
109
442
885 Recovery
incidence11 17/55 16/50 9/50 28/55** 28/55** 24/50
percentage 31% 32% 18% 51% 51% 48%
Covance Laboratories (1998a)
Mouse (B6C3Fi); 70/sex/dose
0, 500, 1,500, 4,000, 8,000 ppm (0, 90,
276, 742,1,560 mg/kg-day in males;
0,112, 336, 910,1,888 mg/kg-day in
females)
Recovery group (55/sex/group):
1,560 mg/kg-day
Diet
Main study: 2 years (interim sacrifice at
79 weeks)
Recovery group: 78 weeks, followed by
a 26-week recovery period with basal
diet alone
Evaluated, but incidences were not reported by study authors
1
2
3
4
5
6
7
8
9
10
11
*Statistically significant from control group (p < 0.05), as reported by study authors.
**Statistically significant from control (p < 0.05), as reported by Chronic Hazard Advisory Panel (CPSC, 2001).
aDINP formulation referenced when the study authors provided the specific formulation.
blncidence data as reported by Pathology Working Group reanalysis (EPL, 1999)
°Spleen weight measured but no difference observed among exposed group (Kwack et al., 2009)
Calculated as follows: [% in diet x intake food/water (mg)] -f body weight (kg) = mg/kg-day.
Percent change compared to control = ([treated value - control value] 4- control value) x 100
Hgb = hemoglobin; Hct = hematocrit; RBC = red blood cell
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 ofDiisononyl Phthalate
I
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (male)
Bio Dynamics, 1986
2 years; Sprague-Dawley, rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
Lington et al., 1997
2 years; F344, rat (male)
Lington et al., 1997
2 years; F344, rat (female)
Covance, 1998b
2 years; F344, rat (male)
Covance, 1998b
2 years; F344, rat (female)
• statistically significant
O not statistically significant
-6 •
10 100
Doses (mg/kg-day)
1000
2
3
Figure 3-14. Exposure-response array of hematopoietic effects following oral
exposure to DINP.
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 ofDiisononyl Phthalate
l 3.4. PRELIMINARY MECHANISTIC INFORMATION FOR DINP
2 The systematic literature search for DINP also identified studies evaluating mechanisms of
3 action considered potentially relevant to effects observed following exposure to DINP. Studies
4 were included if they evaluated mechanistic events following exposure to DINP formulations or
5 metabolites, or contained information relevant to the mechanistic understanding of DINP toxicity.
6 Reviews or analyses that do not contain original data are not included here, but may be considered
7 in later 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 HERO record (contained within a URL
16 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 [http://hero.epa.gov/index.cfm?action=-
20 reference.details&reference id=2 347390]. To access the database, click on the link at the top of the
21 web page and select "download" and then "ok" to view the spreadsheet in Excel. This spreadsheet
22 may also be saved to your desktop by downloading and selecting "save." The resulting inventory of
23 DINP mechanistic studies consists of 60 mechanistic outcomes from 22 in vivo studies, as well as 45
24 mechanistic outcomes from 17 in vitro assays. Table 3-17 presents a summary of the mechanistic
25 outcomes recorded in the database from each study identified.
26 The mechanistic categories developed here are not mutually exclusive and are designed to
27 facilitate the analysis of similar studies and experimental observations in a systematic manner.
28 This process will allow the identification of mechanistic events that contribute to mode(s) of action
29 (MOAs) and/or adverse outcome pathways (AOPs) following DINP exposure. The mechanistic
30 categories assigned to each mechanistic outcome reported by an individual study are as follows: 1)
31 mutation, including investigations of gene and chromosomal mutation; 2) DNA damage, including
32 indicator assays of genetic damage; 3) DNA repair; 4) oxidative stress; 5) cell death and division
33 (this captures a broad range of assays, but it is useful to consider them together as observations
34 resulting from cell cycle alterations; 6) pathology, which includes morphological evaluations
35 pertaining to the dysfunction of organs, tissues, and cells; 7) epigenetic effects, which are
36 observations of heritable changes in gene function that cannot be explained by changes in the DNA
37 sequence; 8) receptor-mediated and cell signaling effects; 9) immune system effects; 10) cellular
38 differentiation and transformation; 12) cellular energetics; and 13) "other," to capture those
39 mechanistic outcomes not easily assigned to a defined category. Mechanistic outcomes in the
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 ofDiisononyl Phthalate
1
2
3
4
5
6
7
8
9
10
11
12
13
14
"other" category include gene expression from mouse liver and rat hypothalamus, rat serum
hormone levels, rat kidney alpha2u globulin, and numerous measurements of rat testicular function
(hormone, protein, and mRNA measurements).
Information summarized in Table 3-17 and Figure 3-15 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.
Table 3-17. Summary of mechanistic outcomes evaluated following DINP
administration
Mechanistic
category
Mutation3
DNA damage
DNA repair
Oxidative stress
Cell death and
division
Pathology
Epigenetics
Receptor-mediated
and cell signaling15
Immune system
Cellular
differentiation and
transformation15
Cellular energetics
Other
Total
Total #
mechanistic
outcomes/
# studies
9/6
1/1
14/7
20/8
26/8
5/3
16/10
14/6
105/35
In vivo (ff mechanistic
outcomes/ft studies)
Total
2/2
0
7/5
20/8
9/5
5/3
3/2
14/6
Primate
0
0
1/1
0
3/2
0
1/1
0
Rat
1/1
0
2/2
19/8
4/3
1/1
1/1
13/5
Mouse
1/1
0
4/3
1/1
2/2
4/2
1/1
1/1
60/22
In vitro (ff mechanistic outcomes/
# studies)
Total
7/5
1/1
7/2
N/A
17/5
0
13/8
0
Human
0
0
4/2
Primate
0
0
0
Rat
0
1/1
3/2
Mouse
3/3
0
0
N/A
5/4
0
2/2
0
3/2
0
1/1
0
6/5
0
1/1
0
2/2
0
8/8
0
45/17
15
16
17
18
19
aDatabase also included three experimental measures in two studies utilizing bacteria, and one experimental
measure from one study using Chinese hamster ovary cells, not listed.
bDatabase also included one experimental measure in one study utilizing primary hepatocytes from Syrian golden
hamsters, not listed.
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 ofDiisononyl Phthalate
1
2
3
4
5
6
7
8
9
Note: The shaded rows represent categories for which no experimental measures were identified in the
database, from any species, in any kind of model system (e.g. in vitro, in vivo, biochemical, etc). Additionally, 10
studies did not have pdfs available to provide the information needed for collection into the spreadsheet (pdfs
have been requested for a future data collection).
Mechanistic Data
(105 mechanistic outcomes from 35 reports)
In vivo • In vitro
Mutation
DNA damage
Cell death and division
Pathology
Receptor-mediated and cell signaling
Immune system
Cellular differentiation and transformation
Other
10
11
12
13
10 15 20
Number of mechanistic outcomes
25
Figure 3-15. Summary of in vivo and in vitro mechanistic data by mechanistic
category
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 ofDiisononyl Phthalate
2
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1 Chou, YY; Huang, PC; Lee, CC; Wu, MH; Lin, SJ. (2009). Phthalate exposure in girls during early puberty. J
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14 repeated dosing in pregnant rats. Reprod Toxicol 35: 5669.
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48 report-pbEUNA20784/
This document is a draft for review purposes only and does not constitute Agency policy.
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1 utero exposure to diethylhexyl phthalate, diisobutyl phthalate, diisoheptyl phthalate, and
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48 http://dx.doi.org/10.1097/01.ede.0000164555.19041.01
This document is a draft for review purposes only and does not constitute Agency policy.
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36
This document is a draft for review purposes only and does not constitute Agency policy.
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