&EPA
United States
Environmental Protection
Agency
Male reproductive toxicity in animal studies of diisobutyl phthalate (DIBP): a case study
application of systematic review approaches
Erin E. Yost1, Susan Y. Euling2, James A. Weaver1, Brandiese E. J. Beverly1, Nagalakshmi Keshava1, Anuradha Mudipalli1 Xabier Arzuaga2, Todd Blessinger2, Laura Dishaw1, Andrew
Hotchkiss1, Susan L. Makris2
1 US EPA National Center for Environmental Assessment, Research Triangle Park, NC;2 US EPA National Center for Environmental Assessment, Washington, DC
Introduction
Diisobutyl phthalate (DIBP) is used as a plasticizer in a variety of industrial and consumer
products. Although DIBP has been less widely studied compared to other phthalates, there is
evidence that DIBP and its primary metabolite, monoisobutyl phthalate (MIBP), cause male
reproductive toxicity. A recent systematic review of endocrine-related low-dose toxicity by
the National Academies of Sciences (NAS) evaluated the effects of DIBP on three anti-
androgenic outcomes [testosterone, anogenital distance (AGD), and hypospadias], and
concluded that DIBP is a presumed human hazard based on decreased fetal testosterone in
rodents exposed during gestation. The Integrated Risk Information System (IRIS) performed
a systematic review of male reproductive effects of DIBP exposure that considered all
outcomes and all life stages of exposure, following recommendations in the 2014 NAS
review of the IRIS program. Here, we use studies that evaluated testosterone in male rodents
exposed to DIBP or MIBP as a case study of the IRIS systematic review process. We also
summarize the overall conclusions for male reproductive effects identified in the IRIS
systematic review of DIBP, and compare these results to the findings of NAS.
Methods
Animal studies for DIBP or MIBP were identified by searching four online databases
(PubMed, Web of Science, Toxline, and TSCATS2), using search terms designed to capture all
potentially pertinent studies. The last update was in July 2017. Title/abstract screening was
used to identify primary health effect studies that exposed non-human mammalian animals to
any administered dose of DIBP or MIBP via oral, dermal, or inhalation routes. These studies
were evaluated by at least two reviewers using the approach in Figure 1.
Individual study level domains
Domain judgments
Judgment
Good
Adequate
Poor
Critically
Deficient
Interpretation
Appropriate study conduct relating to the domain & minor
deficiencies not expected to influence results.
A study that may have some limitations relating to the domain, but
they are not likely to be severe or to have a notable impact on
results.
Identified biases or deficiencies interpreted as likely to have had a
notable impact on the results or prevent reliable interpretation of
study findings.
A serious flaw identified that is interpreted to be the primary
driver of any observed effect or makes the study uninterpretable.
Study is not used without exceptional justification.
Animal
Reporting Quality
Selection or Performance Bias
• Test animal allocation
• Blinding of investigators
Confounding/Variable Control
Reporting or Attrition Bias
Exposure Methods Sensitivity
• Utility of exposure design
• Characterization of exposure
Outcome Measures and Results Display
• Sensitivity, specificity, and usability of results
• Presentation of results
Overall study rating
Rating
High
Medium
Low
Uninformative
Interpretation
No notable deficiencies or concerns identified; potential for bias unlikely or minimal;
sensitive methodology.
Possible deficiencies or concerns noted, but resulting bias or lack of sensitivity would be
unlikely to be of a notable degree.
Deficiencies or concerns were noted, and the potential for substantive bias or inadequate
sensitivity could have a significant impact on the study results or their interpretation.
Serious flaw(s) makes study results unusable for hazard identification
Figure 1. Study evaluation process
After study evaluation, the evidence for each health effect outcome was synthesized
according to the developmental stage of exposure. Based on this synthesis, the evidence was
assigned a conclusion of robust, moderate, slight, indeterminate, or compelling evidence of no
effect. The ratings for individual outcomes were summarized into an overall conclusion for
male reproductive effects using a structured framework (see Poster by Yost et al.).
Unique articles
identified for
title/abstract
screening
(N=1,363)
Eligible for study
evaluation
(N=25)
Included after
study evaluation
(N=19)
Male repro studies
(N=16)
Testosterone
studies
(N=11)
Figure 2. Abbreviated literature flow diagram
U.S. Environmental Protection Agency
Office of Research and Development
Table 1. Animal studies of testosterone and DIBP or MIBP exposure. Of the 11 studies that
evaluated testosterone in male rats or mice, 7 exposed animals during gestation and/or until
weaning, and 4 were postnatal exposures of males near the time of puberty. The postnatal exposure
studies had higher risk of bias because of reporting limitations, including uncertainty about the
pubertal status of the test animals at the time of exposure.
Study description
Study evaluation
Population Exposure Outcome
Borch et al.
2006
Rat (Wistar)
Diet
GD 7-19
Howdeshell et
al. 2008
Rat
(Sprague-
Dawley)
Gavage
GD 8-18
Saillenfait et
al. 2017
Rat
(Sprague-
Dawley)
Gavage
GD 13-19
Furr et al.
2014
Rat
(Sprague-
Dawley)
Gavage
GD 14-18
Hannas et al.
2012
Rat
(Sprague-
Dawley)
Gavage
GD 14-18
Hannas et al.
2011
Rat
(Sprague-
Dawley)
Gavage
GD 14-18
Wang et al.
2017
Mouse (ICR)
Diet Postnatal
GD 0-21; and Adult
GD 0-PND 21 T cone
Oishi and
Hiraga 1980a
Oishi and
Hiraga 1980b
Mouse
(JCLICR)
Mouse
(JCLICR)
Diet
PND 35-42
Diet
PND 35-42
ni
Qj U —
d>
O c
G G High
G ¦ ¦ High
G G G G G G High
G G G High
FetalT
prod
G G G G G
FetalT
prod
G G G High
Medium
Medium
Oishi and
Hiraga 1980c
Rat Diet
(JCL:Wistar) PND 35-42
Postnatal
T cone
NR
Oishi and
Hiraga 1980d
Rat Diet
(JCL:Wistar) PND 35-42
Postnatal
T cone
Abbreviations: Gestation day (GD); Postnatal day (PND); Testosterone (T) production (prod) or concentration (cone)
Study
Borch 2006
Species and Strain
Rat Wistar
Exposure Duration Endpoint
GD7 to GD20/21
Hannas 2011
Hannas 2012
Rat Sprague-Dawley (Harlan) GD14 to GD18
Rat Sprague-Dawley (Harlan) GD14 to GD18
Rat Sprague-Dawley
Howdeshell 2008 Rat Sprague-Dawley
Saillenfait 2017 Rat Sprague-Dawley
GD14 to GD18
GD8 to GD18
GD 13-19
GD 0 to PND 21
in testicular testosterone (T) content (GD 20/21)
in testicular testosterone (T) production (GD 20/21)
in testicular testosterone (T) content (GD 19)
in testicular testosterone (T) production (GD 19)
in testicular testosterone (T) production (GD 18; block 2)
in testicular testosterone (T) production (GD 18; block 14)
in testicular testosterone (T) production (GD 18; block 30)
in testicular testosterone (T) production (GD 18)
in testicular testosterone (T) production (GD 18)
in testicular testosterone (T) production (GD 18)
in testicular androstenedione (AN) production (GD 19)
in testicular testosterone (T) production (GD 19)
in testicular testosterone (T) production (PND 80)
in serum testosterone (T) concentration (PND 21)
in serum testosterone (T) concentration (PND 80)
in testicular testosterone (T) production (PND 21)
in serum testosterone (T) concentration (PND 21)
in testicular testosterone (T) concentration (PND 21)
in serum testosterone (T) concentration (PND 80)
in testicular testosterone (T) concentration (PND 80)
O Not significantly changed
^ Significant decrease
High confidence (H)
Medium confidence (M)
V (")
V
• (H)
• (H)
V
V
V
V
V (H>
•
(M)
V
(M)
•
A
1,000
Log Dose (mg/kg-day)
Figure 4. Summary of exposure-response for testosterone from postnatal exposure studies.
The synthesis of results for testosterone is summarized in an evidence profile table
(Table 2). Gestational exposure studies provided robust evidence for effects on
testosterone, whereas evidence from postnatal exposure studies was found to be
indeterminate. Evidence judgments for other male reproductive endpoints
identified in this systematic review are summarized in Table 3.
Table 2. Evidence profile table for animal studies of testosterone and DIBP
Studies and
interpretation
High confidence
Borch et al. 2006
Furr et al. 2014
Hannas et al. 2011
Hannas et al. 2012
Howdeshell et al.
2008
Saillenfait et al.
2017
Medium confidence
Wang et al. 2017
Medium confidence
Oishi and Hiraga
1980a
Oishi and Hiraga
1980b
Oishi and Hiraga
1980d
Low confidence
Oishi and Hiraga
1980c
Factors that
increase strength
Consistency
Exposure-
response gradient
Effect size
Biological
plausibility
(support from
mechanistic
evidence)
Minimal concern
for bias
Biological
plausibility
Factors that
decrease strength
High risk of bias
Unexplained
inconsistency
Summary of findings
ROBUST
A dose-related decrease in
testicular androgen levels or
production (up to -96% compared
to control) was observed in all
studies in rats and mice that
evaluated this endpoint. Several
of these studies also demonstrate
decreased testicular expression
of genes and proteins in the
steroidogenesis pathway, which
provides support for biological
plausibility.
oco
INDETERMINATE
A dose-related increase in
androgen levels was observed in
two rat studies (Oishi and Hiraga
1980c-d), whereas androgen
levels were decreased or not
changed in mice (Oishi and
Hiraga 1980a-b).
Table 3. Within stream evidence judgments for animal evidence of male
reproductive toxicity following DIBP exposure
Outcome
Includes these endpoints
Evidence following
gestational exposure
Evidence following
postnatal exposure
Testosterone
Androgen levels
Robust
Indeterminate
Male morphological
development
AGD, nipple retention, preputial
separation, hypospadias, cleft
prepuce, exposed os penis,
cryptorchidism
Robust
N/A
Sperm evaluation
and histopathological
effects in testis or
Sperm concentration and motility,
oligospermia, azoospermia,
granulomatous inflammation,
Robust
Moderate
epididymis
tubular degeneration, tubular
necrosis, interstitial hyperplasia
Reproductive organ
weiqht
Testis, epididymis, seminal vesicle
weights
Moderate
Moderate
Male reproductive
overall
Robust
Discussion
Overall, the results from animal studies of male reproductive effects provide robust
evidence of a hazard from DIBP exposure. Conclusions for testosterone are
consistent with those of NAS (2017). The NAS review was limited to gestational
exposure studies and excluded studies that exposed animals to a single high dose
(e500 mg/kg-day); therefore, NAS only considered two fetal testosterone studies,
and had inadequate evidence to evaluate the effects of DIBP on AGD or
hypospadias. The IRIS systematic review included all dose levels and life stages of
exposure, and was able to evaluate a wider range of androgen-dependent and -
independent male reproductive outcomes. Disclaimer: The views expressed in this
poster are those of the author (s) and do not necessarily reflect the views or policies
of the U.S. Environmental Protection Agency.
o
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